MXPA99006688A - Tissue factor methods and compositions for coagulation and tumor treatment - Google Patents

Abstract

The invention embodies the surprising discovery that Tissue Factor (TF) compositions and variants thereof specifically localize to the blood vessels within a vascularized tumor following systemic administration. The invention therefore provides methods and compositions comprising coagulant-deficient Tissue Factor for use in effecting specific coagulation and for use in tumor treatment. The TF compositions and methods of present invention may be used alone, as TF conjugates with improved half-life, or in combination with other agents, such as conventional chemotherapeutic drugs, targeted immunotoxins, targeted coaguligands, and/or in combination with Factor VIIa(FVIIa) or FBVIIa activators.

Description

METHODS OF TISSUE FACTOR AND COMPOSITIONS FOR COAGULATION AND TREATMENT OF TUMORS BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates in general to the fields of blood vessels and coagulation. More particularly, it encompasses the surprising finding that Tissue Factor compositions can localize the tumor vasculature and cause specific coagulation. Methods and compositions are particularly provided for effecting specific coagulation and for treating tumors with Tissue Factor (FT) compositions and combinations of Tissue Factor and other Molecules. 2. Description of the Related Art The resistance of tumor cells to various chemotherapeutic substances represents a major problem in clinical oncology. Therefore, although many advances have been made in the chemotherapy of neoplastic disease over the past 30 years, most of the most prevalent forms of human cancer still resist effective chemotherapeutic intervention. A significant underlying problem that must be addressed in any treatment regimen is the concept of "total cell elimination". This concept holds that • in order to have an effective treatment regimen, be it the surgical or chemotherapeutic approach or both, there must be a total elimination of the cells, of the so-called "clonogenic" malignant cells, that is, cells that have the ability to grow uncontrollably and replace any tumor mass that could have been removed. Due to the final need to develop therapeutic substances and regimens that achieve a total elimination of cells, certain types of tumors have been more docile than others to therapy. For example, soft tissue tumors (eg, lympholas), and tumors of blood and blood-forming organs (e.g., leukemias) have generally responded better to chemotherapeutic therapy than solid tumors such as carcinomas. One reason for the susceptibility of soft and blood-based tumors to chemotherapy is the greater physical accessibility of lymphoma and leukemia cells to the chemotherapeutic intervention. Quite simply, it is much more difficult for most chemotherapeutic substances to reach all the cells of a solid tumor mass than for soft tumors and blood-based tumors, and therefore it is much more difficult to achieve total cell elimination. Increasing the dose of chemotherapeutic substances frequently results in toxic side effects, which generally limit the effectiveness of conventional antitumor substances. It has long been clear that there is a significant need for the development of novel strategies for the treatment of solid tumors. One of these strategies is the use of "immunotoxins", in which an antitumor cell antibody is used to deliver a toxin to the tumor cells. However, as with the chemotherapeutic approach described above, it also suffers from certain disadvantages. For example, antigen-deficient or antigen-deficient cells can survive and re-populate the tumor or lead to other metastases. Also, in the treatment of solid tumors, the mass of the tumor is generally impermeable to molecules of the size of the antibodies and immunotoxins. Therefore, the development of immunotoxins alone does not lead to particularly significant improvements in the treatment of cancer. Some researchers then developed the approach of attacking the vasculature of solid tumors. Targeting the blood vessels of tumors has certain advantages because it is not likely to lead to the development of resistant tumor cells or populations thereof. In addition, the administration of target substances to the vasculature has no problems connected with accessibility, and the destruction of the blood vessels will lead to an amplification of the antitumor effect since many tumor cells depend on a single vessel for their supply of oxygen and nutrients. Strategies with exemplary vascular targets are described in Burrows et al. (1992), Burrows and Thorpe (1993) and WO 93/17715. This targeted administration of anti-cellular substances to the tumor vasculature provides quite promising strategies, however, the use of toxin portions of these molecules still leaves room for improvement in the vascular target. Another approach to targeted destruction of the tumor vasculature has been reported in WO 96/01653, in which antibodies against markers of the tumor vasculature are used to deliver coagulants to the vasculature of solid tumors. The targeted administration of coagulants in this manner has the advantage that significant toxic side effects from any background that fails the target that could result from any low-level cross-reactivity of the targeted antibodies with normal tissue cells are unlikely to result. . Antibody-coagulant constructs for use in this targeted antitumor therapy have been called "coaguligands" (WO 96/01653). Although the specific administration of a coagulant to a tumor vessel marks a surprising breakthrough, the use or manipulation of coagulation in connection with the treatment of various human diseases and disorders has been practiced for some time. By way of example only, Morrissey and Comp have proposed the use of Truncated Tissue Factor (tTF) in combination with Factor Vlla (FVIIa) in the treatment of patients, such as hemophiliacs, in which blood coagulation is prevented. (U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067). Roy and Venar have also developed Tissue Factor mutants that neutralize the endogenous Tissue Factor and can be used as anticoagulants, e.g., in the treatment of myocardial infarction (U.S. Patent Nos. 5,346,991 and 5,589,363). In other studies connected with the Tissue Factor (FT), Edgington and colleagues have shown that, in contrast to normal melanocytes, human melanoma cells with malignant metastasis express high levels of Tissue Factor, the major cellular initiator of the cascades of plasma coagulation protease (WO 94/28017; WO 94/05328; U.S. Patent No. 5,437,864). It was reported that inhibition of Tissue Factor function and subsequent reduction in local protease generation resulted in significantly reduced numbers of tumor cells retained in the vasculature. This leads to the suggestion that there is a direct correlation between the expression of the Tissue Factor and the metastatic phenotype of the tumor cells. Edgington and colleagues proposed that a Tissue Factor function is required for the successful implantation of tumor cells and that interference with tissue factor function, or specific interference with cell surface expression of the Tissue Factor, is useful to inhibit metastasis. These authors have therefore proposed to treat the cancer with antibodies directed against the Tissue Factor. SUMMARY OF THE INVENTION In direct contrast to the previous observations of Edgington and colleagues and the uses of tissue anti-factor antibodies to treat cancer, the present inventors have demonstrated that truncated tissue factor compositions and tissue factor variants can, in themselves, used in the treatment of solid tumors. The present invention was developed, in part, from the inventors' surprising discovery that the truncated Tissue Factor specifically locates blood vessels within a vascularized tumor simply after systemic administration. This location in the absence of any target fraction could not have been predicted from the previous detailed studies of the Tissue Factor molecule. The self-locating nature of the Tissue Factor, as described herein, also contrasts with the above-described uses of the Tissue Factor in the treatment of blood disorders, e.g., in hemophiliacs, in which the administration and the action of the tissue factor either not localized or is limited to topical application in a specific area. Therefore, in certain embodiments, the present invention provides methods for promoting coagulation in prothrombotic blood vessels of an animal or patient, these methods generally comprise administering to the animal a composition comprising a compound of Coagulation Tissue Factor deficient in an amount effective to promote coagulation preferentially, or specifically, in prothrombotic blood vessels. As used throughout the application, the terms "a" and "an" are used in the sense that they mean "at least one (one)" "at least one first or one", "one, one or more "or" a plurality "of the components or steps mentioned, except in cases where an upper limit is specifically set after that. Therefore, "a poor coagulation tissue factor" means "at least a first coagulation tissue factor deficient". The limits and operable parameters of combinations, as well as with the amounts of any single substance, will be known to those of ordinary skill in the field in light of the present disclosure. Prothrombotic blood vessels may be associated with any of a variety of angiogenic diseases, with benign growth or with a vascularized tumor. In the context of the present invention, the term "a vascularized tumor" means a vascularized malignant tumor. The present invention is particularly advantageous for treating vascularized tumors of at least approximately medium size and for treating large vascularized tumors. The composition is generally pharmaceutically acceptable and preferably will be administered to the animal systemically, such as via intravenous injection. The methods of the invention are further described as methods for treating an animal or human patient having a disease associated with prothrombotic blood vessels, which comprises administering to the animal an amount of at least one first coagulant composition comprising at least one first factor compound. Effective deficient coagulation tissue for preferentially, or specifically, promoting coagulation in the prothrombotic blood vessels associated with the site of benign or malignant disease. The essence of the invention can also be defined as a composition comprising at least a biologically effective amount of at least one first coagulation tissue factor compound deficient for use in the preparation of a medicament for use in promoting coagulation preferentially , or specifically, in prothrombotic blood vessels of an animal, particularly those associated with a site of benign or malignant disease. In the methods, medicaments and uses of the present invention, one of the advantages is in the fact that the mere provision of the coagulant composition in the systemic circulation of the animal results in the specific or preferential localization of the Tissue Factor compound to the site of the disease. Preferred methods described herein are for use in promoting coagulation in the tumor vasculature of an animal or human subject having a vascularized tumor, these methods generally comprise administering to the animal one or more compositions comprising • one or more compounds of deficient coagulation tissue factor in an amount sufficient to specifically or preferentially promote coagulation in the tumor vasculature. The treatment of vascularized tumors of medium or large size is particularly advantageous. Treatment methods can be described as methods for treating an animal having a vascularized tumor, comprising administering to the animal a biologically effective amount of at least one coagulant composition comprising an amount of at least one first Tissue Factor compound of coagulation deficient enough to specifically or preferentially promote coagulation in the tumor vasculature. Another description is of a method for treating an animal or patient having a vascularized tumor which comprises systemically administering to the animal one or more compositions comprising one or a plurality of deficient coagulation tissue factor compounds in an amount (amounts) and for an effective period of time to promote coagulation specifically or preferentially in the vasculature of the vascularized tumor. The antitumor effects of the present invention are particularly described in the methods characterized in that they comprise administering to the animal with a tumor a composition comprising at least one clotting factor compound deficient in an effective amount to promote coagulation in the vasculature of the tumor and to specifically or preferentially cause tissue necrosis in the tumor. These aspects of the invention also provide a composition comprising at least a biologically effective amount of at least one first coagulation tissue factor compound deficient for use in the preparation of a medicament for use in promoting coagulation preferentially, or specifically , in the prothrombotic blood vessels associated with a malignant vascularized tumor of an animal; wherein the medicament is thus destined for use to treat an animal with cancer causing coagulation of the blood vessels of the tumor and tumor necrosis. The terms "preferentially" and "specifically", as used herein in the context of promoting coagulation in prothrombotic blood vessels or the vasculature of a tumor, and / or as used in the context of promoting sufficient coagulation to cause tissue necrosis at a disease site such as a tumor, means that the tissue factor compound or the combination of (tissue factor) - (second substance) functions to achieve coagulation and / or tissue necrosis that is substantially confined to the site of the disease, such as the tumor region, and does not extend substantially causing clotting or tissue necrosis in healthy, normal tissues. The deficient coagulation tissue factor compound or combinations thereof thereby exert coagulatory and / or tissue destroying effects at a disease or tumor site and therefore have little or no clotting or tissue destroying effect on cells. or normal, healthy tissues. The coagulation and / or destruction of the tissue is therefore located at the disease site or tumor and does not extend substantially or significantly to other large or important blood vessels or tissues. Therefore, the methods of the invention are maintains the function of healthy cells and tissues substantially undamaged "Poor coagulation tissue factors" of the invention will generally be tissue factor compounds that are at least one hundred times less active than the original full-size tissue factor. , e.g., when tested in an appropriate phospholipid environment The Tissue Factor compounds will still have activity, and are preferably described as being about one hundred times and about one million times less active than the original Tissue Factor of full size, eg, when tested in an appropriate phospholipid environment The compounds of coagulation tissue factor deteriorate preferably, they will be at least about one thousand times less active than the original, full-length Fabric Factor, - more preferably they will be at least ten thousand times less active than the original full-length Fabric Factor; even more preferably they will be at least about one hundred thousand times less active than the full length natural Tissue Factor, e.g., when tested in a suitable phospholipid environment. The "at least approximately one hundred thousand times less active" is not the minimum, and the Tissue Factor compounds can be at least about five hundred thousand times or about one million times less active than the original full-length Tissue Factor, v .gr., when tested in an appropriate phospholipid environment. Human tissue factor compounds will generally be preferred for human uses, but the use of other Tissue Factor species, which include E. coli Tissue Factor, is not excluded. For ease of preparation, deficient coagulation tissue factor compounds will also preferably be prepared by recombinant expression, although this is not essential. The Tissue Factor may render the coagulation deficient to be deficient in binding to a phospholipid surface and / or deficient in inserting into a phospholipid or lipid bilayer membrane. Preferred examples are the "truncated tissue factors". As defined in U.S. Patent No. 5,504,064, in which the compounds are used for different purposes, "truncated tissue factors" generally have an amino acid sequence that differs from that of the original Tissue Factor because sufficient Transmembrane amino acids that function to bind the original Tissue Factor with the phospholipid membranes are missing from the truncated Tissue Factor protein so that the truncated Tissue Factor protein does not bind to the phospholipid membranes. Particular examples of truncated Tissue Factors are Tissue Factor compounds comprising approximately the first 219 contiguous amino acids of the original Tissue Factor sequence, as further exemplified by a Tissue Factor compound consisting essentially of the amino acid sequence of Tissue Factor. Identification Sequence No. 1 (SEQ ID N0: 1). Although intended for use in different methods, U.S. Patent No. 5,504,067 defines truncated Tissue Factors as Tissue Factor proteins having an amino acid sequence beginning at position one and terminating near position 219 of the defined Tissue Factor sequence. Dimeric deficient coagulation tissue factors, including homodimeric and heterodimeric tissue factors, may also be employed. Exemplary tissue factor dimers are described herein as those consisting essentially of dimers of the amino acid sequence of Identification Sequence No.3 (dimer H6-tTF219-cys-C), Identification Sequence No.6 ( dimer H6-tTF220-cys-Cf), Sequence Identification No.7 (dimer H6-tTF221-cys-C), or Sequence Identification No.2 (dimer H6-N'-cys-tTf219). Chemically conjugated dimers, as described in detail hereinafter, are preferred for use in certain aspects of the present invention, although dimers produced recombinantly, in structure with linkers within a structure, are also contemplated for its use in particular modalities. The impaired coagulation tissue factor compounds for use herein may also be polymeric or multimeric tissue factors. In certain embodiments, the Tissue Factor compound will be a mutant Tissue Factor deficient in the ability to activate Factor VII. Although useful alone, the most preferred uses of these mutants will be in conjunction with the co-administration of a biologically effective amount of at least one Vlla Factor or a Vlla Factor activator, such as when used with a sufficient amount of Factor Vlla to increase the coagulation of the vasculature of the tumor and the necrosis of the tumor in the animal. These mutants can be those that include a mutation in the amino acid region between about position 157 and about position 167 of Identification Sequence No. 1. Exemplarily, but in no way limiting are the mutants where, within the sequence of Identification No.l, Trp in position 158 is changed by Arg; where Being in position 162 is changed to Ala; where Gly at position 164 is changed to Ala; or where Trp at position 158 is changed to Arg and Ser at position 162 is changed to Wing. Defined examples of these mutants are those consisting essentially of the amino acid sequence of Identification Sequence No. 8 or Sequence Identification No.9. Any of the truncated, dimeric, multimeric and / or mutant deficient coagulation tissue factor compounds can also be modified to increase the longevity, half-life or "biological half-life" of the tissue factor molecule. Various modifications of the polypeptide structure can be made in order to effect this change in properties. Particular examples of the Tissue Factors modified to increase their biological half-life are the Tissue Factor compounds that have been operatively linked, and preferably covalently linked, to a carrier molecule, such as a protein carrier. The carriers are preferably inert carriers, such as, by way of example only, an albumin or a globulin. Non-protein carriers such as polysaccharides and synthetic polymers are also considered. The operative binding of a Tissue Factor construct to an antibody or portion thereof is a currently preferred form of deficient Coagulation Tissue Factor with increased biological half-life. However, in the context of the first substance for use in the cancer treatment strategies provided herein, the Tissue Factor will be bound to an antibody that does not exhibit significant specific binding to a component of a tumor cell, vasculature. of tumor or tumor stroma. That is, wherein the Tissue Factor compound is not bound to an "anti-tumor" antibody, and when the resulting Tissue Factor compound is not a "Tissue Factor directed to the tumor" compound. In these Tissue Factor-antibody conjugates, the Tissue Factor compound can be operably linked to an IgG molecule of so-called "irrelevant specificity", ie, one that has no immunoenhancing affinity for a component of a tumor cell. , tumor vasculature or tumor stroma. The Tissue Factor compounds can also be operatively linked to an Fc portion of an antibody, which has no specific objective function in the context of the antibody specificity. Other contemplated constructions are those in which the Tissue Factor compound has been introduced into an IgG molecule instead of the CH3 domain. The surprisingly effective Tissue Factor treatments of the present invention can be advantageously combined with one or more other treatments. For example, the methods of treatment may further comprise administering to an animal or patient a biologically or therapeutically effective amount of at least one second therapeutic compound, such as at least one of a second therapeutic compound selected from the group consisting of Factor Vlla, an activator of Factor Vlla and at least a first anticancer substance. The at least one first anticancer substance can be a "chemotherapeutic substance". As used herein, the term "chemotherapeutic substance" is used to refer to a chemotherapeutic substance or drug used in the treatment of malignancies. This term is used for simplicity irrespective of the fact that other compounds, including immunotoxins, can be described technically as a chemotherapeutic substance because they exert an anticancer effect. However, "chemotherapeutic" has come to have a different meaning in the art and is being used in accordance with this standard meaning. "Chemotherapeutic" in the context of the present application therefore does not generally refer to immunotoxins, radiotherapeutic substances and the like, despite their operational overlap. Several exemplary chemotherapeutic substances are listed in Table II. Technicians with ordinary skill in the field will readily understand the uses and appropriate doses of the chemotherapeutic substances, although the doses may well be reduced when used in combination with the present invention. A currently preferred chemotherapeutic substance is etoposide. A new class of drugs that can also be called "chemotherapeutic substances" are substances that induce apoptosis. One or more of these drugs, including genes, vectors, and antisense constructs, as appropriate, may also be used in conjunction with the present invention. Suitable anticancer substances also include specifically targeted toxic substances. For example anti-cancer antibodies and, preferably, conjugated antibody constructs comprise an antibody that specifically binds to a component of a tumor cell, tumor vasculature or tumor stroma, wherein the antibody is operatively linked or conjugated with at least one first cytotoxic or anticellular substance or, e.g., at least a first coagulation factor. By way of example only, the target construct or conjugate may be an antibody construct or conjugate that specifically binds to a tumor cell surface molecule; to a tumor vasculature component, such as E-selectin, P-selectin, VCAM-1, ICAM-1, endoglin or an integrin; to a component adsorbed or located in the vasculature or stroma, such as VEGF, FGF or TGFβ; to a component whose expression is induced naturally or artificially in the tumor environment, such as E-selectin, P-selectin or a MHC Class II antigen. Target substances that are not antibody include growth factors, such as VEGF and FGF; peptides containing the tripeptide R-G-D, which bind specifically to the vasculature of the tumor, and other target components such as annexins and related ligands. The constructs and conjugates of antibodies can be operatively linked to at least one first cytotoxic or otherwise anticellular substance. They can also be operatively linked to at least one first coagulation factor. In conjunction with coagulants, bispecific constructs (e.g., using two antibody binding regions) may also be advantageously employed, although covalent bonds are generally preferred for use with the toxins. One or more of the toxic or coagulant substances known in the art can be used in these "immunotoxins" or "coaguligands", and Tissue Factor or Tissue Factor derivatives can also be used as part of the coaguligands, where the coaguligand is the second "anticancer substance". The present invention therefore further provides methods for treating an animal or patient having a vascularized tumor, these methods generally comprising administering to an animal systemically one or more compounds of deficient coagulation tissue factor and one or more anticancer substances in an animal. effective combined amount to coagulate the vasculature of the tumor and specifically induce tumor necrosis. The anticancer substance may be a chemotherapeutic substance, such as exemplified by etoposide, an antibody, or an antibody or conjugate construct comprising an antibody that specifically binds to a component of a tumor cell, a tumor or stromal vasculature of tumor operatively linked to a cytotoxic substance or a coagulation factor. Whether the anticancer substance is a chemotherapy or an antibody-based construct, the one or more anticancer substances can be administered to the animal simultaneously, e.g., from a single composition or from two or more different compositions. The stepwise or sequential administration of one or more tissue factor compounds and one or more anticancer substances is also considered. "Sequential administration" requires that the Tissue Factor and anticancer substance be administered to the animal in "biologically effective time intervals". For example, the Tissue Factor compounds can be administered to the animal at a biologically effective time before the anticancer substance or substances, or the anticancer substance or substances can be administered to the animal at a biologically effective time before the compound (s). Fabric Factor. When a Tissue Factor compound is first administered, it will generally be given at a sufficiently biologically effective time to allow the Tissue Factor compound to preferentially be located within the tumor vasculature prior to the administration of the anticancer substance or substances. . The present invention further includes methods for using at least one Factor Vlla or a Factor Vlla activator to increase the effectiveness of one or more of the deficient coagulation tissue factor compounds that define primary therapy. These methods generally comprise administering in addition to an animal or patient a therapeutically effective amount of Factor Vlla or an activator of Factor Vlla. In these modalities, the use of the Factor Vlla alone will generally be preferred. The Factor Vlla employee can, consist essentially of the amino acid sequence of Identification Sequence No.14. Again, Factor Vlla or Factor Vlla activator can be administered to the animal simultaneously with the deficient coagulation tissue factor compound. As such, Factor Vlla can be administered to the animal or patient in a preformed tissue Factor-Factor Vlla complex. In certain modalities, the Tissue Factor-Factor Vlla complex will be an equimolar complex. In addition, the deficient coagulation tissue factor compound and the Factor Vlla compound can be administered to the animal using stepwise or sequential administration. The first administration of the Tissue Factor compound will generally be preferred and it will be preferable to administer it to the animal in a biologically effective time before the Factor Vlla compound. This effective prior administration of the Tissue Factor compound will generally be at a biologically effective time to allow the Tissue Factor compound to preferentially be located within the tumor vasculature prior to the administration of the Factor Vlla compound. These methods of the invention can thus be described as methods for promoting coagulation in the vasculature of the tumor of an animal or patient having a vascularized tumor, comprising systemically providing the animal or patient with a deficient coagulation tissue factor compound. and Factor Vlla or an activator of Factor Vlla in a combined amount sufficient to preferentially or specifically promote coagulation in the tumor vasculature. The subject animal will preferably be provided with a deficient coagulation tissue factor compound at a time prior to providing the Vlla Factor, wherein the time interval prior to the administration of Factor Vlla is effective for the tissue factor compound to preferentially specifically located within the vasculature of the tumor.

Other methods are described as methods for treating an animal having a vascularized tumor, comprising the systemic administration to the animal of a deficient coagulation tissue factor compound and Factor Vlla in a combined amount effective to promote coagulation in the vasculature. of the tumor and specifically cause necrosis in the tumor. The pre-administration of the Tissue Factor is generally preferred so that the compound of Tissue Factor is preferentially located within the vasculature of the tumor and forms a reservoir for the subsequent combination of Factor Vlla. All of these combination Vlla Factor treatments can be used with any deficient coagulation tissue factor compound, such as truncated, bimeric, and / or mutant and / or those with increased half-life. These methods are particularly useful for combination with Tissue Factor compounds that are deficient in their ability to activate Factor VII. The combined treatment methods of the invention also encompass triple combinations using one or more compounds of poorly clotting tissue factor, one or more anticancer substances and factor Vlla or an activator of the Vlla Factor The present invention also provides novel compositions in the form of compositions comprising one or more deficient coagulation tissue factor compounds that have been modified to increase their half-life, different from others in which the modification consists in binding the compound of Factor Tissue to an antibody that binds to a component of a tumor cell, tumor vasculature or tumor stroma. "Increased half-life tissue factor compounds" encompass all of the deficient coagulation tissue factor compounds described above, such as truncated, dimeric, polymeric, and / or mutant tissue factors. The augmented half-life tissue factor compounds preferably comprise a deficient coagulation tissue factor compound that is operatively linked, e.g., covalently linked, to a carrier molecule. Protein carriers are currently preferred, as exemplified by albumins or globulins, although non-protein carriers are also considered carriers. A class of increased half-life deficient coagulation tissue factor compounds are those that are operably linked to an antibody or portion thereof, such as a hemoglobin molecule or an Fc portion of an antibody. Tissue factors introduced into a contiguous portion of an IgG molecule, e.g., the locus of CH3 domain are also considered.

The invention still further provides a series of novel therapeutic kits for use in conjunction with the methods of the invention. These games will comprise, preferably in convenient container elements, at least one first coagulation tissue factor compound deficient in combination with at least one first anticancer substance. The deficient coagulation tissue factor compounds may be one or more of the deficient coagulation tissue factors described herein, such as truncated, dimeric, polymeric, and / or mutant tissue factors, which include mutant tissue factors. deficient in the ability to activate Factor VII. When the Factor VII activation mutants are employed in the game, the game optionally may further comprise a biologically effective amount of Factor Vlla. The term "anticancer substance" is used as described above and covers chemotherapeutic substances, such as etoposide; and anti-cancer substances based on antibodies, such as antibody conjugates comprising an antibody that specifically binds to a component of a tumor cell, tumor vasculature or tumor stroma operatively linked to a cytotoxic substance or to a coagulation factor., which includes a Tissue Factor or a Tissue Factor derivative. Other therapeutic games of the invention generally comprise, preferably in convenient container elements, a mutant tissue factor compound that is deficient in its ability to activate Factor VII in combination with Factor Vlla. Previously, the mutants of this category had been thought to both lack activity that could not be used therapeutically to induce coagulation, but only to act as an antagonist to the wild-type Tissue Factor and inhibit coagulation. Only the combination of substantially active truncated tissue factor with Factor Vlla has been previously proposed, this in connection with the treatment of hemorrhages. The present invention thus provides the novel combination of a mutant Tissue Factor compound that is more significantly impaired in its coagulant capacity than the truncated Tissue Factor, preferably by virtue of being deficient in the ability to activate Factor VII, together with the Factor Vlla. Factor Vlla will become "exogenous Vlla Factor" after administration to an animal. These sets therefore preferably comprise, in a convenient container element, a biologically effective amount of a mutant Tissue Factor compound deficient in its ability to activate Factor VII in combination with a biologically effective amount of at least one Factor Vlla or a Factor Vlla activator. Activators of the Vlla Factor can be replaced by the Vlla Factor in these games, or they can be used in addition with the Factor Vlla. Supplemental substances can also be added. Tissue Factor mutants for use in these kits are exemplified by those that include a mutation in the amino acid region between approximately position 157 and approximately position 167 of Identification Sequence No.l. This is exemplified by the mutants in which, within Identification Sequence No.l, the Trp at position 158 changes to Arg.; where to be in position 162 Ccimbia to Ala; where Gly at position 164 changes to Ala; or where Trp at position 158 changes to Arg and Ser at position 162 changes to Ala. Other examples are mutant tissue factors consisting essentially of the amino acid sequence of Identification Sequence No.8 or Identification Sequence No.9. The combination treatment kits comprising, preferably in a convenient container element, at least the first coagulation tissue factor compound deficient, at least one first anticancer substance and Factor Vlla or an activator of the Factor Vlla are also provided by the invention. BRIEF DESCRIPTION OF THE DRAWINGS The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein. Figure 1: Induction of plasma coagulation by full-length tissue factor. The blood (A) is shown in contact with the cell membrane (B). Figure 2: The domain structure of the Fabric Factor. The extracellular domain (A, amino acids 1-219) and the cell membrane (B) are represented. The NH2 domain (10) are represented as cross shading. The binding region factor VlI / VIIa (20) is represented as cross shading. The transmembrane domain (40) starts at amino acid 220 (30) and spans the cell membrane. The transmembrane domain of the Tissue Factor is deleted or becomes otherwise non-functional to generate a functional (FTt) of the present invention. In certain compositions of (FTt) the NH2 domain can also be deleted or rendered non-functional. Figure 3: Model for coagulation induced by Truncated tissue factor of tumor vasculature. The blood (A) is represented in contact with the cell membrane (B) of the vascular endothelium of the tumor (C). The endothelium of the prothrombotic tumor has factor IX, X (shown) or TFPI / Xa plus serine phosphatidyl (PS ") on its surface, which binds tTF-VII or tTF-VIIa, leading to coagulation, Figures 4A and 4B. Figure 4A: Induction of coagulation by the truncated tissue factor bound to the cell A20 cells (105 cells, 100 μl) were incubated with antibodies (0.33 μg) and truncated tissue factor (0.17 μg) for one hour at 4 ° C. Calcium chloride (12.5 mM) and citrated mouse plasma were added to the cells and time was recorded for the first fibrin strands to form (coagulation time, seconds, horizontal axis) The sample number is shown on the axis vertical Sample 1 does not include added antibodies or truncated tissue factor (control), sample 2 includes antibody B21-2 / 10H10, sample 3 includes truncated tissue factor, sample 4 includes antibody B21-2 / OX7 plus Factor of truncated tissue, sample 5 includes CA antibody MPATH-2 / 10H10 plus truncated Tissue Factor, sample 6 includes antibody 10H10 F (ab ') 2 plus truncated Tissue Factor, sample 7 includes antibody 10H10 Fab' plus truncated Tissue Factor, sample 8 includes antibody B21- 2F (ab ') 2 plus truncated Tissue Factor, sample 9 includes antibody B21-2 Fab' plus truncated Tissue Factor, sample 10 includes antibody B21-2 / 10H10 plus truncated Tissue Factor. Figure 4B: Relationship between the number of bound truncated tissue factor molecules and the plasma coagulation time. A20 cells (105 cells, 100 μl) were incubated with varying concentrations of B21-2 / 10H10 plus an excess of truncated Tissue Factor for one hour at 4 ° C in the presence of sodium azide and then washed, warmed to 375C. . Calcium chloride (12.5mM) and citrated mouse plasma (a different lot from that of A) were added to the cells and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). The number of truncated tissue factor molecules bound to the cells (0) was determined in a parallel study with 1 5I-tTf (scale log, horizontal axis). The values represent the average of the three measurements, with standard deviation. Figure 5: Coagulation of mouse plasma by tTF219, H6-N'-cys-tTf219 and H6-tTF219-cys-c 'associated with the cells. A20 lymphoma cells (positive I-Ad) were treated at room temperature with the "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and truncated Tissue Factor. The cells were washed and two different preparations of Truncated Tissue Factor 219 [tTf219 (0) [standard and tTF219 (A)], H6-N'-cys-tTf219 (D) or H6-tTf219-cys-c 'were added. (•) at a range of truncated tissue factor concentrations (concentration, M, horizontal axis). The cells were washed and heated to 37 SC. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis).

Figure 6: Coagulation of mouse plasma by K6-tTf220_cys-C 'and tTF22o-cys-C' associated with the cell. A20 lympho- na cells (positive I-Ad) were treated at room temperature with the "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and truncated Tissue Factor. The cells were washed and standard tTf2i9 (M), H6-tTF220-cys-C '(0) and tTf220-cys-C' (•) were added at a concentration range (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma was added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 7: Coagulation of mouse plasma by H6-tTF221-cys-C ', tTF221-cys-C' and dimer of H6-tTF22? -cys-C 'associated with the cells. A20 lymphoid cells were treated (Positive I-Ad) at room temperature with "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and truncated tissue factor. The cells were washed and standard tTf21 (O), H6-tTF221-cys-C '(•), tTf221-cys-C' (D), or dimer of H6-tTf22i-cys-C '(A) were added to a range of concentrations (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 8: Coagulation of mouse plasma by H6-N'-cys-tTF219 and dimer of H6-N'-cys-tTF219 associated with the cell. A20 lympho- na cells (positive I-Ad) were treated at room temperature with the "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and truncated Tissue Factor. The cells were washed and standard tTF219 (0), H6 ~ N '-cys-tTF219 (I) and dimer of H6-N'-cys-tTF219 (•) were added in a range of concentrations (concentration, M, horizontal axis ). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 9: Thrombosis of vessels in large tumors C1300 Mu? through tTF219. Nu / Nu mice that had C1300 Mu tumors? large subcutaneous (> 1000 cm3) were injected intravenously with 16-20 μg of tTF219. Twenty-four hours later, the mice were anesthetized, exsanguinated and tumors and organs were removed. Paraffin sections of the tissues were evaluated to determine the presence of vessels with thrombosis. The number of vessels with thrombosis and open vessels in sections of tumors were counted. The percentage of tumor vessels with thrombosis is shown on the vertical axis. The shaded bar represents mice injected with tTF219, the open bar represents mice injected with phosphate buffered saline. Figure 10: Thrombosis of vessels in large tumors C1300 by tTF219. Nu / Nu mice that had large subcutaneous C1300 tumors (> 1000 cm3) were injected intravenously with 16-20 μg of tTF219. Twenty-four hours later, the mice were anesthetized, exsanguinated and tumors and organs were removed. Paraffin sections of the tissues were evaluated to determine the presence of vessels with thrombosis. The number of vessels with thrombosis and open vessels in sections of tumors were counted. The percentage- of tumor vessels with thrombosis is shown on the vertical axis. The shaded bar represents mice injected with tTF219, the open bar represents mice injected with phosphate buffered saline. Figure 11: Thrombosis of vessels in large 3LL tumors by tTF219. C57BL / 6 mice that had large subcutaneous 3LL tumors (> 800 cm3) were injected intravenously with 16-20 μg of tTF219. Twenty-four hours later, the mice were anesthetized, exsanguinated and tumors and organs were removed. Paraffin sections of the tissues were evaluated to determine the presence of vessels with thrombosis. The number of vessels with thrombosis and open vessels in sections of tumors were counted. The percentage of tumor vessels with thrombosis is shown on the vertical axis. The shaded bar represents mice injected with tTF219, the open bar represents mice injected with phosphate buffered saline. Figures 12A and 12B: Inhibition of tumor growth C1300 Mu? in mice using tTF219. Figure 12A: Mice with C1300 tumors (Mu?) Of 0.8 to 1.0 centimeters in diameter were given two intravenous injections of coaguligand B21-2 / 10H10-tTF (•) separated by six days (arrows). The mice in the control group were given equivalent doses of Truncated Tissue Factor alone (D), CAMPATH-2 / 10H10 plus tTf (?) Or phosphate buffered saline (0). Mice that received B21-2 / OX7 and tTF had similar tumor responses to animals that received tTF alone. Administration of B21-2 / 10H10 alone did not affect tumor growth. Each group contained 12 to 27 mice. The points represent the mean volume of the tumor per group (± SEM). The average volume of the tumor (cm3) is shown on the vertical axis, days after the first treatment is shown on the horizontal axis. Figure 12B: Nu / Nu mice that had C1300 Mu tumors? small subcutaneous (350 mm3) were injected intravenously with 16-20 micrograms of tTF219 (I) or phosphate buffered saline (O). The treatment was repeated a week later. Tumors were measured daily and tumor volumes were calculated (+ one standard deviation). The number of mice per treatment group was 8-10. The average volume of the tumor (cpr) is shown on the vertical axis, days after the first treatment is shown on the horizontal axis. Figure 13: Growth inhibition of H460 tumors in mice by tTF2i9- Nu / Nu mice having small subcutaneous H460 tumors (350 mitr) were injected intravenously with 16-20 micrograms of tTF2i9 (B) or phosphate buffered saline ( O). The treatment was repeated a week later. The time of the injections is designated by arrows. Tumors were measured daily and tumor volumes were calculated (+ one standard deviation). The number of mice per treatment group was 8-10. The average volume of the tumor (cnr) is shown on the vertical axis, days after the first treatment is shown on the horizontal axis. Figure 14: Inhibition of HT29 tumor growth in mice by tTF2i9- A Nu / nu mice that had large subcutaneous HT29 tumors (1200 mm3) were injected intravenously with 16 micrograms or 64 micrograms of tTF2i9 (D) or phosphate buffered saline (•). The tumors were measured daily (days after the injection; horizontal axis), and tumor volumes were calculated (+ one standard deviation) (tumor volume, mm3, vertical axis). The number of mice per treatment group was 3-4. Figure 15: Coagulation of mouse plasma by IgG-H6-N '-cys-tTF219 associated with cells. A20 lympho cells (positive AI) were treated with the "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and tTF2i9-IgG-H6-N '-cys-tTF219 (A), H6 was added -N '-cys-tTF219 (I) or tTF219 (0) at a range of concentrations at room temperature (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma was added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 16: Coagulation of the mouse plasma by IgG-H6-N '-cys-tTF219 and IgG-H6-tTF219-cys-C' associated with the cells.

Immunoglobulin-tTF conjugates were prepared by linking B21-2 IgG (against I-Ad) to H6-N '-cys-tTF219 (A) or H6-tTF219-cys-C' (B).

The conjugates were added at a range of concentrations to lycorin A20 cells (positive I-Ad) at room temperature, and compared with tTF2j9 (•) (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium and mouse plasma were added citrated. Time (seconds) was recorded for the first fibrin strands to form. The vertical axis shows the coagulation time as a percentage of the control. Figure 17: Conversion of Factor X to Factor Xa by IgG-H6-N '-cys-tTF219 associated with cells and Fab'-H6-N'-cys-tTF219, measured by a chromogen test. A20 cells (positive I-Ad) were treated with the bispecific "capture" antibody, B21-2 / 10H10, recognizing both I-Ad and tTF, added with IGg-H6-N '-cys-tTF219 (O) or FAb'-H6-N'-cys-tTF219 (?), Which were added at a range of concentrations at room temperature. B21-2 / 10H10 plus H6-N '-cys-tTF219 (X) and MAc51 / 10H10 plus H6-N'-cys-tTF219 (control, I) (concentration, M, horizontal axis) were also added. The cells were washed and heated to 37 ° C. Calcium and "proplex t" were added (the proplex t contains factors II, VII, IX and X). The production of Xa was measured by adding the substrate that releases chromophore, S-2765, and measuring the optical density at 409 nm (OD409 nm, vertical axis). Figure 18: Inhibition of the growth of C1300 μg tumors in mice by immunoglobulin-tTF conjugate. Nu / Nu mice having small subcutaneous C1300 μg tumors (300 mm3) were injected intravenously with 16-20 micrograms of tTF219 complexed with bispecific "carrier" antibody 0X7 Fab '/ loHlO Fab (?). Other mice received tTF219 alone (I), or diluent (PBS, O). The treatment was repeated a week later. The days in which the treatments were given are designated by arrows. Tumors were measured daily and tumor volumes were calculated (plus one standard deviation). The number of mice per treatment group was 7-10. The average volume of tumor (cm3) is shown on the vertical axis, days after the first treatment is shown on the horizontal axis. Figure 19: Increase in antitumor activity of immunoglobulin-tTF by etoposide. SCID mice having human subcutaneous L540 Hodgkin tumors were given a single intravenous injection of a complex of tTF219 and the bispecific antibody "carrier" Mac51 Fab '/ l? HIO Fab1 (I). Other mice received 480 μg of etoposide intraperitoneally two days later, one day after and on the day of treatment with the immunoglobulin-tTF conjugate (?). Other mice received etoposide alone (O) or diluent (phosphate buffered saline,?). Tumors were measured daily and tumor volumes were calculated (+ one standard deviation). The average volume of the tumor (cm3) is shown on the vertical axis, days after the treatment is shown on the horizontal axis. Figure 20: Increase in plasma coagulation by Factor Vlla. A20 lymphoma cells were treated (Positive I-Ad) at room temperature with the "capture" bispecific antibody, B21--2 / 10H10, recognizing both I-Ad- and tTF. The cells were washed and tTF219 alone (O) or tTF219 were added with Factor Vlla in a range of concentrations of Factor Vlla, as follows: 0.1 nM (I); 0.3 nM (•); 0.9 nM (?); 2.7 nM (A); and 13.5 nM (+) (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 21: Weak coagulation of mouse plasma by tTF219 (W158R) and tTF2i9 (G164A) mutants associated with cells. A20 lymphoma cells (positive I-Ad) were treated at room temperature with the "capture" bispecific antibody, B21-2 / 10H10, recognizing both I-Ad and 'tTF. Cells were washed and tTF2α 9 (O), tTF2α9 (W158R) (•) or tTF219 (G164A) (D) were added at a range of concentrations (concentration, M, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 22: Restoration of the coagulation inducing activity of the mutant tTF219 (G164A) and (W158R) by the Factor Vlla. A20 lymphoma cells (positive I-Ad) were treated at room temperature with the bispecific "capture" antibody, B21-2 / 10H10, recognizing both I-Ad and tTF. The cells were washed and not treated (?) Or treated with: tTF219 (0); tTF219 (G164A) (B) O tTF219 (W158R) (•); each with the addition of Factor Vlla to a range of concentrations (concentration, nM, horizontal axis). The cells were washed and heated at 37 ° C. Calcium mouse calcium and plasma were added and time was recorded for the first fibrin strands to form (coagulation time, seconds, vertical axis). Figure 23: Antitumor activity of tTF219 complexes: Vlla and tTF219 (G164A), - Vlla in mice that have HT29 human colorectal carcinomas. From left to right, the bars represent: saline solution (1); tTF (2); Vlla factor (3); tTF plus Factor Vlla (4); G164A (5); and G164A plus Factor Vlla (6). The vertical axis shows the average percentage of necrosis in the tumors examined. SEQUENCE SUMMARY: SEQ ID NO: 1 Amino Acid Sequence of tTF2i9 SEQ ID NO: 2 Amino Acid Sequence of H6-N'-cys-tTF219 SEQ ID NO: 3 Amino Acid Sequence of H6-tTF219-cys-C 'SEQ ID NO: 4 Amino Acid Sequence of N '-cys-tTF219 SEQ ID NO: 5 Amino Acid Sequence of tTF219-cys-C' SEQ ID NO: 6 Amino Acid Sequence of H6-tTF220-cys-C 'SEQ ID NO: 7 Amino Acid Sequence of H6-tTF22-cys-C 'SEQ ID NO: 8 Amino Acid Sequence of tTF2? G (W 158 R) SEQ ID NO: 9 Amino Acid Sequence of tTF2i9 (G 164 A) SEQ ID NO: 10 Sequence cDNA for tTF SEQ ID NO: 11 Complete genomic sequence of Tissue Factor SEQ ID NO: 12 Amino Acid Sequence of Tissue Factor SEQ ID NO: 13 Factor VII DNA SEQ ID NO: 14 Factor VII Amino Acids SEQ ID NO: 15 5 'primer for tTF amplification SEQ ID NO: 16 3' primer for tTF amplification SEQ ID NO: 17 5 'primer GlytTF complementary DNA amplification primer SEQ ID NO: 18 5' primer pa Preparation of tTF and the 5 'half of the linker DNA SEQ ID NO: 19 3' primer for the preparation of tTF and the 5 'half of the linker DNA SEQ ID NO: 20' primer for the preparation of the 3 'half of the AND linker and DNA tTF SEQ ID NO: 21 3 'primer for the preparation of the 3' half of the linker DNA and the tTF DNA SEQ ID NO: 22 5 'primer for Cys [tTF] linker [tTF] construct SEQ ID NO : 23 3 'primer for Cys [tTF] linker [tTF] construct SEQ ID NO: 24 5' primer for [tTF] linker [tTF] cys SEQ ID NO: 25 3 'primer for [tTF] linker [tTF] cys SEQ ID NO: 26 Primer for the formation of [tTF] G164A SEQ ID NO: 27 Primer for [tTF] W158R S162S DESCRIPTION OF THE ILLUSTRATIVE MODALITIES Solid tumors and carcinoma are more than 90 percent of all cancers in humans ( Shockley et al., 1991). Therapeutic uses of monoclonal antibodies and immunotoxins have been investigated in lymphoblast and leukemia therapy (Lowder et al., 1987; Vitetta et al., 1991), but they have been disappointingly ineffective in clinical trials against carcinomas and other solid tumors (Byers et al. Baldwin, 1988; Abrams and Oldham, 1985). A major reason for the ineffectiveness of antibody-based treatments is that macromolecules are not easily transported to solid tumors (Sands, 1988; Epenetos et al., 1986). Even when these molecules reach the tumor mass, they fail to distribute evenly due to the presence of strong bonds between the tumor cells (Dvorak et al., 1991), fibrous stroma (Baxter et al., 1991), interstitial pressure gradients (Jain, 1990) and binding site barriers (Juweid et al., 1992). To develop new strategies for treating solid tumors, methods that involve targeting the tumor vasculature, rather than the tumor cells themselves, offer distinct advantages. Inducing a blockage of blood flow through the tumor, eg, through the specific fibrin formation of the tumor vasculature, would interfere with the influx and exlux processes at a tumor site, resulting in a antitumor effect. Stopping the blood supply to a tumor can be carried out by changing the procoagulant, fibrillilitic balance in the vessels associated with the tumor in favor of coagulant processes by specific exposure to coagulant substances. In accordance with the foregoing, constructs of coagulant antibodies and bispecific antibodies have been generated and used in the specific administration of a coagulant to the tumor environment (WO 96/01653).

However, the requirement for specificity, although not as strict as in immunotoxins, is still important. To achieve specificity, it has generally been believed that an effector molecule, either a toxin or a coagulant, needs to be conjugated or functionally associated with a target molecule, such as an antibody or other ligand with specificity for the tumor environment. These target entities can be targeted to the same tumor cells, although it is now believed that it is preferable to use target molecules directed against components of the tumor vasculature or tumor stroma. Several suitable target molecules have been identified that are specifically or preferentially expressed, localized, adsorbed to or indudate in the cells or in the environment of the tumor vasculature and / or stroma. Although methods targeting the tumor vasculature and stroma can be quite effective, it will be recognized that the practice of this objective methodology still requires certain knowledge and requires the preparation of convenient conjugates or coordinated molecular complexes. For example, to direct a coagulant to the tumor vasculature, a suitable vascular antigen must be identified, an antibody or ligand that binds to the target antigen be selected, a suitable coagulant chosen, the coagulant bound to the antibody or ligand or another way a functional association of the two components, and conduct the location protocols using doses that do not result in a significant incorrect direction of the substance. Although these methods can be easily and successfully practiced, it can be seen that the advantages would be the result of the development of a methodology that included fewer preparatory steps and therefore could be carried out more cost-effectively. The present invention provides these new methods for effecting specific blood coagulation, as exemplified by tumor specific coagulation, without the need for target molecules, such as antibodies. This is achieved by administering compositions comprising deficient coagulant tissue factor, which has been found to specifically promote coagulation in the tumor vasculature, despite the fact that it lacks any recognized tumor target component. The present invention provides that these impaired coagulation tissue factor compositions can be administered alone, as Tissue Factor conjugates with improved half-life, in combination with conventional chemotherapy, in combination with immunotoxins or target coaguligands, in combination with Factor Vlla (FVIIa ) or activators of the Factor Vlla or any of the above combinations. A. Fabric Factor. The Tissue Factor (TF) is the most important initiation receptor for thrombogenic cascades (blood coagulation) (Davie, et al., 1991). The Tissue Factor is a single strand, membrane glycoprotein of 263 amino acids (SEQ ID N0: 12), and its primary sequence has structural similarity to the chemokine receptor family (Edgington et al., 1991). The Tissue Factor is a transmembrane cell surface receptor and functions as the receptor and cofactor for the Vlla Factor. The Tissue Factor binds the Vlla Factor to form a proteolytically active complex on the cell surface (Ruf and Edgington, 1991b, 1994; Ruf et al., 1991, 1992a, 1992b). This complex rapidly activates the serine protease zylogens Factors IX and X by limited proteolysis, leading to the formation of thrombin and, finally, to a blood clot (Figure 21). In this way, the Tissue Factor is an activator of the extrinsic path of blood coagulation and is not in direct contact with blood under physiologically normal conditions (Osterud et al., 1986; Nemerson, 1988; Broze, 1992; Ruf et al. Edgington, 1994). In vascular damage or activation by certain cytokines or endotoxin, however, the tissue factor will be exposed to the blood, either by (sub) endothelial cells (Weiss et al., 1989) or by certain blood cells (Warr et al. collaborators, 1990). The tissue factor will be coupled with the Factor Vlla, which under normal conditions circulates at low concentrations in the blood (Wildgoose et al., 1992), and the TF / Factor Vlla complex will start the coagulation cascade through the activation of the Factor X in Factor Xa. The cascade will eventually result in the formation of fibrin (Figure 1). For this sequence of events to occur, the TF: Vlla complex has to be associated with a phospholipid surface on which coagulation initiation complexes with Factors IX or X can be assembled (Ruf and Edgington, 1991a; Ruf et al 1992c; Paborsky et al. 1991; Bach et al. 1986; Krishnaswamy et al. 1992; ten Cate et al. 1993). A limited number of cells constitutively express the tissue factor. Lung and central nervous system tissues contain high levels of tissue factor activity, tissue factor being found in bronchial mucosa and alveolar epithelial cells in the lung and in glial cells and astrocytes in the nervous system. Tissue Factor expression has also been reported in cardiac myocytes, renal glomeruli, and in certain epithelial or mucosal tissues of the intestine, bladder, and airways. In this way it can be seen that the Tissue Factor is generally expressed constitutively as tissue barriers between the tissues of the body and the external environment (Drake et al., 1989; Ruf and Edgington, 1994).

The Tissue Factor is also present as tissue boundaries between organs, such as in the liver, spleen and kidney organ capsules, and is also present in the adventitia of arteries and veins. The expression of the Tissue Factor in this way allows the Tissue Factor to work to stop the internal bleeding. Therefore it is important to note that the tissue factor is absent in the joints and the skeletal muscle of hemophiliacs, which are the first bleeding sites in these patients. The Tissue Factor is typically not expressed in any degree in blood cells or on the surface of endothelial cells that form the vasculature under normal conditions, but its expression by (sub) endothelial cells and monocytes within the vasculature can be induced by Infectious agents. Monocytes, for example, are induced to express Tissue Factor by cytokines and T cells. The expression of Tissue Factor in the vasculature will typically result in disseminated intravascular coagulation or localized initiation of blood clots or thrombogenesis. In this context, it is important to note that the Tissue Factor must be available in all body sites where coagulation would be necessary after tissue infection damage or other aggressions. Therefore, the Tissue Factor should be equally available in all those tissue sites and should not be reserved generally within a particular localized area of the body. Certain studies have led to the delineation of a connection between the tissue factor and the development of the neoplastic phenotype in certain types of tumors (Ruff and Edgington, 1994). In fact, increasing levels of tissue factor have been reported as an indicator of metastatic potential for malignant melanoma (Mueller, et al., 1992). It has been argued that a generalized activation of the coagulation cascade could damage the vasculature leading to access of tumor cells or vesicles derived from tumor cells to the general circulation, allowing these tumor cells to flare and cause tumor growth. metastatic Regardless of the underlying mechanism, the studies described above have led Edgington et al to propose the use of antibodies directed against the Tissue Factor in the treatment against cancer (WO 94/05328). These authors have therefore proposed that antibodies with binding affinity for Tissue Factor have therapeutic utility in the treatment against cancer, particularly in connection with patients believed to be at risk of the development of metastatic tumors. This attempt has led to the development of hybridomas that produce monoclonal antibodies that react with human tissue factor (U.S. Patent No. 5,223,427). In addition to the use in cancer treatment, tissue anti-factor antibodies have also been proposed for use in the inhibition of excessive coagulation, which can also be used in connection with the treatment of septic shock and in moderating inflammatory responses (Morrissey et al., 1988; U.S. Patent No. 5,223,427), or in the treatment of myocardial infarction, wherein the antibodies are used as tissue factor antagonists (U.S. Patent No. 5,589,173). The combined use of tissue anti-factor antibody and other thrombolytic substances to dissolve occluding thrombi is particularly disclosed in U.S. Patent No. 5,589,173. A specific method for using these antibodies is in the inhibition of coagulation in the extracorporeal circulation procedure in which blood is removed from a patient during a surgical procedure, such as a cardiopulmonary bypass procedure (US Pat. No. 5,437,864). As developed more fully below (Section B), the Human Tissue Factor has been cloned and is available for some time (Morrissey et al., 1987; Edgington et al., 1991; Patent of the United States of North America number 5,110,730). In certain previous studies, the same protein currently identified as a human tissue factor is disclosed as a heavy chain protein of human tissue factor or the heavy chain of tissue factor. The gene encodes a polypeptide precursor of a length of 295 amino acids, which includes a forward peptide with alternative dissociation sites, which is known to drive the formation of a protein of 263 amino acids in length. Recombinant expression of human tissue factor in CHO cells has been reported to lead to the production of tissue factor at a level which is described as being one of the highest expression levels reported for a recombinant transmembrane receptor after the production in mammalian cells (Rehemtulla et al., • 1991). A recombinant form of tissue factor containing only the cell surface or the extracellular domain has been constructed (Stone, et al., 1995) and lacks the transmembrane and cytoplasmic regions of the tissue factor. This "truncated" tissue factor (tTF) has a length of 219 amino acids and is a soluble protein with approximately 105-fold less X-activating factor activity relative to the original transmembrane tissue factor in a suitable phospholipid membrane environment (Ruf. , et al., 1991b). This difference in activity is due to the fact that the TF: VIIa complex binds and activates Factors IX and X much more efficiently when associated with a negatively charged phospholipid surface (Ruff, et al., 1991b; Paborsky, et al., 1991). . In spite of the significant deterioration of the coagulant capacity of the truncated Tissue Factor, the truncated Tissue Factor can promote blood coagulation when tied or functionally associated by some other means with a phospholipid or membrane environment. For example, it is demonstrated herein that using a bispecific antibody that binds the truncated Tissue Factor to a plasma membrane antigen allows the restoration of useful coagulant activity. This led one of the present inventors to develop methods for specific vascular tumor coagulation in vivo using target constructs to administer truncated Tissue Factor or variants thereof specifically to tumor or stromal vasculature (WO 96/01653). Intravenous administration of this "coaguligand" leads to localization of the coagulants within the tumor, thrombosis of the tumor vessels, and necrosis resulting from the tumor. The development of intelligent, directed coagulant administration to the tumor vasculature, as exemplified using a bispecific target antibody composition and truncated tissue factor, can be seen to represent an improvement over classical immunotoxin therapy. In fact, this coaguligand treatment induces thrombosis of the tumor vessels in less than 30 minutes, compared with approximately 6 hours necessary to achieve the same effect after the administration of an immunotoxin. In addition, there are no notable side effects as a result of the coaguligand treatment. Although the targeted administration of a coagulant such as the truncated Tissue Factor was surprisingly effective, it still requires the preparation of the "target construct". Other tissue factor studies with very different objectives have also been reported to identify uses of the truncated tissue factor that do not depend on their association with the target substance. With regard to this, the truncated tissue factor has lately been considered as a candidate for use in the treatment of disorders such as hemophilia. This work may have developed from attempts to use tissue apo-factor in these treatments. Apo-TF is a delipidated preparation of the Tissue Factor that was proposed for infusion in hemophiliacs, based on the hypothesis that this molecule would spontaneously and preferably be incorporated or associate with membrane surfaces exposed and available at injury sites. In this way, it was reasoned that apo-TF could be useful in these treatments, without leading to significant side effects (O'Brien et al., 1988; Patent of the United States of America number 5,017,556). Apo-TF therapy has been proposed for use in chronic bleeding disorders characterized by a tendency towards bleeding, both hereditary and acquired. U.S. Patent No. 5,017,556 describes these disorders as those connected with the deficiency of Factors VIII, IX or XI; or those connected with the acquisition of inhibitors to Factors V, VIII, IX, XI, XII and XIII. The use of apo-TF, characterized as substantially devoid of the lipid that occurs naturally from the Tissue Factor and possessing substantially no procoagulant activity prior to administration, is recognized to be in contrast to the expected results, which has been reasoned that lead to toxicity. It now appears that the results described in US Pat. No. 5,017,556 generally represent an anomaly in the art, and these studies have been contradicted by other researchers working in the field. In fact, during attempts to carry out studies based on what was described above, experimental animals were observed to develop side effects such as disseminated intervascular coagulation (DIC). This led to the conclusion that the intravenous administration of apo-TF is too dangerous to be used (Sakai and Kisiel, 1990, Patents of the United States of America numbers 5,374,617, 5,504,064, and 5,504,067). The development of a soluble, truncated form of Tissue Factor has not been recognized as resolving the problems associated with the Tissue Factor or apo-TF. For example, the truncated Tissue Factor has been rejected as an alternative to the Tissue Factor, due to the fact that it has been characterized as having almost no procoagulant activity when tested with normal plasma (Paborsky et al., 1991; United States of America number 5,374,617). Potential uses for the truncated Tissue Factor possible prior to the present invention were then confined to targeted administration of truncated Tissue Factor, e.g. , using antibodies, and the possible use of truncated Tissue Factor to treat a limited amount of disorders used in combination with other accessory molecules necessary for the restoration of sufficient activity (U.S. Patent No. 5,374,617). This second possibility has been exploited in certain limited circumstances by combining the use of truncated tissue factor with the administration of the coagulation factor, Factor Vlla. The combined use of Vlla Factor with the truncated Tissue Factor results in the restoration of sufficient coagulant activity for this combination to be in use to treat blood disorders, such as hemophilia. However, in contrast to the targeted administration of coagulants such as the truncated Tissue Factor discussed in WO 96/01653, the combination therapy of the truncated Tissue Factor and the Vlla Factor does not include specific targeting concepts. This therapy has therefore been proposed for use only in relation to patients in whom the coagulation is impaired (U.S. Patent Nos. 5,374,617, 5,504,064, and 5,504,067). The group of patients most easily identified with these impaired coagulation mechanisms are hemophiliacs, including those who have hemophilia A and hemophilia B, and those who have high counts of antibodies targeting coagulation factors. In addition, this combined treatment of truncated Tissue Factor and Vlla Factor has been proposed for use in relation to patients suffering from severe thromboembolism, postoperative bleeding or even cirrhosis (U.S. Patent Nos. 5, 374, 617; 5,504,064 and 5,504,067). Both systemic administration by infusion and topical application have been proposed as useful in these therapies. These therapies can thus be seen to supplement the body with two "Factors" of the type of coagulation in order to overcome any natural limitation of these or other related molecules in the coagulation cascade in order to stop bleeding at a specific site. Roy and colleagues have also proposed the use of certain Tissue Factor mutants in the treatment of myocardial infarction, particularly in the prevention of reocclusion of the coronary arteries (U.S. Patent No. 5,346,991). As such, Tissue Factor mutants are being used as "thrombolytic substances," and are described as drugs capable of lysing a fibrin-platelet thrombus in order to allow blood to flow back through an affected blood vessel. . The described Tissue Factor mutants are designated with the intention of being able to neutralize the effects of the endogenous Tissue Factor. Its use in connection with myocardial infarction therapy is said to allow repercussion, avoid reocclusion and therefore limit tissue necrosis. Artificial elements to recreate the natural environment in the context described above are linked to natural processes, where the Tissue Factor is described as being constitutively present in the boundaries between the organs in order to allow them to function as an initiating molecule to stop the bleeding. However, this limitation of bleeding episodes in hemophiliacs naturally needs to be achieved without tipping the balance of the coagulation trajectories to broad coagulation, which would be detrimental to these patients and would inhibit the oxygen supply to the particular tissue or organ in question. Therefore, the broad circulation and activity of the truncated Tissue Factor would be undesirable and in fact would not be expected to occur from the studies described above. Although the truncated Tissue Factor has not previously been shown to have any ability to preferentially locate within a given site, and although the coagulant capacity relative to the full-length, original Tissue Factor is known to be greatly diminished, the present invention demonstrates that when routinely administered to animals with solid tumors, the truncated Tissue Factor induces specific coagulation of the tumor blood supply, resulting in regression of the tumor. The various aspects of the present invention are therefore based on the discovery of the selective thrombosis of the tumor vessels by the truncated tissue factor. Al. Deficient Coagulation Tissue Factor The surprising finding of the inventors that the truncated Tissue Factor specifically located within the tumor sufficiently to cause antitumor effect was discovered during the studies using the truncated Tissue Factor as a control in studies directed at tumors. with antibody • coagulant ("coaguligating"). From this initial discovery, the inventors developed the various aspects of the invention described herein. The Tissue Factor compounds or constructs for use in the present invention have been developed in this way from the original truncated Tissue Factor employed first. Accordingly, several Tissue Factor constructions can now be employed, including many different forms of truncated Tissue Factor, longer but still deteriorated Tissue Factors, mutant Tissue Factors. Any truncated tissue factor, variant or mutant modified or otherwise conjugated to improve its half-life, and all functional equivalents thereof. However, it will be understood that each construction of the Tissue Factor for use in the invention is unified by the need to be "deficient coagulation". As detailed hereinafter, there are several structural considerations that can be employed in the design of the candidate clotting Factors of poor coagulation, and several tests are available to confirm that the truly candidate Tissue Factors are suitable for use. in the aspects of the treatment of the present invention. Since the technological skills to create a variety of compounds, e.g., using molecular biology, and routine for those with ordinary skill in the art, and since there is extensive structural and functional guidance provided herein, the ordinary technician would easily be able to make and use various factors. Deficient coagulation tissue in the context of the present invention. Also described in significant detail herein, one or more of a variety of Tissue Factors may also be combined with other substances for use in the advantageous treatment of solid tumors and other diseases associated with vessels of prothrombotic fluid. In addition to combination with standard treatments, such as surgery and radiotherapy, the coagulation approach of the present invention can also be combined with the administration of classical chemotherapeutic drugs other than immunotoxins or coaguligands, or with coagulation factors, as exemplified by the Factor Vlla. Since the combined treatments of the invention are expected to give an additive, increased or even synergistic antitumor effect, those skilled in the art will also readily appreciate that the Tissue Factor constructs that have less than optimal properties in the types of assays in vi and live described herein may still be used in the context of the present invention. For example, if a candidate clotting factor deficient coagulation construct has coagulant activity towards the lower end of the scale recommended herein, such a molecule can still demonstrate that it is useful in combination with coagulation factors, chemotherapies or other anticancer substances. . Likewise, candidate deficient coagulation tissue factor constructs that can be considered to have a coagulant activity high enough to cause problems with respect to side effects can still be shown to be useful after careful in vivo studies using experimental animals and in clinical studies starting with low doses. Therefore, the following guidelines concerning deficient coagulation tissue factor molecules are provided only as exemplary teachings, and technicians with ordinary field experience will readily appreciate that the tissue factor molecules that do not fit directly into the structural and quantitative lineaments presented herein may still have significant therapeutic utility in the context of the present invention. Although determining this fact frequently may generally require in vivo testing in animals, these tests are routine for those of ordinary skill in the art. and simply require administration and supervision A2 Structural Considerations for the Deficient Coagulation Growth Factor Technicians skilled in the art will readily appreciate that the Tissue Factor molecules for use in the present invention can not be their essentially the original Fabric Factor. This is evident since the original Tissue Factor and the close variants thereof are particularly active in promoting coagulation. Therefore after the administration to an animal or patient, this would lead to extend coagulation and be lethal. Therefore, the formulations of the original tissue factor, intact, should be avoided. Similarly, attempts to modulate the activity of the Tissue Factor by manipulating its physical environment are not believed to be particularly productive in the context of the present invention. For example, the apo-TF approach of O'Brien et al. (1988) should be avoided because disseminated intervascular coagulation is expected to result. Figure 2 is provided herein as an instructional model concerning the domain of the original Tissue Factor molecule. It is an object of the invention to provide Tissue Factor molecules that do not substantially associate with the plasma membrane. Naturally, the truncation of the molecule is the most direct way in which a modified tissue factor that does not bind to the membrane is achieved. These types of truncated constructions are described more fully below. However, the actual truncation or shortening of the molecule is not the only mechanism by which variants of operative Tissue Factor can be created. By way of example only, mutations can be introduced into the C-terminal region of the molecule that normally traverses the membrane in order to avoid proper membrane insertion. It is contemplated that the insertion of several additional amino acids, or the mutation of these residues already present, can be used to effect this membrane ejection. Therefore, modifications with respect to this which reduce the hydrophobicity of the C-terminal portion of the molecule can be considered so that the thermodynamic properties of this region are no longer favorable to the insertion of the membrane. Considering making structural modifications to the original Tissue Factor molecule, those skilled in the art will realize the need to maintain significant portions of the molecule sufficient for the resulting Tissue Factor variant to be able to function to promote at least some coagulation. An important consideration is that the Tissue Factor molecule should substantially retain its ability to bind Factor VII or Factor Vlla. Referring to Figure 2, it will be seen that the VII / VIla binding region is generally central to the molecule and this region should therefore be maintained substantially in all the proposed Tissue Factor variants for use in the present invention. The particular location of this binding region and the optional use of mutants, either alone or in combination with other substances, are discussed in more detail below. However, certain portions of sequences from the N-terminal region of the original Tissue Factor are also considered dispensable. Therefore, mutations can be introduced into this region or deletion mutants (truncated from the N-terminus) can be employed in the candidate Tissue Factor molecules for use herein. Given these guidelines, those skilled in the art will appreciate that the following exemplary truncated, dimeric, multimeric and mutant tissue factor constructs are in no way limiting and that many other functionally equivalent molecules can be readily prepared and used. . A3. Deficient Coagulation Tissue Factor Constructions. The following exemplary tissue factor compositions, including the truncated, dimeric, multimeric and mutated versions, may exist as distinct polypeptides or may be conjugated to inert carriers, such as immunoglobulins, as described hereinafter. i. Truncated Tissue Factor As used herein, the term "truncated" when used in relation to the Tissue Factor means that the particular Tissue Factor construct lacks certain amino acid sequences. The term truncated in this way means constructions of Tissue Factor of shorter length, and differentiates these compounds from other Tissue Factor constructions having reduced membrane binding or association. Although the modified but substantially full length Tissue Factors can thus be regarded as functional equivalents of truncated ("functionally truncated") Fabric Factors, the term "truncated" is used in the present in its classical sense to mean that the Tissue Factor molecule becomes deficient in membrane binding by removing enough amino acid sequences to effect this change in property. In accordance with the above, a truncated Tissue Factor protein or polypeptide is one that differs from the original Tissue Factor in that a sufficient amount of the transmembrane amino acid sequence has been removed from the molecule, compared to the original Fabric Factor. In this context a "sufficient amount" is a quantity of transmembrane amino acid sequence originally sufficient to introduce the Tissue Factor molecule into the membrane, or otherwise bind the functional middle membrane of the Tissue Factor protein. The removal of this "sufficient amount of transmembrane extension sequence" therefore creates a protein or a truncated Tissue Factor polypeptide deficient in phospholipid membrane binding capacity, so that the protein is substantially a soluble protein that does not bind significantly to phospholipid membranes, and that it substantially fails to convert Factor VII to Factor Vlla in a standard tissue assay, and thus retains the so-called catalytic activity that includes activation of Factor X in the presence of Factor Vlla. . U.S. Patent No. 5,504,067 is specifically incorporated herein by reference for the purpose of further describing these truncated Tissue Factor proteins. The preparation of the particular truncated Tissue Factor constructs are described hereinafter. Preferably, the Tissue Factors for use in the present invention generally lack the transmembrane and cytosolic regions (amino acids 220-263 of SEQ ID NO: 12) of the protein. However, there is no need for truncated Tissue Factor molecules to be limited to molecules of the length of 219 amino acids. Therefore, constructs of between about 210 and about 230 amino acids in length can be used. In particular, the constructions may have approximately 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or approximately 230 amino acids in length. Of course, it will be understood that the intention is to substantially suppress the transmembrane region of approximately 23 amino acids from the truncated molecule. Therefore, in truncated Tissue Factor constructions that are longer than 218 to 222 amino acids in length, portions of significant sequences thereafter will generally be comprised of approximately 21 amino acids that form the cytosolic domain of the molecule. Original Fabric Factor. In this respect, the truncated Tissue Factor constructions may have between about 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, or about 241 amino acids in length. In certain preferred embodiments, the truncated Tissue Factor can be designated as the extracellular domain of the mature Tissue Factor protein. Therefore, in exemplary preferred embodiments, the truncated Tissue Factor may have the amino acid sequence of SEQ ID NO: 1, comprising residues 1-219 of the mature protein (SEQ ID NO: 12). SEQ ID NO: 1 may be encoded by, for example, SEQ ID NO: 10. Of course, SEQ ID NO: 1 is only an exemplary truncated Tissue Factor and any Tissue Factor protein derived from the nucleic acid sequence of SEQ ID NO: 11, or the related sequences, which possesses the desirable properties of high affinity binding to Factor VII or Factor Vlla and possesses a reduced procoagulation cofactor activity will generally also be useful as described herein. ii. Dixinic Tissue Factor Constructions It has previously been shown that it is possible that the original Tissue Factor on the surface of J82 bladder carcinoma cells exists as a dimer (Fair et al., 1987). The binding of a Factor VII or Vlla Factor molecule to a Tissue Factor molecule can also facilitate the binding of another Factor VII or Factor Vlla to another Tissue Factor (Fair et al., 1987; Bach et al., 1986). In addition, the Tissue Factor shows structural homology to members of the cytokine receptor family (Edgington et al., 1991), some of which dimerizes to form active receptors (Davies and Wlodawer, 1995). It is thus contemplated that the truncated Tissue Factor compositions of the present invention may be useful as dimers. In accordance with the foregoing, any of the truncated, mutated or otherwise deficient coagulation tissue Factor constructs described herein, or an equivalent thereof, may be prepared in a dimeric form for use in the present invention. . As will be known to those skilled in the art, these Tissue Factor dimers can be prepared using standard molecular biology and recombinant expression techniques, in which two coding regions are prepared in frame and expressed from a vector expression. Similarly, various chemical conjugation technologies can be employed in connection with the preparation of Tissue Factor dimers. The individual Tissue Factor monomers can be derived before conjugation. All of these techniques would be readily known to those skilled in the art. If desired, the Tissue Factor dimers or multimers can be linked by a biologically releasable linkage, such as a selectively dissociable amino acid link or sequence. For example, peptide linkers that include a dissociation site for a preferentially localized or active enzyme within a tumor environment are contemplated. Exemplary forms of these peptide linkers are those that are dissociated by urokinase, plas ina, thrombin, Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase, gelatinase or estro elisine. In certain embodiments, the Tissue Factor dimers may further comprise an hindered hydrophobic membrane insertion fraction, to then encourage the functional association of the Tissue Factor with the phospholipid membrane, but only under certain defined conditions. As described in the context of truncated tissue factors, hydrophobic membrane association sequences are generally stretches of amino acids that promote association with the phospholipid environment due to their hydrophobic nature. Likewise, fatty acids can be used to provide the potential membrane insertion fraction. These membrane insertion sequences can be located either in the N-terminus or in the C-terminus of the Tissue Factor molecule, or generally attached to any other point in the molecule as long as their binding does not impede the functional properties of the construction of the Fabric Factor. The intent of the prevented insertion fraction is to remain nonfunctional until the tissue factor construction is located within the tumor environment, and allows the hydrophobic appendage to become accessible and still further promote physical association with the membrane. Again, it is contemplated that biologically releasable linkages and selectively dissociable sequences will be particularly useful in this regard, only being the bond or sequence dissociated or otherwise modified after its location within the tumor environment and exposure to particular enzymes or other bioactive molecules. By way of example only, the inventors have constructed dimeric truncated tissue factor corresponding to a dimer of C-cys-tTF219 (dimer of SEQ ID NO: 3); a dimer of C * -cys-tTF220 (dimer of SEQ ID N0: 6); a dimer of C »-cys-tTF221 (dimer of SEQ ID NO: 7); and a dimer of H6-N'-cys-tTF219 (dimer of SEQ ID NO: 2). However now it will be understood that each of the foregoing sequences are exemplary and in no way limiting the dimeric structures that may be created and used in accordance with the present invention. iii. Tri-Factor and Multimeric Factor Constructions In other embodiments, the truncated-tissue factor constructions of the present invention may be multimeric or polymeric. In this context a "polymeric construction" contains 3 or more Fabric Factor constructions of the present invention. A "multimeric or polymeric tissue factor construct" is a construct comprising a first tissue factor molecule or derivative operably linked to at least a second and a third tissue factor molecule or derivative, and preferably, wherein the resulting multimeric or polymeric construct is still deficient in coagulant activity as compared to the native-type Tissue Factor. In preferred embodiments, the mutimeric and polymeric tissue factor constructs for use in this invention are multimers or polymers of truncated Tissue Factor molecules, which may optionally be combined with other constructs or variants of Deficient Coagulation Tissue Factor. The multimers may comprise between about 3 and about 20 of these Tissue Factor molecules, with between about 3 and about 15 or about 10 being preferred and between about 3 and about 10 being more preferred. Naturally, the Tissue Factor multimers of at least about 3, 4, 5, 6, 7, 8, 9 or 10 or so are included within the present invention. The individual Tissue Factor units within the multimers or polymers can also be linked via selectively dissociable peptide linkers or other releasable biological linkages as desired. Again, as with the Tissue Factor dimers presented above, the constructions can be easily made using either recombinant manipulation and expression or using standard synthetic chemistry. iv. Factor VII Activation Mutants Still other tissue factor constructs useful in the context of the present invention are. mutants deficient in the ability to activate Factor VII. The basis of the utility of these mutants lies in the fact that they are also "deficient coagulation". These "Factor VII activating mutants" are generally defined herein as Tissue Factor mutants that bind functional factor VlI / VIIa, proteolytically activate Factor X, but are substantially free of the ability to proteolytically activate Factor VII. In accordance with the foregoing, these constructs are Tissue Factor mutants that lack the activation activity of Factor VII.

The ability of these Factor VII activation mutants to function by promoting specific tumor coagulation is based on both the location of the tissue factor construct to the tumor vasculature, and the presence of Factor Vlla at low levels in the plasma. After administration of this factor VII activating mutant, the mutant would generally localize within the vasculature of a vascularized tumor, as would any tissue factor construct of the invention. Prior to localization, the Tissue Factor mutant would be unable to promote coagulation elsewhere in the body, based on its inability to convert Factor VII to Factor Vlla. However, after localization and accumulation within the tumor region, the mutant will then find sufficient Plasma Factor Vlla in order to initiate the extrinsic coagulation path, leading to tumor-specific thrombosis. As developed more fully below, the most preferred use of Factor VII activating mutants is in combination with the co-administration of Factor Vlla. Although useful inside and outside thereof, as described above, these mutants will generally have less than optimal activity since the Vlla Factor is known to be present in plasma only at low levels (approximately 1 nanogram / milliliter, in contrast to approximately 500 - nanogram / milliliter of Factor VII in plasma; Patents of the United States of America numbers 5,374,617; 5,504,064; and 5,504,067). Therefore, the co-administration of exogenous Factor Vlla together with the Factor VII activation mutant is considerably preferred over the administration of the mutants alone. Since these mutants are expected to have almost no side effects, their combined use with simultaneous, preceding, or subsequent administration of the Vlla Factor is a particularly advantageous aspect of the present invention. One or more of a variety of Factor VII activation mutants can be prepared and used in connection with some aspect of the present invention. There is a significant amount of scientific knowledge related to the recognition sites of the Tissue Factor molecule for Factor VlI / VIIa. By way of example only, reference may be made to the articles by Ruf and Edgington (1991a), Ruf et al. (1992c), and WO 94/07515 and WO 94/28017, each specifically incorporated herein by reference for guidance. additional on these issues. Thus it will be understood that the activation region of Factor VII is generally between approximately amino acid 157 and approximately amino acid 167 of the Tissue Factor molecule. However, it is contemplated that residues outside this region may also demonstrate that they are important for Factor VII activation activity, and one may consider introducing mutations into one or more of the residues generally located between about amino acid 106 and approximately amino acid 209 of the Faztor sequence of Wound (WO 94/07515). In terms of the preferred region, one or more of amino acids 147, 152, 154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and / or 167 can be generally considered to mutate. With reference to preferred candidate mutations generally outside this region, reference may be made to the following amino acid substitutions: S16, T17, S39, T30, S32, D34, V67, L104, B105, T106, R131, R136, V145, V146, F147, V198, N199, R200 and K2D1, amino acids A34, E34 and R34 are also considered (WO 94/28017). As mentioned, preferably the Factors of Tissue become deficient in its ability to activate Factor VII by altering one or more amino acids in the region generally between approximately position 157 and approximately position 167 in the amino acid sequence, when reference is made to SEQ ID NO: 12. Mutants examples are those where the Trp at position 158 is changed to Arg (SEQ ID NO: 8); where Being in position 162 is changed to Ala; wherein Gly at position 164 is changed to Ala (SEQ ID NO: 9); and the double mutant where Trp at position 158 changes to Arg and Ser at position 162 changes to Ala. These are then exemplary mutations and it is considered that any Tissue Factor mutant having an altered amino acid composition that has the desirable characteristics of binding to Factor VlI / VIIa but not activating the coagulation cascade will be useful in the context of this invention. A4. In Vitro Quantitative Assessment of Coagulant Deficiency The tissue factor constructs of the present invention, whether they are truncated, mutated, truncated and mutated, dimers: .cas, multimeric, conjugated with inert carriers to increase their half-life, or any combination of the above, each one is deficient coagulation in comparison with the original natural type tissue factor. By the term "deficient coagulation", as used herein, it is understood that the Tissue Factor constructs have a deteriorated ability to promote coagulation so that their administration into the systemic circulation of an animal or a human patient does not lead to for significant collateral effects or limiting toxicity. A Tissue Factor construct can be easily analyzed in order to determine if it satisfies this definition, simply by conducting a test on an experimental animal. However, the following detailed guidance is provided to assist those skilled in the art in the above characterization and selection of appropriate candidates for deficient coagulation tissue factor constructions, so that any animal study can be carried out. experimentally efficient and cost efficient. In quantitative terms, the factors of deficient coagulation tissue will be a hundred times or more or less active than the original, full-length tissue factor, that is, they will be a hundred times or more or less capable of inducing plasma coagulation than the factor of original, full-length tissue, when tested in a suitable phospholipid environment. More preferably, the impaired Tissue Factors should be one thousand times or more or less capable of inducing plasma coagulation than the full-length original type Tissue Factor in a suitable phospholipid environment; even more preferably, the Tissue Factors should be ten thousand times or more or less capable of inducing plasma coagulation than the full-length, full-length Tissue Factor in such an environment; and more preferably, the impaired Tissue Factors should be one hundred thousand times or more or less capable of inducing plasma coagulation than their original, full-length Tissue Factor in a suitable phospholipid environment. It will be appreciated that this expression "one hundred thousand times" generally corresponds to one of the currently preferred constructions, the truncated Tissue Factor of 219 • amino acids in length (SEQ ID NO: 1). Inherent within the definition of "X times or more or less capable of inducing plasma coagulation" is the concept that the tissue factor object of the research is still capable of inducing plasma coagulation. Obviously, a tissue factor that has been modified to become completely incapable of inducing coagulation will generally not be useful in the context of the present invention. Tissue Factors that are less active than the Tissue Factor of the wild type in controlled phospholipid tests for approximately 500 thousand times are still considered to be useful in connection with the present. Similarly, all variants and mutants of Tissue Factor that are between approximately 500 thousand times and approximately one million times less capable of inducing plasma coagulation than their original full-length tissue factor in a suitable phospholipid environment yet it is considered that they have utility in certain modalities. Nevertheless, it is generally considered that an inability to activity a million times (106) will generally be approximately the least active that could be considered for use in the present invention. In addition, those Tissue Factor constructions that are towards the less active end of the established range may find more utility in relation to certain defined treatment regimens or in combination therapies. The particular choice of tissue factor variant and therapeutic strategy will be readily determined by one skilled in the art. Regardless of whether there are certain preferred and / or optimal uses and combinations of the various elements of Tissue Factors, the factors of poor coagulation tissue for use in the present invention will generally be approximately between 100 times and approximately one million times less active. that the Tissue Factor of the natural type; more preferably, they will be between about 1000 times and about 100 thousand times less active; and can be categorized as less active by any number within the established ranges, including by approximately ten thousand times. The same ranges can also be varied between approximately 1000 times and a million times, or between 10 thousand times and 500 thousand times, or something similar. One or more of several in vitro plasma coagulation activity assays may be employed in connection with the quantitative test of poor coagulation tissue factors. For example, a method of conducting an assay of innate plasma coagulation activity is as follows: 1) add approximately 50 microliters of plasma (human or mouse) to plastic tubes at approximately 37 ° C; 2) adding approximately 50 microliters of relipidated full-length tissue factor (preferably from a commercial source, such as American Diagnostics Inc., Greenwich, CT) to a range of concentrations in a convenient regulator such as calcium free phosphate or saline Regulated HEPES, pH 7.4 at 37 ° C. To other tubes add the Tissue Factor candidate in truncated or mutant version to a range of concentrations in the same regulator. 3) add approximately 50 microliters of 30 mM CaCl 2 at about 37 ° C; 4) record the time for the first fibrin strands to form; and 5) construct a standard curve of the concentration of full-length tissue factor (mol per liter) against coagulation time. Construct a curve of the concentration of candidate mutant tissue factor (mol per liter) against coagulation time. Calculate the activity difference between the full length Tissue Factor and the Tissue Factor "test" by comparing the concentration of each one necessary to give a coagulation time equivalent to approximately half of the maximum decrease in coagulation time. The "test" mutant tissue factor should be more than 100 times less capable than the full-length tissue factor on a molar basis to induce plasma coagulation. Variations of this type of assay can be carried out as would be evident to a person with ordinary skill in the art. For example, tests based on the binding of the Tissue Factor and the tissue factor construct candidate to a cell membrane or phospholipid surface can be carried out, for example, using an antibody or another ligand to effect this binding. In these tests, the candidate or Truncated Tissue Factor or Tissue Factor mutant should be more than 100 times less effective in inducing plasma coagulation than the wild-type Tissue Factor when bound by an antibody. or another ligand to a cell membrane or phospholipid surface. With Tissue Factor mutants that do not allow Factor VII to efficiently convert to Vlla Factor, it may be necessary to add Factor Vlla to the plasma to obtain this level of activity. In an exemplary assay, this activity can be measured using the following method: 1) Cells such as mouse lymphoma cells A20 (positive I-Ad) (e.g., 4 x 106 cells / milliliter, 50 microliters) are incubated in a regulator such as phosphate buffered saline for about one hour at about room temperature with a binding promoting substance, such as a bispecific antibody (50 micrograms / milliliter, 25 microliters), e.g., in terms of cells A20, which consist of a Fab 'arm of an antibody such as the B21-2 antibody directed against I-Ad, bound to the Fab' arm of an antibody such as the 10H10 antibody directed against a non-inhibitory epitope in the Tissue Factor; 2) Prepare an identical set of tubes containing cells, but no bispecific antibody or other binding substance; 3) Wash the cells effectively, e.g., twice at room temperature, and resuspend the cells in approximately 50 microliters of phosphate buffered saline; 4) Add varying concentrations of the candidate Tissue Factor mutants in phosphate buffered saline (approximately 50 microliters) at about room temperature. The bispecific antibody or other binding substance captures the Tissue Factor mutant and puts it in close proximity with the cell surface. Vlla Factor (1-10 nM) is added in addition to the Tissue Factor mutant when desired to determine activity in the presence of Vlla Factor. The total volume per tube is adjusted to approximately 150 microliters with phosphate buffered saline. The tubes are incubated for approximately one hour at about room temperature; 5) The cells are heated to approximately 37 ° C. 6) Calcium chloride (approximately 50-mM, 50 microliters) and citrated mouse or human plasma (approximately 50 microliters) are added at about 37 ° C. 7) The time is recorded for the first fibrin strands to form; and 8) The coagulation time (in seconds) is plotted against the concentration of Tissue Factor mutant (mol per liter) for cells coated or not coated with binding substance, e.g., bispecific antibody. The concentration of Tissue Factor mutant is calculated, which gives a coagulation time equivalent to approximately half of the maximum decrease in clotting time (usually of 50 to 100 seconds). The increase in coagulation activity given by the bispecific antibody is calculated and should be 100 times more. It is considered that the compositions of Factor of Tissue candidates prepared by the present invention can be tested using assays similar to those described above to confirm that their functionality has been maintained, but that their ability to promote coagulation has deteriorated by at least the required amount of approximately 100 times and preferably by approximately 1,000 times, more preferably by approximately 10,000 times, and more preferably by approximately 100,000 times. In modalities where it is contemplated that an additional substance should ultimately be used in combination with the candidate weak coagulation tissue factor, it is important that the additional factor or substance be included in the in vi tro assay. A particularly relevant example is the analysis of an activating mutant of Factor VII, which preferably should be analyzed together with the addition of Factor Vlla. However, the Vlla Factor is not the only additional component that can be tested in this way. In general, additional substances can be called "additional candidates". To identify an additional candidate, or to optimize preferred amounts of the candidates for use in the present invention, tests such as those described above in parallel can be carried out. That is, coagulation would be measured or determined in the absence of the additional candidate, and then the candidate substance would be added to the composition and would redetermine the time and / or duration of blood coagulation. An additional candidate substance that functions in combination with a Tissue Factor mutant or variant would result in an overall level of coagulation that is between approximately 100 times and approximately 1,000,000 times lower than that observed with the original Tissue Factor again would be a combination suitable for use in the context of the present invention. The technicians with ordinary experience in the field will understand that each of the previous tests in vi tro and variations thereof are relatively simple to establish and perform. In this way, a panel of candidate Tissue Factor variants or combinations of Tissue Factors can be tested with other substances and the most promising candidates can be selected for further studies, particularly for experimental tests in an animal or human assay. Regardless of whether the above assays are believed to be particularly useful in connection with the present invention, the in vitro tests contemplated for use herein are not limited to these assays. In accordance with the above, one can carry out any type of coagulation or procoagulation test that one wishes. For example, for more details regarding truncated Tissue Factor and procoagulation assays, experienced practitioners refer to U.S. Patent Nos. 5,437,864; 5,223,427; and 5,110,730 and the International Patent with publication numbers WO 94/28017; WO 94/05328; and WO 94/07515, each of which is specifically incorporated by reference herein for purposes of further complementing the present disclosure with respect to the tests. TO 5. In Vivo Confirmatory Studies It will be understood by those skilled in the art that candidate deficient coagulation tissue mutants, variants or combinations thereof with additional substances should generally be tested in a live setting before being used in a human subject. These preclinical tests on animals are routine in the art. To carry out these confirmatory tests, all that is required is an animal model accepted in the technique of the disease in question, such as an animal that has a solid tumor. Any animal can be used in this context, such as, e.g., a mouse, rat, guinea pig, hamster, rabbit, dog, chimpanzee, or the like. In the context of cancer treatment, studies using small animals such as mice are widely accepted because they are predictive of clinical efficacy in humans, and these animal models are therefore preferred in the context of the present invention as they are easily available and relatively inexpensive, at least in comparison with other experimental animals. The way to carry out an experimental animal test will be directly that of the ordinary experience in the technique. All that is required to carry out this test is to establish equivalent treatment groups, and administer the test compounds to a group while carrying out several parallel control studies on the equivalent animals in the remaining group or groups. The animals are monitored during the course of the study and, finally, the animals are sacrificed to analyze the effects of the treatment. One of the most useful features of the present invention is its application to the treatment of vascularized tumors. In accordance with the foregoing, antitumor studies can be carried out to determine specific thrombosis within the tumor vasculature and overall antitumor effects. As part of these studies, you can also monitor the specificity of the effects, including evidence of coagulation in other vessels and tissues and good general condition. of animals should be monitored carefully. In the context of the treatment of solid tumors, it is contemplated that the effective Tissue Factor constructs and the effective amounts of the constructions will be those constructs and amounts that generally result in at least about 10% of the vessels within a tumor. vascularized exhibiting thrombosis, in the absence of significant thrombosis in vessels outside the tumor, - preferably thrombosis will be observed in at least approximately 20%, approximately 30%, approximately 40%, or approximately 50% also of blood vessels within the solid tumor mass, without significant thrombosis not localized. In the treatment of large tumors, the present inventors have routinely observed these positive effects. Undoubtedly, tumors have been analyzed in which at least approximately 60%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95% or even and including approximately 99% of the tumor vessels have become thrombotic Naturally, the greater the number of vessels that exhibit thrombosis, the more preferred the treatment is, as long as the effect remains specific, relatively specific or preferential to the vasculature associated with the tumor and as long as coagulation is not apparent in other tissues to a degree enough to cause significant damage to the animal. After the induction of thrombosis within the blood vessels of the tumor, the tumor tissues become necrotic. The successful use of the constructs of the invention, or the doses thereof, can thus also be assessed in terms of the expansion of necrosis induced specifically in the tumor. Again, the expansion of cell death in the tumor will be assessed in relation to the maintenance of healthy tissues in all other areas of the body. Tissue Factor substances, combinations or optimal doses will have therapeutic utility in accordance with the present invention when their administration results in at least about 10% of the tumor tissue becoming necrotic (10% necrosis). Again, it is preferable to obtain at least about 20%, about 30%, about 40% or about 50% of necrosis in the tumor region, without significant adverse side effects. These beneficial effects have again been observed by the present inventors. Naturally, it will be preferable to use constructs and doses capable of inducing at least 60%, about 70%, about 80%, about 85%, about 90%, about 95% to including 99% of the necrosis of the tumor, as long as the constructs and doses used do not result in significant side effects or some other unwanted reactions in the animal. All the above determinations can be easily made and properly assessed by technicians with ordinary experience in the field. For example, scientists and attending physicians could use these data from experimental animals in optimizing doses suitable for human treatment. In subjects with advanced disease, some degree of side effects can be tolerated. However, patients in the early stages of the disease can be treated with more moderate doses in order to obtain a significant therapeutic effect in the absence of side effects. The effects observed in these experimental animal studies should preferably be statistically significant over the control levels and should be reproducible from the study under study.

Those of ordinary skill in the art will further understand that the Tissue Factor constructs, combinations, and dosages that result in specific thrombosis of the tumor and necrosis toward the lower end of the effective ranges mentioned above may nevertheless be useful in connection with the present invention. For example, in embodiments where a continuous application of the active substances is contemplated, an initial dose of a construct that only results in approximately 10% thrombosis and / or necrosis will nevertheless be useful, particularly as it is often observed that it is Initial reduction "primes" the tumor for a subsequent destructive assault after the subsequent reapplication of the therapy. In any case, even if it exceeds approximately 40% or so the inhibition of the tumor is not finally achieved (which is the general goal), it will be understood that any induction of thrombosis and necrosis is useful because it represents an advance on the patient's condition before treatment As discussed above in relation to the in vi tro testing system, it will naturally be understood that combinations of substances intended for use together should be tested and used together. By way of example only, the factor Vlla activating mutant of the present invention falls into this category and should generally be tested in conjunction with the simultaneous, prior or subsequent administration of the exogenous Vlla Factor. Similarly, the individual Tissue Factor constructs of the present invention can be analyzed directly in combination with one or more chemotherapeutic drugs, immunotoxins, coaguligands or the like. The analysis of the combined effects of these substances would be determined and evaluated in accordance with the guidelines presented above. A6. Biologically Functional Equivalents As discussed, the compositions of Factor of Truncated tissue useful in the present invention are those that will generally promote coagulation at least 100 times less effectively than the natural type of Tissue Factor. In other modalities the truncated tissue factor promotes coagulation at least 1,000 times less effectively, in other modalities the truncated tissue factor promotes coagulation at least 104 or up to 105 times less effectively than the tissue factor of the natural type, being the factors of Fabric that are approximately 106 times or less active than the natural-type Tissue Factor approximately the minimum required activity required. Exemplary tissue factors are those that lack a transmembrane and cytosolic region (amino acids 220-263). A truncated Tissue Factor of the present invention is given in SEQ ID NO: 1 and contains amino acids 1-219 of the wild-type Tissue Factor (SEQ ID NO: 12). Of course this is only an exemplary truncated Tissue Factor and other truncated Tissue Factor constructs are contemplated, for example, a construct comprising amino acids 1-220; 2-219, 3-219 or any other truncation of the identification sequence number 12 which leaves the molecule lacking the transmembrane domain and / or cystosolic domains of the wild-type Tissue Factor otherwise resulting in a functionally comparative molecule . Mutants are also contemplated, as described in detail above. Using the detailed guidance provided above, still other equivalents of the Fabric Factors can be made. Modifications and changes in the structure of the Tissue Factor can be made and still obtain a molecule having similar or otherwise desirable characteristics. For example, certain amino acids can be substituted by other amino acids in a protein structure without appreciable loss of interactive binding capacity, such as, for example, binding to Factor Vlla. Since this is the interactive and natural capacity of a protein that defines this biological functional activity of the protein, certain substitutions of amino acid sequences can be made in a protein sequence (or of course, the underlying DNA sequence) and yet obtain a protein with equal (agonistic) properties. In this way it is contemplated that various changes can be made in the sequence of the Tissue Factor (SEQ ID NO: 12) proteins and peptides (or underlying DNA sequence, SEQ ID NO: 11) without appreciable loss of their usefulness or biological activity . It also comprises the skilled artisan that inherent in the definition of an equivalent biologically functional protein or peptide is the concept that there is a limit to the number of changes that can be made within a defined portion of the molecule and still result in a molecule with an acceptable level of equivalent biological activity. The equivalent biologically functional peptides are thus defined herein as the peptides in which certain amino acids, not the majority or all, can be substituted. Of course, a plurality of different proteins / peptides with different substitutions can easily be made and used according to the invention. Amino acid substitutions are generally based on the relative simplicity of the amino acid side chain substituents, for example, their hydrophobicity, hydrophilicity, charge, size, and the like. An analysis of the size, shape and type of amino acid side chain substituents reveals that arginine, usin and histidine are positively charged residues; that alanine, glycine and serine are of a similar size; and that phenylalanine, tryptophan and tyrosine generally have a similar form. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and 'irosine; they are defined herein as biologically functional equivalents. Making more quantitative changes, we can consider the hydropathic index of amino acids. Each amino acid has been assigned a hydropathic index based on its hydrophobicity and loading characteristics, these are: isoleucine (+4.5); valina (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5). The importance of the hydropathic amino acid index to confer interactive biological function on a protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted by other amino acids that have a similar hydropathic rating or rating and still retain a similar biological activity. When making changes based on the hydropathic index, substitution of amino acids whose hydropathic indices are within ± 2 is preferred, those that are within ± 1 are particularly preferred, and those within ± 0.5 are still more particularly preferred. It is understood that an amino acid can be substituted by another that has a similar hydrophilicity value and still obtains a biologically equivalent protein. As detailed in U.S. Patent No. 4,554,101 (incorporated herein by reference), the following hydrophilicity values have been assigned to the amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); Alanine (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); tryptophan (-3.4). When making changes based on hydrophilicity values, substitution of amino acids whose hydrophilicity values are within ± 2, those within ± 1 are particularly preferred, and those within ± 0.5 are still more particularly preferred. . B. Tissue Factor Polynucleotides Bl. DNA segment. The polynucleotides, which encode the Tissue Factors of the present invention can encode a whole Tissue Factor protein, as long as it is deficient coagulation, a functional Tissue Factor protein domain, or any mutant Tissue Factor polypeptide or variant. according to the detailed guide presented in this. If one wishes to prepare a Tissue Factor, a Tissue Factor mutant or a truncated and mutated Tissue Factor, the underlying useful DNA and gene will generally be the same. Since human DNA is available for the entire Tissue Factor molecule, it will generally be preferred to use this human construct since clinical treatment in humans is intended. However, in no way is the use of other Tissue Factor genes excluded, as long as the produced protein does not produce adverse or immunological reactions after administration to a human patient. The methods and compositions described in U.S. Patent No. 5,110,730 are specifically incorporated herein by reference for the purposes of further complementing the description of applicants with reference to the genes and DNA segments for use therewith. . Polynucleotides can be derived from genomic DNA, that is, cloned directly from the genome of a particular organism. In other embodiments, however, the polynucleotides can be complemented DNA (cDNA). The cDNA is DNA prepared using messenger RNA (mRNA) as a standard. Thus, a cDNA does not contain any interrupted coding sequence and generally contains almost exclusively the coding region (s) for the corresponding protein. In other embodiments, the polynucleotide can be produced synthetically. As is known to those skilled in the art, it is generally preferred to use the cDNA construct in recombinant expression since those constructs are easy to manipulate and use. The use of longer genomic clones, up to and including full length sequences, is by no means excluded. Although a surprising feature of the present invention is that the Tissue Factor constructs are preferentially located specifically in the vasculature of a solid tumor and induce specific antitumor effects therein, it is also contemplated that the Tissue Factor proteins and the polypeptides are can be administered to the tumor environment using a recombinant vector that expresses the products of the Tissue Factor. These "gene therapy" approaches to cancer treatment can be easily practiced by reference to certain scientific references that refer to adequate constructions and protocols. By way of example only, a viral vector, such as a retroviral vector, herpes simplex virus, HSV (U.S. Patent No. 5,288,641), cytomegalovirus, can be used.; adeno-associated virus, AAV (U.S. Patent No. 5,139,941); and / or an adenoviral vector. The genomic human DNA sequence for the Tissue Factor is provided in SEQ ID NO: 11, with the corresponding amino acid sequence provided in SEQ ID NO: 12. If one wishes to express Factor VII, the DNA and the sequences of amino acids are provided in SEQ ID NO: 13 and SEQ ID NO: 14 respectively. It is contemplated that the natural variants of the Tissue Factor exist and that they have different sequences than those described herein. Thus, the present invention is not limited to the use of the polynucleotide sequence provided for the Tissue Factor but, instead, includes the use of any variant that occurs naturally. The present invention also encompasses chemically synthesized mutants of these sequences, intelligently designed after an application of the quantified structural and functional considerations detailed above. Another class of sequence variants are the result of codon variation. Because there are several codons for most of the 20 normal amino acids, many different DNAs can code for the Tissue Factor. The reference to Table 1 will allow these variants to be identified TABLE I Amino Acids Codons Alanine Wing A GCA GCC GCG CUC Cysteine Cys C UGC UGU A cid Asp D GAC GAU Aspartic Acid Glu E GAA GAG Glutamic Phenylalanine Phe F UUC UUU Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAU Isoleucine He I AUA AUC AUU Lysina Lys K AAA AAG Leucina Leu L UUA UUG CUA CUC CUG CUU Methionine Met M AUG Asparagine Asn N AAC AAU Proline Pro P CCA CCC CCG CCU Glutamine Gln Q CAÁ CAG Arginine Arg R AGA AGG CGA CGC CGG CGU Serina Ser S AGC AGU UCA UCC UCG UCU Threonine Thr T ACA ACC ACG ACU Valine Val V GUA GUC GUG GUU Tryptophan Trp W UGG Tyrosine Tyr and UAC UAU B2. Mutagenesis Site-specific mutagenesis is a useful technique in the preparation of individual peptides, or equivalent biologically functional proteins or peptides, through specific mutagenesis of the underlying DNA. The technique further provides an easy ability to prepare and test sequence variants, which incorporates one or more of the foregoing considerations, by introducing one or more nucleotide sequence changes into the DNA. Site-specific mutagenesis allows the production of mutants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation, as well as a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and complexity of sequence to form a stable duplex on both sides of the suppression junction that is being traversed. Typically, a primer of about 17 to 25 nucleotides in length with about 5 to 10 residues is preferred on both sides of the junction of the sequence being altered. The technique of site-specific mutagenesis is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both single chain and double chain form. Typical vectors useful in site-directed mutagenesis include vectors such as M13 phage. These phage vectors are commercially available and their use is generally well known to those skilled in the art. Double-stranded plasmids are also routinely employed in site-directed mutagenesis, which eliminates the step of transferring the gene of interest from a phage to the plasmid. In general, site-directed mutagenesis is performed by first obtaining a single chain vector, or by mixing two double-stranded vector chains that include within their sequence a DNA sequence encoding the desired protein. An oligonucleotide primer having the desired mutated sequence is prepared synthetically. This primer is fixed with the single-stranded DNA preparation, and is subjected to DNA polymerization enzymes such as a Klenow fragment of E. coli polymerase. in order to complete the synthesis of the chain that carries the mutation. In this way, a heteroduplex is formed in which one strand encodes the original non-mutated sequence and the second strand carries the desired mutation. This heteroduplex vector is then used to transform suitable cells, such as E.coli cells, and clones are selected that include recombinant vectors that carry the configuration of the mutated sequence.

The preparation of sequence variants of the selected gene using site-directed mutagenesis is provided as a means of producing potentially useful species and is not presumed to be limiting, since there are other ways in which sequence variants of the genes can be obtained. For example, recombinant vectors encoding the desired gene can be treated with mutagenic substances, such as hydroxylamine, to obtain sequence variants. Suitable techniques are also described in U.S. Patent No. 4,888,286, incorporated herein by reference. Although the above methods are convenient for use in mutagenesis, the use of the polymerase chain reaction (PCRMR) is now generally preferred. This technology offers a fast and efficient method to introduce the desired mutations into a given DNA sequence. The following text particularly describes the use of the polymerase chain reaction to introduce point mutations in a sequence, since it can be used to change the amino acid encoded by the given sequence. The adaptations of this method are also convenient for introducing restriction enzyme sites into a DNA molecule. In this method, synthetic oligonucleotides are designed to incorporate a point mutation at one end of an amplified segment. After the polymerase chain reaction, the amplified fragments are blunted by treating them with Klenow fragments, and the blunt fragments are ligated and subcloned into a vector to facilitate sequence analysis. To prepare the standard DNA to be mutagenized, the DNA is subcloned into a high copy number vector, such as pUC19, using the restriction sites flanking the area to be mutated. The template DNA is prepared using a plasmid minipreparation. Suitable oligonucleotide primers that are based on the parent sequence, but that contain the desired point mutation and that are flanked at the 5 'end by a restriction enzyme site, are synthesized using an automated synthesizer. Generally, the primer is required to be homologous to the standard DNA for about 15 bases or so. The primers can be purified by denaturing polyacrylamide gel electrophoresis, although this is not absolutely necessary for use in the polymerase chain reaction. The 5 'end before the oligonucleotides should then be phosphorylated. The standard DNA should be amplified by polymerase chain reaction, using the oligonucleotide primers containing the desired point mutations. The concentration of MgCl2 in the regulated amplification solution will generally be about 15 mM.

Generally, 20 to 25 cycles of polymerase chain reaction should be carried out as follows: denaturation, 35 seconds at 95 ° C; Hybridization, 2 minutes at 50 CC; and extension, 2 minutes at 62 ° C. The polymerase chain reaction will generally include a last extension cycle of approximately 10 minutes at 72 ° C. After the final extension step, approximately 5 units of Klenow fragments should be added to the reaction mixture and incubated for another 15 minutes at approximately 30 ° C. The exonuclease activity of the Klenow fragments is required to make the ends wash and be convenient for blunt cloning. The resulting reaction mixture should generally be analyzed by agarose or non-denaturing acrylamide gel electrophoresis to verify that the amplification has produced the predicted product. Then we would proceed to the reaction mixture by removing most of the mineral oils, extracting with chloroform to remove the remaining oil, extracting with regulated phenol and then concentrating by precipitation with 100% ethanol. Next, approximately half of the amplified fragments should be digested with a restriction enzyme that cleaves all flank sequences used in the oligonucleotides. The digested fragments are purified on a low gelling / melting agarose gel. To ubclone the fragments and verify the point mutation, the two amplified fragments would be subcloned into an appropriate digested vector by blunt ligation. This would be used to transform E. coli, from which the plasmid DNA could subsequently be prepared using a minipreparation. The amplified portion of the plasmid DNA would be analyzed by DNA sequencing to confirm that the correct point mutation was generated. This is important since the TAQ DNA polymerase can introduce additional mutations into the DNA fragments. The introduction of a point mutation can also be carried out using sequential polymerase chain reaction steps. In this procedure, the two fragments encompassing the mutation are fixed to one another and extended by mutually initiated synthesis. This fragment is then amplified by a second polymerase chain reaction step, thereby avoiding the blunt ligation required in the previous protocol. In this method, the preparation of the standard DNA, the generation of the oligonucleotide primers and the first amplification of the polymerase chain reaction are carried out as described above. However, in this process, the chosen oligonucleotides must be homologous to the standard DNA for a stretch of approximately 15 to 20 bases and must also overlap with each other for about ten bases or more.

In the second polymerase chain reaction amplification, each amplified fragment and each flank sequence primer would be used and chain reaction of p > olimerase for between about 20 and about 25 cycles, using the conditions described above. Again two fragments would be subcloned and it would be verified that the point mutation was correct by using the steps indicated above. Using any of the above methods, it is generally preferred to introduce the mutation by amplifying a fragment as small as possible. Of course, parameters such as the melting temperature of the oligonucleotide will generally be influenced by the GC content and the length of the ol: .go, should also be carefully considered. Execution of these methods, and their optimization if necessary, will be known to those skilled in the art, and is further described in several publications, such as Current Protocols in Molecular Biology, 1995, incorporated herein by reference. B3 Expression constructions and protein production In this whole application, the term "expression construction" means that it includes any type of genetic construct that contains a nucleic acid encoding a gene product in which part or all of the sequence encoding the nucleic acid is capable of being transcribed. The transcript will usually be translated into a protein. Thus, the expression preferably includes both the transcription of a Tissue Factor gene and the translation of a Factor of a Tissue Factor mRNA into a Tissue Factor protein product. A technique frequently used by experts in the field of protein production today is to obtain the so-called "recombinant" version of the protein, to express it in a recombinant cell and obtain the protein from these cells. These techniques are based on the "cloning" of a DNA molecule that encodes the protein from a DNA library, that is, obtaining a specific DNA molecule different from other portions of the DNA. This can be achieved by, for example, cloning a cDNA molecule, or cloning a genomic-like DNA molecule. Techniques such as these would be suitable for the production of particular Tissue Factor compositions according to the present invention. Recombinant fusion proteins are discussed in greater detail hereinafter, and in US Pat. No. 5,298, E >.; 99, incorporated herein by reference for purposes of further exemplification of production and use of fusion protein. For the expression of the Tissue Factor, as soon as a suitable clone or clones have been obtained (full length if desired), whether they are based on genomic or cDNA, an expression system for the recombinant preparation can be continued. of the Fabric Factors. The engineering of the DNA segments for expression in a prokaryotic or eukaryotic system can be carried out by techniques generally known to those skilled in the field of recombinant expression. It is believed that virtually any expression system can be employed in the expression of these proteins. These proteins can be expressed successfully in eukaryotic expression systems, e.g., CHO cells, as described by Rehemtulla et al. (1991), however, bacterial expression systems, such as pQE-60, are considered. of E. coli will be particularly useful for the large-scale preparation and subsequent purification of proteins or peptides. The cDNAs for the Tissue Factor can be expressed in bacterial systems, the encoded proteins being expressed as fusions with 3-galactosidase, ubiquitin, glutathione S-transferase from Schistoso to iaponicum and the like. It is believed that bacterial expression will have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained therefrom. The techniques of U.S. Patent Nos. 5,298,599 and 5,346,991 are also incorporated herein by reference to further complement the soluble Tissue Factor production methods described herein, with particular reference to the U.S. Pat. No. 5,346,991 for the purposes of further complementing the description with respect to the creation and production of mutants and variants of the Fabric Factor. In order for the construct to effect the expression of a Tissue Factor transcript, the polynucleotide encoding the Tissue Factor polynucleotide will be under the control of transcription of a promoter. A "promoter" refers to a DNA sequence recognized by the synthetic machinery of the host cell, or the synthetic machinery introduced, which is required to initiate the specific transcription of a gene. The phrase "under transcription control" means that the promoter is in the correct place in relation to the polynucleotide to control the initiation and expression of polymerase of the polynucleotide RNA. The term "promoter" will be used herein to refer to a group of transcription control modules that are nested around the initiation site of RNA polymerase II. In terms of microbial expression, U.S. Patent Nos. 5,583,013; 5,221,619; 4,785,420; 4,704,362; and 4,366,246 are incorporated herein by reference for the purposes or to further complement the present disclosure in relation to the expression of genes in recombinant host cells. B4 Purification of Tissue Factor and related compositions As soon as the peptides have been expressed they can be isolated and purified using protein purification techniques well known to those skilled in the art. These compositions will be used alone or in combination with antibodies, chemotherapies and effector ligands as therapeutic substances in the treatment of tumors as detailed hereinafter. The exemplary peptides of the present invention are shown in SEQ ID NO: l-SEQ ID NO: 9, it is understood that these are only examples and any mutation, alteration or variants that occur naturally of these are also contemplated sequences as useful in conjunction with the present invention. Protein purification techniques are well known to those skilled in the art. These techniques tend to involve the fractionation of the cell medium to separate the protein of interest from other components of the mixture. Particularly suitable analytical methods for the preparation of a pure peptide are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, isoelectric focusing and the like. A particularly efficient method for purifying peptides is rapid protein liquid chromatography or even high pressure liquid chromatography. Several other convenient techniques for use in protein purification will be well known to those skilled in the art. These include, for example, precipitation with ammonium sulfate, PEG, antibodies and the like or by heat denaturation, followed by centrifugation.; chromatography steps such as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focus; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that the order of conducting the different steps of purification can be changed, or that certain steps can be omitted, and would anyway result in a convenient method for the preparation of a protein or peptide. substantially purified. As described herein in detail, the generally preferred techniques for purifying Tissue Factor constructs expressed for use in the present invention involve the generation of a Tissue Factor molecule that includes an affinity purification tag and the use of an affinity separation matrix to obtain the free tissue factor construction of most or all contaminating species. Many of these fusion protein tags are known to those of ordinary skill in the art and these expression and separation protocols can be easily carried out. There is also technology available to dissociate the original affinity tag prior to the use of the released protein or polypeptide, which can be effected by inserting a sensitive protease linker between the affinity tag and the protein of interest. This methodology is undoubtedly employed in relation to aspects of the present invention. U.S. Patent No. 5,298,599 is also instructive regarding this. However, it is also known that many of these labels do not impede the ability of the expressed protein to carry out its biological functions, and the removal of the label is not necessarily required before the use of the construction of the Tissue Factor in the present invention. C. Pharmaceutical Compositions and Games The pharmaceutical compositions of the present invention will generally comprise an effective amount of truncated Tissue Factor dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce an adverse, allergic or otherwise adverse reaction when administered to an animal, or to a human, as appropriate. As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal substances, isotonic and absorption retardant substances and the like. The use of these media and substances for active pharmaceutical substances is well known in the art. Except insofar as some conventional substance or substance is incompatible with the active ingredient, its use in the therapeutic compositions is considered. Supplementary active ingredients can also be incorporated into the compositions. Cl. Parenteral Formulations The truncated Tissue Factor of the present invention will often be formulated for parenteral administration, e.g., formulated for intravenous, intramuscular, subcutaneous or other injection of these routes, including direct instillation into a tumor or diseased site. The preparation of an aqueous composition containing a coagulant substance directed to the tumor as an active ingredient will be known to those skilled in the art in light of the present disclosure. Typically, these compositions can be prepared as injectables, either as liquid solutions or suspensions; it is also possible to prepare solid forms suitable for use in preparing solutions or suspensions after the addition of a liquid before injection; and the preparations can also be ulsified. The solutions of the active compounds as free base or pharmacologically acceptable salts in water conveniently mixed with a surfactant, such as hydroxypropylcellulose. Dispersions can be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions; formulations that include sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that there is easy injectability. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The truncated Tissue Factor compositions can be formulated in a salt-like or neutral form composition. The pharmaceutically acceptable salts include the acid addition salts (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandélico, and similars. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or ferric hydroxides, and organic bases such as isopropylamine, trimethylamine, histidine, procaine and the like. The carrier can also be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and the like), convenient mixtures thereof, and vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating, such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. The prevention of the action of microorganisms can be applied with respect to various antibacterial and antifungal substances, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic substances, for example, sugars or sodium chloride. The prolonged absorption of the injectable compositions can be applied by the use of compositions of substances that retard absorption, for example, aluminum monostearate and gelatin. Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients listed above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle containing basic dispersion medium and the other ingredients required from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying techniques, and lyophilization which produce a powder of the active ingredient plus some additional desired ingredient from the previously sterile filtered solution thereof. . After formulation, the solutions will be administered in a manner compatible with the formulation of the dose and in an amount that is therapeutically effective. The formulations are easily administered in a variety of dosage forms, such as the type of injectable solutions described above, but drug release capsules and the like can also be employed. Suitable pharmaceutical compositions according to the invention will generally include a quantity of the deficient coagulation tissue Factor mixed with a pharmaceutically acceptable diluent or excipient, such as sterile aqueous solution, to give a range of final concentrations, depending on the intended use. Preparation techniques are generally well known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980, incorporated herein by reference. It should be noted that endotoxin contamination should be kept to a minimum at a safe level, for example, less than 0.5 nanogram / milligram of protein. Furthermore, for human administration, the preparations must meet sterility, pyrogenicity, general safety and purity standards as required by the FDA Office of Biological Standards (Food and Drug Administration). Therapeutically effective doses are readily determinable using an animal model, as shown in the studies detailed herein. Experimental animals that have solid tumors are often used to optimize appropriate therapeutic doses before moving to a clinical setting. These models are known to be very reliable in predicting effective anticancer strategies. For example, mice that have solid tumors, such as those used in the Examples, are widely used in preclinical tests. The inventors have used these mouse models accepted in the art to determine the ranges of truncated tissue factor that give beneficial anti-tumor effects with minimal toxicity. In addition to compounds formulated for parenteral administration, such as intravenous or intramuscular injection, other pharmaceutically acceptable forms are also contemplated, e.g., tablets or other solids for oral administration, delayed-release capsules, liposomal forms and the like. Other pharmaceutical formulations may also be used, depending on the condition to be treated. For example, topical formulations are suitable for treating pathological conditions such as dermatitis and psoriasis; and ophthalmic formulations for diabetic retinopathy. As described in detail herein it is contemplated that certain benefits will result from the manipulation of the deficient coagulation tissue factor constructs to provide them with a longer live half-life. These techniques include, but are not limited to, the manipulation or modification of the Tissue Factor molecule itself, and also the conjugation of Tissue Factor constructs to inert carriers, such as different proteins or non-protein components, including immunoglobulins and Fc portions. . These compositions are called Fabric Factor constructions with a longer half-life here. It will be understood that the longer half-life is not the same as the pharmaceutical compositions for use in "slow release". Slow release formulations are generally designed to give a constant drug level over an extended period. By increasing the half-life of a drug, such as a Tissue Factor construct according to the present invention, it is intended that it results in a high plasma level after administration, and that these levels be maintained for a longer time. long, but these levels generally decline depending on the drug-kinetics of the construct. Although not currently preferred, the slow release formulations of the Tissue Factor construct and combinations thereof are in no way excluded from use in the present invention. C2. Therapeutic Games The present invention also provides therapeutic kits comprising the truncated tissue factor constructs described herein. These sets will generally contain, in a convenient container means, a pharmaceutically acceptable formulation of at least one deficient coagulation tissue factor construct according to the invention. The kits may also contain other pharmaceutically acceptable formulations, such as those containing components to address the truncated tissue factor constructs; extra coagulation factors, particularly Factor Vlla; bispecific antibodies, T cells, or other functional components for their use, e.g., in antigen induction; components for use in antigen suppression, such as a cyclosporin, if necessary; antibodies or immunotoxins from different antitumor sites; and any one or more of a range of chemotherapeutic drugs. The games may have a single container element containing the truncated Fabric Factor, with or without additional components, or may have different container elements for each desired substance. Also considered are games that comprise the separate components necessary to make a bispecific coagulant ligand or immunotoxin. Certain preferred games of the present invention include a deficient coagulation tissue factor construct that is impaired in its ability to activate Factor VII, packaged in a kit for use in combination with the coadministration of exogenous Factor Vlla. In these games the Tissue Factor mutant and the Vlla Factor can be precomplexed, either in a combination of molar equivalent, or with one component in excess of the other; or each of the tissue factor and Vlla Factor components of the kit can be maintained separately within different containers prior to administration to a patient. Other preferred games include a deficient coagulation tissue factor in combination with a "classical" chemotherapeutic substance. This is exemplary of the considerations that are applicable to the preparation of these games of Factor of Tissue and combinations of games in general.

When the components of the kit are provided in one or more liquid solutions, the liquid solution is an aqueous solution, being a particularly preferred sterile aqueous solution. However, the components of the game can be provided as dry powders. When the reactants or components are provided as dry powder, the powder can be reconstituted by the addition of a convenient solvent. It is considered that the solvent can also be provided in another container element. The container element of the kit will generally include at least one bottle, test tube, flask, bottle, syringe or other container element, in which the truncated Tissue Factor, and any other desired substance can be placed and, preferably, in sufficient quotas. . When additional components are included, the kit will also generally contain a second vial or other container in which they are placed, enabling the administration of separate designated doses. The kits may also comprise a second / third container element to contain a sterile, pharmaceutically acceptable regulator, or other diluent. The games may also contain an element by which the Tissue Factor is administered truncated to an animal or patient, eg, one or more needles or syringes, or even an eye dropper, pipette, or other such device, from which the formulation can be injected into the animal or applied to a desired area of the body. The kits of the present invention will also typically include an element for containing the jars, or the like, and another component, in narrow confinement for commercial sale, such as, e.g., plastic containers molded by injection or by blowing into the which are placed and hold the bottles and other desired devices. D. DI treatment. Prothrombotic vessels The composition and methods provided by this invention are broadly applicable to the treatment of any disease, such as a benign or malignant tumor, having as a component of the disease "prothrombotic vessels". These diseases associated with the vasculature more particularly include solid, malignant tumors, and also benign tumors, such as BPH. However, the treatment of diabetic retinopathy, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, neovascular glaucoma and psoriasis; and also angiofibrona, arthritis, atherosclerotic plaques, neovascularization of corneal graft, hemophilic joints, hypertrophic scars, Osler-Weber syndrome, pyogenic granuloma, retrolental fibroplasia, scleroderma, trachoma, vascular adhesions, synovitis, dermatitis and even endometriosis are not excluded either. The present invention is based on the use of Tissue Factor or Tissue Factor constructs in combination with other substances, wherein the Tissue Factor construct or combination has sufficient trogenic activity to disrupt the procoagulant environment within the associated specific vessels with the disease, such as those of a vascularized tumor, in the direction of thrombosis. The environment in vessels in normal tissues is fibrinolytic, whereas in tumor vessels it is procoagulant, that is, it is predisposed towards thrombosis. The procoagulant changes in the tumor vessels are partly the result of a local release of the endothelial cytokines that activate the cells, IL-1 and TNFa. Interleukin 1 is secreted by most tumor cells and by activated macrophages. TNFa is secreted by host cells that have infiltrated the tumor, including activated lymphocytes, macrophages, NK cells and LAK cells. Interleukin 1 and TNFa induce a variety of changes in the vascular endothelium, including upregulation of tissue factor, down regulation of plasminogen activators and upregulation of the plasminogen activator inhibitor PAI-1 (Nawroth and Stern, 1986; Nawroth et al., 1988). These effects are further magnified by factors derived from the tumor (Murray et al., 1991; Ogawa et al., 1990), possibly VEGF. The collective result of these and other changes is that the endothelium becomes more able to support the formation of thrombi and less able to dissolve fibrin, producing a predisposition towards thrombosis. Therefore, in light of the scientific phenomena described above, the inventors contemplate that when poor clotting tissue factors are administered, they have sufficient residual thrombogenic activity to tilt the balance of the coagulation cascade towards thrombosis in the vessels that generally they are prothrombotic in nature (Figure 3). Although a mechanistic understanding of scientific reasoning is not necessary in order to practice the present invention, it will be understood that the foregoing explanation is a mechanism by which the invention can operate. This mechanism is based less on the specific location of the tissue factors within the vessels of a vascularized tumor, as opposed to other vessels, but nevertheless it is surprising that an equal biodistribution of the tissue factor, if this occurs, can result in to an uneven effect on coagulation within disease sites such as within solid tumors. Given that it is, of course, an inherent property of the tumor to maintain a network of blood vessels and to continue in the angiogenic process, it is evident that the blood vessels associated with the tumor can not be as predisposed to thrombosis as they do spontaneously or easily support coagulation. , since coagulation would necessarily result in the arrest of oxygen and nutrients to the tumor cells and cause the tumor to self-destruct. Obviously, this does not happen. It will be readily apparent that the present invention has significant utility in the treatment of diseases, such as vascularized tumors, independently of an understanding of the mechanisms by which specific coagulation can be induced in vessels associated with the disease. However, the inventors further reason that another mechanism that underlines the possible surprising action of the Tissue Factor constructs is that the Tissue Factors selectively bind to certain vascular endothelial cells in preference to those in other tissues or sites of the body. (Figure 3). In accordance with the above, if the Truncated Tissue Factor selectively binds to the vascular endothelium of the tumor after injection, this would put it in contact with a lipid surface and promote the assembly of coagulation initiation complexes in the tumor vessels. . Perhaps, due to the prothrombotic nature of the tumor vessels, there is an increase in the local concentration of the Factors Vlla, IXa, Xa, the tissue factor pathway inhibitor (ITFT) or other molecules that interact with the Factor ' Weaving, thus encouraging the location. The methods of the present invention can be used to test the location of the truncated Tissue Factor by labeling the truncated Tissue Factor, injecting it into mice that have tumors, and determining whether it is actually located within the tumor vessels. Although of scientific interest, carrying out these studies is not necessary to practice the present invention, since the administration of deficient coagulation tissue factor constructions advantageously results in specific antitumor effects independent of the precise mechanism of action underlying this. phenomenon. The present uses of the deficient coagulation tissue factor molecules to promote coagulation in prothrombotic blood vessels are different from the previous uses proposed for the tissue factor constructs, such as the truncated tissue factor in combination with the Vlla Factor. Truncated Tissue Factor and Vlla Factor have been proposed for combined use in the treatment of blood disorders, such as hemophilia (U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067). U.S. Patent Nos. 5,346,991 and 5,589,363 also disclose the use of the K165A and K166A mutants to inhibit coagulation in the treatment of myocardial infarction, and to provide recombinant DNA sequences and vectors for their production. It will immediately be appreciated that the objectives of the above methodology are in direct contrast to the objective prothrombotic blood vessels of the present invention. The "prothrombotic" blood vessels are in a dynamic state that predisposes them to coagulation, but in which the coagulation does not occur in the natural environment. This is exemplified by blood vessels within a vascularized tumor categorized as prothrombotic, but where the tumor maintains a sufficient blood supply and is necessary to support the maintenance and growth of the tumor. In contrast, target sites within an individual with a blood disorder are by their very nature significantly deficient in their ability to withstand coagulation. The combined methodology of Truncated Tissue Factor and Factor Vlla directed mainly for use in hemophiliacs has been proposed for use together with the control of postoperative bleeding or severe trauma in which an external injury has prevented the necessary coagulation process from being effective . This in turn is different than the intention of the present invention. The most important use of the present invention is believed to be related to the treatment of malignant, vascularized tumors. However, in addition to the various diseases and disorders listed above, the invention is also contemplated for use in therapy of other benign growths. A particular example of this is benign prostatic hyperplasia (BPH), which can be treated according to the particular doses and treatment regimens presented below. For the treatment of benign prosthetic hyperplasia it can also be combined with other treatments currently practiced in the art. For example, the attack of immunotoxins on markers localized within benign prostatic hyperplasia, such as PSA, is certainly contemplated. D2. Cancer and Treatment The location of the truncated Tissue Factor and the specific coagulation of the invention is more preferably exploited for therapeutic uses of truncated Tissue Factor in the treatment of cancers and tumors. These uses may employ truncated tissue factor alone or in combination with chemotherapeutic substances and / or immunotoxins or coaguligands. The compositions and methods provided by this invention are broadly applicable to the treatment of any malignant tumor having a vascular component. Typical vascularized tumors are solid tumors, particularly carcinomas, which require a vascular component for the supply of oxygen and nutrients. Exemplary solid tumors that can be treated using the invention include, but are not limited to, carcinomas of the lung, chest, ovary, stomach, pancreas, larynx, esophagus, testes, liver, parotid, bile ducts, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, and the like. The present invention is contemplated for use in the treatment of any patient who presents a solid tumor. However, in what the present invention is particularly successful is in the treatment of solid tumors of moderate or large sizes, patients in these categories are likely to receive more significant benefits of treatments according to the methods and compositions provided herein. . In general, the invention can be used to treat tumors of about 0.3-0.5 centimeters and more, although it is a better use of the invention to treat tumors larger than 0.5 centimeters in size. From studies already conducted in acceptable animal models, it is believed that tumors of about 1.0 or about 1.2 centimeters represent the size of solid tumors that are most effectively attacked by the Tissue Factor constructs of the present invention. Therefore, patients with tumors between 1.0 and about 2.0 centimeters in size will be in the preferred treatment group of patients in relation to the present tissue factor therapies, although tumors up to and including the more massive tumors found in humans can also be treated. Although the present invention is not generally intended as a preventive or prophylactic treatment, the use of the invention is also not confined to the treatment of patients having tumors of moderate or large sizes. There are many reasons under this aspect of the breadth of the invention. For example, a patient presenting with a primary tumor of moderate or greater size may also have several other metastatic tumors that are considered small in size or even in the early stages of implanting metastatic tumors. Since the Tissue Factor constructs and combinations of the invention are generally administered in the systemic circulation of a patient, it will naturally have effects on the smaller, metastatic secondary tumors, although this should not be the primary intention of the treatment. Furthermore, even in situations where the mass of the tumor as a whole is a single small tumor, certain benefits of antitumor effects will result from the use of the present treatments. The guidance provided above with respect to the patients most convenient for use in connection with the present invention is intended as teaching that certain patient profiles can aid in the selection of patients that can be treated by the present invention, or as such. Once, it is better to treat them using other anti-cancer treatment strategies. However, the fact that a more effective or otherwise preferred treatment is perceived to exist in relation to a certain category of patients, in no way negates the basic utility of the present invention in relation to the treatments of all patients who have a vascularized tumor. Another consideration is the fact that the initial assault on a tumor, provided by the Tissue Factor Therapy of the present invention, may be minor in any immediate and measurable effect, but may predispose the tumor to other therapeutic treatments so that the Further treatment results in a global synergistic effect or even leads to total remission or cure. It is not believed that any particular type of tumor should be excluded from the treatments using the present invention. Since the attempt of the therapy is to coagulate the vasculature of the tumor, and since the vasculature is substantially or entirely the same in all solid tumors, it will be understood that the present methodology is broadly or entirely applicable to the treatment of all solid tumors, regardless of the phenotype or particular genotype of the same tumor cells. However, the type of tumor cells may be important for the use of the invention in combination with secondary therapeutic substances, particularly immunotoxins and / or antitumor cell coaguligrants. Technicians with ordinary skill in the art will understand that certain types of tumors may be more amenable to the induction of thrombosis and necrosis using the present invention. The phenomenon is observed in experimental animals, and can occur in human treatments. For example, it is known that the tumor sulfate model HT29 is relatively difficult to coagulate; while the C1300 tumor model is generally more amenable to the induction of thrombosis and subsequent necrosis. These considerations will be taken into account to conduct both preclinical studies in experimental animals and to optimize the doses for use in the treatment of any particular patient or group of patients. As detailed earlier in the discussions concerning live quantitative studies, there are real objectives that can be used as guidelines in connection with preclinical testing before proceeding with clinical treatment. However, this is more a matter of cost effectiveness than global utility, and is a mechanism for selecting the most advantageous compounds and doses. With respect to its basic utility, any construction or combination thereof that results in consistent detectable thrombosis and antitumor effects will still define a useful invention. Thrombotic and necrotic effects may be observed between about 10% and about 40-50% of the blood vessels of the tumor and of the tumor tissues, with up to about 50% and about 99% of these effects being observed. It will also be understood that even in circumstances where the antitumor effects of the construction of tissue factor and combinations are towards the lower end of this range, it may be that this therapy is equally or even more effective than all the other therapies known in the context. of the particular tumor targets. Unfortunately it is evident to a physician that certain tumors can not be effectively treated on a medium or long term basis, but this does not negate the usefulness of the present therapy, particularly when it is as effective as other generally proposed strategies. In order to design appropriate doses of the constructs and combinations of deficient coagulation tissue factor, it can easily be extrapolated from the animal studies described herein in order to arrive at suitable doses for clinical administration. To achieve this conversion, the mass of the substances administered per unit mass of the experimental animal would be considered, and then the differences in the body surface area between the experimental animal and the human patient would be considered. All these calculations are well known and routine for those skilled in the art. For example, taking the successful dose of 16 micrograms per mouse (total body weight of approximately 20 grams), and applying the calculation indicated above, the equivalent dose for use in a human patient would be approximately 2 milligrams. In accordance with the foregoing, using this information, the inventors contemplate that useful doses of deficient coagulation tissue factor for use in human administration would be between about 0.2 milligrams and about 200 milligrams of the tissue factor construct per patient. Notwithstanding this established range, it will be understood that, given the parameters and the detailed guidance presented above, other variations in the active or optimum ranges within the present invention will still be covered. The doses contemplated will therefore generally be between about 0.2 milligrams and about 180 milligrams; between 0.5 and approximately 160 milligrams; between l and approximately 150 milligrams; between about 5 and about 125 milligrams; between approximately 10 and approximately 100 milligrams; between about 15 and about 80 milligrams; between about 20 and about 65 milligrams; between about 30 and about 50 milligrams; approximately 40 milligrams; or in any particular range using any of the above-mentioned exemplary doses or any intermediate value between the particular set ranges. Although doses around 1 milligram, 2 milligrams, 3 milligrams, 4 milligrams and 5 milligrams are currently preferred, it will be understood that lower doses may be more suitable in combination with other substances, and that higher doses can still be tolerated, particularly given the fact that the Tissue Factor substances for use in the invention are not themselves cytotoxic and even if there is an adverse side effect, it would not necessarily result in coagulation that could not be counteracted by normal homeostatic mechanisms, which is believed to decrease the chances of significant toxicity in healthy tissues. The intention of the therapeutic regimens of the present invention is generally to produce the maximum antitumor effects and at the same time to keep the dose below the levels associated with unacceptable toxicity. In addition to varying the same dose, the administration regimen can also be adapted to optimize the treatment strategy. A currently preferred treatment strategy is to administer between about 0.2 milligrams, and about 200 milligrams of the Tissue Factor construct or combination thereof about 3 times in about a 7 day period. For example, doses would be given - approximately on day 1, day 3 or 4 and day 6 or 7. To administer the same particular doses, it would be preferred to provide a pharmaceutically acceptable composition to the patient systemically. Intravenous injection is generally preferred, and the most preferred method is to employ a continuous infusion for a period of time approximately 1 or 2 hours or so. Although it is not required to determine these parameters prior to treatment using the present invention, it should be noted that the studies detailed herein result in at least some thrombosis being observed specifically in the blood vessels of a solid tumor within approximately 30 minutes of the injection. , and the tumor cells themselves begin to die within approximately 3 to 4 hours. The extent of the tumor necrosis is usually observed in the following approximately 24 hours, up to and including more than 90% of necrosis. E. Combination Therapies The methods of the present invention can be combined with any other method generally employed in the treatment of the particular disorder or disorder that the patient presents. For example, in relation to the treatment of solid tumors, the methods of the present invention can be used in combination with classical approaches, such as surgery, radiotherapy and the like. As long as a therapeutic approach is not known to be harmful in itself, or counteract the effectiveness of the Tissue Factor therapy, its combination with the present invention is contemplated. When one or more substances are used in combination with the Tissue Factor therapy, the combined results are not required to be added to the effects observed when each treatment is carried out separately, although this is evidently desirable, and there is no requirement particular for the combination treatment to exhibit synergistic effects, although this is certainly possible and advantageous. In terms of surgery, any surgical intervention can be practiced in combination with the present invention. In connection with radiotherapy, any mechanism for inducing DNA damage locally within the tumor cells is contemplated, such as gamma irradiation, X-rays, ultraviolet irradiation, microwaves and even electronic emissions and the like. Targeted administration of radioisotopes to tumor cells is also contemplated, and this can be used in relation to a targeted antibody or other objective elements. Cytokine therapy has also been shown to be an effective partner for combined therapeutic regimens. Several cytokines may be employed in these combined approaches. Examples of cytokines include IL-IL-lβ, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-iS, GM-CSF, M-CSF, G-CSF, TNFa, TNF / 3, LAF, TCGF, BCGF, TRF, BAF, BDG , MP, LIF, OSM, TMF, PDGF, IFN-Qi, IFN-β, IFN-7. Cytokines are administered according to standard regimens, consistent with clinical indications such as the condition of the patient and the relative toxicity of the cytokine. The. Chemotherapeutic Combinations and Treatment In certain embodiments, the present invention shows that the antitumor activity of truncated Tissue Factor increases when it is administered in combination with a chemotherapeutic substance. The mechanisms by which drugs increase the antitumor activity of truncated Tissue Factor have not been precisely defined, but the inventors believe that the drug kills proliferating tumor cells creating necrotic areas that cause phagocytic cells to infiltrate the tumor. IL-1, TNFa and other cytokines released by the infiltrating cells then activate the vascular endothelium of the tumor making it more possible to withstand coagulation by the truncated Tissue Factor, a generally weak thrombogen. The drug thus increases the thrombotic action of the truncated tissue factor. Another possibility for the increased actions of the tissue factor and anticancer drugs is that the truncated tissue factor induces the formation of thrombi in the tumor vessels, thereby trapping the drug within the tumor. Although the drug is removed from the rest of the body, it stays inside the tumor. The tumor cells are then exposed to a higher concentration of the drug for a longer period of time. This entrapment of the drug within the tumor can also make it possible to reduce the dose of the drug, making the treatment safer as well as more effective. Regardless of the mechanisms by which increased tumor destruction is achieved, aspects of the combined treatment of the present invention have obvious utility in the effective treatment of the disease. In order to use the present invention in combination with the administration of a chemotherapeutic substance, a deficient coagulation tissue factor construct would simply be administered to an animal in combination with the chemotherapeutic substance in an effective manner to result in its combined antitumor actions within the animal These substances would therefore be provided in an effective amount and for an effective period of time to result in their combined presence within the tumor vasculature and their combined actions in the tumor environment. To achieve this goal, the Tissue Factor and the chemotherapeutic substances can be administered to the animal simultaneously, either in a single composition or as two different compositions using different routes of administration. Alternatively, the Tissue Factor treatment may precede or follow the treatment of the chemotherapeutic substance in intervals ranging from minutes to weeks. In embodiments where the chemotherapeutic factor and the Tissue Factor are applied separately to the animal, it would generally be ensured that a significant period of time did not expire between the time of each administration, so that the chemotherapeutic substance and the composition of Tissue Factor they would still be able to exert a combined effect advantageously over the tumor. In these cases, it is contemplated that the tumor would be contacted with both substances within about 5 minutes to about a week of each and, more preferably, within 12-72 hours of the other, with a delay of only about 12-48 hours. In some situations, it may be desirable to extend the time period for treatment significantly, where several days (2, 3, 4, 5, 6 or 7) or up to several weeks (1, 2, 3, 4, 5, 6, 7 or 8) pass between the respective administrations. It is also conceivable that more than one administration of, either the tissue factor or the chemotherapeutic substance. To achieve tumor regression, both substances are administered in an effective combined amount to inhibit their growth irrespective of the times for administration. A variety of chemotherapeutic substances are intended for use in the combined treatment methods described herein. Chemotherapeutic substances contemplated as examples include, e.g., etoposide (VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and even hydrogen peroxide. In certain embodiments, the use of etoposide has already been shown to be effective in tumor size regression when administered in combination with the truncated tissue factor compositions of the present invention. As will be understood by those skilled in the art, an adequate dose of chemotherapeutic substances will generally be around those already employed in clinical therapies wherein the chemotherapeutic substances are administered alone or in combination with other chemotherapeutic substances. By way of example only, substances such as cisplatin, and other DNA alkylating substances can be used. Cisplatin has been widely used to treat cancer, with effective doses used in clinical applications of 20 mg / m2 for 5 days every three weeks for a total of three courses. Cisplatin is not orally absorbed and therefore must be applied via injection intravenously, subcutaneously, intratumorally, or intraperitoneally. Other useful substances include compounds that interfere with DNA replication, mitosis, and chromosomal segregation. These chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, verapamil, podophyllotoxin, and the like. Widely used in a clinical setting for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg / m2 at 21-day intervals for adriamycin, up to 35-50 mg / m2 for etoposide intravenously or twice the intravenous dose orally. Substances that interrupt the synthesis and fidelity of polynucleotide precursors can also be used. Particularly useful are substances that have undergone extensive testing and are readily available. As such, substances such as 5-fluorouracil (5-FU) are preferentially used for the neoplastic tissue, making this substance particularly useful for targeting neoplastic cells. Although quite toxic, 5-fluorouracil is applicable in a wide range of carriers, including topical, however intravenous administration with doses ranging from 3 to 15 mg / kg / day is commonly used. Exemplary chemotherapeutic substances that are useful in connection with the combination therapy are listed in Table II. Each of the substances listed in it are exemplary and in no way limiting. The skilled artisan is referred to "Remington's Pharmaceutical Sciences" eleventh edition, chapter 33, in particular pages 624-652. Some dose variation will necessarily arise depending on the condition of the subject being treated. The person responsible for the administration, in any case, will determine the appropriate dose for the individual subject. Moreover, for human administration, the preparations must meet the standards of sterility, pyrogenicity, general safety and purity required by the Biological Standards Office of the Food and Drug Administration of the United States of America. TABLE II USEFUL CHEMOTHERAPEUTIC SUBSTANCES IN NEOPLASTIC DISEASE Substances Alkylating Sulfonates Busulfan Granulocytic Leukemia (continued) Chronic alkyl Carmustine (BCNU) Hodgkin's disease, non-Hodgkin lymphomas, primary brain tumors, multiple myeloma, malignant melanoma Nitrosoureas Lomustine (CCNU) Hodgkin's disease, non-Hodgkin lymphomas, primary brain tumors, small cell lung Semustine Tumors of the brain (methyl-CCNU) primary, stomach, colon Streptozocin Pancreatic insulin (streptozotoci malignant, carcinoid Triazines na) malignant Dacarbazine (DTIC; malignant melanoma, dimethyltriazene-Hodgkin's disease, i m i d a z o l e - carboxamide tissue sarcomas) soft Methotrexate analogues Lymphocytic leukemia Anti-imeta Folic acid (ametopterin) acute, choriocarcinoma, mycosis fungoides pellets, breast, head and neck, lung, osteogenic sarcoma Fluoracil analogs (5- Sine, colon, stomach, fluorouracil pyrimidine, 5- pancreas, ovary, FU) Floxuridine head and neck, bladder (f 1 uorodeoxy-urinary, uridine lesions; FUdR) premalignant skin (topical) Citarabine Granulocytic leukemia (acute cytosine and lymphocytic arabinoside) acute Mercaptopurine (6- Mercaptopurine lymphocytic leukemia, acute, granulocytic 6-MP) acute and chronic granulocytic Thioguanine analogues Lymphocytic leukemias P u r i n a e (6 - ioguanine, - TG) acute, granulocytic Acute and granulocytic inhibitors Related chronic Pentostatin Cell leukemia (2-deoxicoforfoluda, mycosis mycin) fungoides, chronic lymphocytic leukemia Vinblastine (VLB) Hodgkin's disease, non-Hodgkin's lymphomas, breast, testicles Alkaloids Vincristine Acute vinca lymphocytic leukemia, neuroblastoma, Wilms tumor, rhabdomyosarcoma, Hodgkin's disease, non-Hodgkin lymphomas, small cell lung Epipodofilo- Etoposida Testis, lung of Products toxins Tertiposide small cell and other natural lungs, breast, Hodgkin's disease, non-Hodgkin lymphomas, acute granulocytic leukemia, Kaposi's sarcoma Dactinomycin Choriocarcinoma, Wilms' tumor (actinomycin D), rhabdomyosarcoma, testicles, Kaposi's sarcoma Daunorru icina Acute granulocytic (daunomycin, acute and lymphocytic rubidomycin) leukemia E2. Immunotoxin and Coaguligand Combinations and Therapy One or more deficient coagulation tissue factor constructs of the invention may be used in combination with immunotoxins (ITs) and / or coaguligands in which the target portion thereof (e.g. antibody or ligand) is directed to a relatively specific marker of the tumor cells, the tumor vasculature or the tumor stroma. In common with the chemotherapeutic substances discussed above, it is possible that the use of a deficient coagulation tissue factor construct in combination with a toxic substance targeted (IT) immunotoxin or coagulant (coaguligating) will result in different substances being directed against different objectives within the tumor environment. This should lead to additive results, markedly greater than the additives or even synergists. In connection with the preparation and use of exemplary immunotoxins and coaguligands, the following descriptions of patent applications are specifically incorporated herein by reference for purposes as well as to supplement the teachings herein: US Patent applications with numbers of series 07 / 846,349; 08 / 295,868; 08 / 205,330; 08 / 350,212; 08 / 273,567; and 08 / 482,369. At least one linking region of the second substances employed in combination with the truncated tissue factor constructs of the present invention will be a component that is capable of delivering a toxin or coagulant substance to the tumor region, i.e., capable of be located within a tumor site. Since a somewhat wider distribution of the coagulating substance will not be associated with severe side effects, there is a less stringent requirement imposed on the target element of the coaguligands than with the immunotoxins. The target substance can be targeted to components of the tumor cells; components of the tumor vasculature; components that bind to, or are usually associated with, tumor cells; components that bind to, or are generally associated with, the tumor vasculature; components of the extracellular matrix of the tumor or stroma or those attached thereto; and even types of cells found within the tumor vasculature. With coaguligands, the burden of target restrictions, eg, that are imposed when immunotoxins are used, decreases. Therefore, achieving specific objectives means that coagulation is promoted in the vasculature of the tumor in relation to the vasculature in non-tumor sites. Thus, the specific objective of a coaguligand is a functional term instead of a purely physical term in relation to the properties of the biodistribution of the target substance, and it is not unlikely that useful objectives can not be completely restricted to the tumor, and that the objective ligands that are effective in promoting specific tumor coagulation, however, may be found in other sites of the body after administration. i. Targets of the tumor cells The malignant cells that make up the tumor can be attacked using a ligand or a bispecific ligand having a region capable of binding to a relatively specific marker of the tumor cell. The toxins kill the tumor cells and, in that bond with the tumor cells, a coagulant substance associated with the tumor will be localized, and specific coagulation will be achieved. Many so-called "tumor antigens" have been described, any of which can be used as an objective in connection with the combined aspects of the present invention. A large number of antigens associated with exemplary solid tumors are listed below. The preparation and use of antibodies against these antigens is very well within the skill of the art, and exemplary antibodies include gynecological tumor sites: OC 125; OC 133; IMO; Mo saw; Mo v2; 3C2; 4C7; ID3; DU-PAN-2; F 36/22; 4F7 / 7A10; OV-TL3; B72.3; DF3; 2C8 / 2F7; MF 116; MOV18; CEA 11-H5; CA 19-9 (1116NS 19-9); H17-E2; 791T / 36; NDOG2; H317, 4D5, 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; breast tumor sites: DF3; NCRC-11; 3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2; 3.15.C3; M3, M8, M24; M18; 67-D-ll; D547Sp, D75P3, H222; Anti-EGF; LR-3; TAI; H59; 10-3D-2; HmABl, 2; MBR 1,2,3; 24-17-1; 24-17-2 (3E1.2); F36 / 22.M7 / 105; CU, G3, H7; B6-2; Bl-1; Cam 17-1; SM3; SM4; C-Mul (566); 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2; DU-PAN-2; 4F7 / 7A10; DF3; B72-3; cccccCEA 11; H17-E2; 3-14-A3; F023C5; of colorectal tumor sites B72-3; (17-1A) 1083-17-1A; C017-1A; ZCE-025; AB2; HT-29-15; 250-30.6; 44X14; A7; GA73-3; 791T / 36; 28A32; 28.19.8; X MMC0-791; DU-PAN-2; ID3; CEA 11-H5; 2C8 / 2F7; CA-19-9 (1116NS 19-9); PR5C5; PR4D2; PR4D1; of melanoma sites 4-1; 8.2M17, 96-5; 118-1, 132.2, (113-2); L,, L10, R10 (R19); I12; K5; 6-1; R24; 5-1; 225.28S; 465.12S; 9-2-27; FU; 376.96S; 465.12S; 15-75; 15-95; Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M148; of gastrointestinal tumors: ID3; DU-PAN-2; OV-TL3; B72-3; CEA 11-H5; 3-14-A3; CCOLI; CA-19-9 (1116NS 19-9) and CA50; OC125; of lung tumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2HÜ, 3E8, 5B8, 7D3, SB8; MO v2; B72-3; DU-PAN-2; CEA 11-H5; MUC 8-22; MUC 2-63; MUC 2-39; MUC 7-39; and of miscellaneous tumors: PAb 240; PAb 246; PAb 1801; ERIC-1; M148; FMH25; 6-1; CAÍ; 3F8; 4F7 / 7A10; 2C8 / 2F7; CEA 11-H5. Another means of defining a targeting tumor is in terms of the characteristics of the tumor cell itself, rather than describing the biochemical properties of an antigen expressed by the cell. In accordance with the foregoing, the skilled artisan refers to the ATCC catalog for the purpose of exemplifying human tumor cell lines that are available to the public (from the ATCC catalog). Exemplary cell lines include J82; RT4; Scaber; T24; TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088; SW 1783; U-87 MG; U-118 MG; U- 138 MG; U-373 MG; Y79; BT-20; BT-474; MCF7; MDA-MB-134-VI; MDA- MD-157; MDA-MB-175-VII; MDA-MB-361; SK-BR-3; C-33 A; HT-3; ME- 180; MS751; SiHa; JEG-3; Caco-2; HT-29; SK-CO-1; HuTu 80; A- 253; FaDu; A-498; A-704; Caki-1; Caki-2; SK-NEP-1; SW 839; SK- HEP-1; A-427; Calu-1; Calu-3; Calu-6; SK-LU-1; SK-MES-1; SW 900; EB1; EB2; P3HR-1; HT-144; Malme-3M; RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3; SK-MEL-5; SK-MEL-24; SK-MEL-28; SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW 626; Capan-1; Capan-2; DU 145; A- 204; Saos-2; SK-ES-1; SK-LMS-1; SW 684; SW872; SW982; SW1353; U-2 OS; Malme-3; KATO III; Cate-IB; Tera-1; Tera-2; SW579; AN3 CA; HEC-1-A; HEC-1-B; SK-UT-1; SK-UT-1B; SW 954; SW 962; NCI-- H69; NCI-H128; BT-483; BT-549; DU4475; HBL-100; Hs 578Bst; Hs 578T; MDA-MB-330; MDA-MB-415; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; T-47D; Hs766T; Hs 746T; Hs 695T; Hs 683; Hs 294T; Hs 602; JAR; Hs 445; Hs 700T; H4; Hs 696; Hs913T; Hs 729; FHs 738Lu; FHs 173We; FHs 738 Bl; NIH: 0VCAR-3; Hs 67; RD-ES; ChaGo K-l; WERI-Rb-1; NCI-H446; NCI-H209; NCI-H146; NCI-H441; NCI-H82; H9; NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820; NCI-H520; NCI-H661; NCI-H510A; D283 Med; Daoy; D341 Med; AML- 193 and MV4-11. The ATCC catalog of any subsequent year can be consulted to identify other suitable cell lines.

Also, if a particular cell type is desired, the means for obtaining these cells, and / or their instantaneous available source, will be well known to those skilled in the particular art. An analysis of the scientific literature will easily reveal an appropriate choice of cell for any type of tumor cell desired to be attacked. (a) Anti-tumor cell antibodies A direct element for recognizing a tumor antigen target is through the use of an antibody that has a binding affinity for the particular antigen. A large number of antibodies are known to be directed against solid tumor antigens. Certain useful antitumor antibodies are listed above. However, as those skilled in the art will readily realize, certain antibodies listed will not have adequate biochemical properties, or may not have sufficient tumor specificity, to be therapeutically useful. An example is MUC8-22 which recognizes a cytoplasmic antigen. Antibodies such as these will generally be useful only in research environments, such as in model systems or assay assays. Generally speaking, antibodies useful in these aspects of the present invention will preferably recognize antigens that are accessible on the cell surface and that are preferentially, or specifically, expressed by tumor cells. These antibodies will preferably also exhibit high affinity properties, such as exhibiting a K¿ of <preferably, of < 100 nM, and will not show significant reactivity with vital normal tissues, such as one or more tissues selected from the heart, kidney, brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenal, muscle, fibers nerves, pancreas, skin, or other vital organs or tissues in the human body. The "vital" tissues that are most important for the purposes of the present invention, from the point of view of low reactivity, include heart, kidney, tissues of the central and peripheral nervous system and liver. The term "significant reactivity", as used herein, refers to an antibody or antibody fragment, which, when applied to the particular tissue under conditions suitable for immunohistochemistry, will obtain either no staining or a negligible staining with only a few positive cells scattered among a field of most negative cells. Particularly promising antibodies contemplated for use in the present invention are those that have high selectivity for the solid tumor. For example, antibodies that bind to TAG_72_and proto-oncogene protein HER-2, which are selectively found on the surfaces of many breast, lung and colorectal cancers (Thor et al., 1986; Colcher et al., 1987).; - Shepard et al., 1991), - M0vl8 and 0V-TL3 and antibodies that bind to the mucin nucleus protein of milk and to the milk fat globule (Miotti et al., 1985, Burchell et al., 1983); and the 9.2.27 antibody that binds to the high Mr melanoma antigens (Reisfeld et al., 1982). Other useful antibodies are those against the folate binding protein, which is known to be homogenously expressed in almost all ovarian carcinomas; those against the erJ family of oncogenes that are overexpressed in squamous cell carcinomas and most gliomas, - and other antibodies known to be subject to preclinical and continuous clinical evaluation. Antibodies B3, KSI / 4, CC49, 260F9, XMMCO-791, D612 and SM3 are believed to be particularly suitable for use in clinical modalities, following the standard preclinical testing routine practiced in the art. B3 (U.S. Patent No. 5,242,813; Brinkmann et al., 1991) has accession number ATCC HB 10573; KS1 / 4 can be made as described in U.S. Patent 4,975,369; and D612 (U.S. Patent No. 5,183,756) has the accession number of ATCC, HB 9796. Other means to define the objectives associated with the tumor are in terms of the characteristics of the tumor cell, rather than describing the biochemical properties of an antigen expressed by the cell. In accordance with the foregoing, the inventors contemplate that any antibody that preferentially binds to a tumor cell can be used as the target component of an immunotoxin or coaguligand. The preferential tumor cell linkage is again based on the antibody that exhibits high affinity for the tumor cell and that has no significant reactivity with the vital normal cells or tissues, as defined above. The invention also provides various means for generating an antibody for use in the methods of targeted coagulation described herein. To generate a tumor cell-specific antibody, an animal with a composition comprising a tumor cell antigen would be immunized and, as described more fully below, a resulting antibody with appropriate specificity is selected. The immunizing composition may contain a purified, or partially purified, preparation of any of the antigens listed above; a composition, such as a membrane preparation, enriched for any of the antigens listed above; any of the cells listed in the previous list; or a mixture or population of cells that include any of the cell types listed above. Of course, regardless of the source of the antibody, in the practice of the invention in human treatment, it is preferred to ensure in advance that the target tumor clinically expresses the finally selected antigen. This is achieved by a fairly direct assay, which involves antigenically testing a sample of tumor tissue, for example, a surgical biopsy, or perhaps testing the occult circulation of the antigen. This can easily be carried out in an immunological assay assay such as a binding enzyme immunosorbent assay (ELISA), wherein the binding affinity of antibodies from a "bank" of hybridomas is tested to determine their reactivity against the tumor. Antibodies demonstrating adequate tumor selectivity and affinity are selected for the preparation of the bispecific antibodies of the present invention. Due to the well-known phenomenon of cross-reactivity, it is contemplated that useful antibodies may be the result of immunization protocols in which antigens originally used were derived from an animal, such as a mouse or a primate, in addition to those in which obtained original antigens from a human cell. When antigens of human origin are used, can be obtained from a human tumor cell line, or can be prepared by obtaining a biological sample from a particular patient in question. Undoubtedly, methods are known for the development of antibodies that are "tailored to the client" of the patient's tumor (Stevenson et al., 1990) and contemplated for use in connection with this invention. (b) Other targets of tumor cell and binding ligands. In addition to the use of antibodies, other ligands may be used to direct a coagulant substance to a tumor site by binding to a tumor cell antigen. For tumor antigens that have overexpressed receptors (estrogen receptor, EGF receptor), or mutant receptors, the corresponding ligands could be used as target substances. In a manner analogous to the endothelial cell receptor ligands, there may be many components that bind specifically, or preferentially, to the tumor cells. For example, if a tumor antigen is an overexpressed receptor, the tumor cell can be coated with a specific ligand in vivo. It appears that the ligand could then be directed either with an antibody against the ligand, or with a form of the same receptor. Specific examples of this type of target substances are antibodies against TIE-1 or TIE-2 ligands, antibodies against platelet factor 4, and leukocyte adhesion binding protein. ii. Other target diseases In other embodiments, tissue factors may be employed in combination with immunotoxins or coaguligands that bind to a target molecule that is specifically or preferentially expressed at a disease site other than a tumor. Exemplary target molecules associated with other diseased cells include, for example, leukocyte adhesion molecules, which are associated with psoriasis; FGF, which is associated with proliferative diabetic retinopathy; platelet factor 4, which is associated with the activated endothelium of various diseases; and VEGF, which is associated with vascular proliferative disease. It is believed that an animal or patient having any of the above diseases would benefit from the specific induction of coagulation at the site of the disease and optionally from the administration of targeted toxin. Diseases known to have a common angiodependent pathology, as described in Klagsburn and Folkman (1990), can also be treated as described herein. In particular, an endothelial cell directed ligand or a stromal targeting ligand will be used to achieve coagulation at the disease site. The treatment of BPH, diabetic retinopathy, vascular restenosis, vascular adhesions, AVM, meningioma, hemangioma, neovascular glaucoma, rheumatoid arthritis and psoriasis are particularly contemplated at this time. iii. Objectives of vasculature cells associated with the disease The cells of the vasculature are intended as targets for use in the present invention. In these cases, at least one binding region of the immunotoxin or coaguligand will be able to bind to a preferentially accessible marker expressed by the endothelial cells of the vasculature associated with the disease. The exploitation of vascular markers is possible due to the proximity of vascular endothelial cells to the area of the disease and to the products of local aberrant physiological processes. For example, tumor vascular endothelial cells are exposed to tumor cells and tumor-derived products that change the phenotypic profile of endothelial cells. It is known that tumor cells make tumor-derived products, such as lymphokines, monocins, colony-stimulating factors, growth factors and angiogenic factors, which act on nearby vascular endothelial cells (Kandel et al., 1991; Folkman et al. 1985a, 1985b) and cytokines (Burrows et al., 1991; Ruco et al., 1990; Borden et al., 1990). The tumor products bind to the endothelial cells and serve to selectively induce the expression of certain molecules. In these induced molecules that can be attacked using the specific tumor endothelial toxin and / or the coagulant administration provided by certain aspects of the present invention. Vascular endothelial cells in tumors proliferate at a rate 30 times greater than those in miscellaneous normal tissues (Denekamp et al., 1982), suggesting that the determinants linked to proliferation would also serve as markers for vascular endothelial tumor cells. In certain embodiments of the invention, the target component of the immunotoxins or coaguligands will be a component that has a relatively high degree of specificity for the tumor vasculature. These objective components can be defined as components that bind to molecules expressed in the tumor endothelium, but that have little or no expression on the surface of normal endothelial cells. This specificity can be assessed by standard methods of immunostaining tissue sections, which are routine for those skilled in the art. In terms of coaguligands, it is generally proposed that the molecules to be attacked using the bispecific ligands or antibodies of this invention will be those that are expressed in the tumor vasculature at a higher level than on normal endothelial cells. (a) Markers of Vascular Endothelial Cells in Disease Molecules that are known to be preferentially expressed on the surface of vascular endothelial cells in a site or disease environment are herein called "vascular endothelial cell markers associated with the natural disease." This term is used for simplicity to refer to the components of endothelial cells that are expressed in diseases connected with inadequate augmentation or angiogenesis or endothelial cell proliferation. A particular example is the components of tumor endothelial cells that are expressed in situ in response to tumor-derived factors. These components are also called "naturally induced tumor endothelial cell markers." Both the vascular endothelial growth factor / vascular permeability factor and the components of the fibroblast growth factor family are concentrated in the tumor vasculature. The corresponding receptors therefore provide a potential target for attack on the tumor vasculature. For example, it is known that vascular endothelial growth factor receptors are upregulated in tumor endothelial cells, contrary to endothelial cells in normal tissues., both in rodents and in humans (Thieme et al., 1995). Possibly, this is a consequence of hypoxia - a characteristic of the tumor microenvironment (Leith et al., 1992). Fibroblast growth factor receptors are also upregulated 3-fold in endothelial cells exposed to hypoxia, and are thought to upregulate in tumors (Bicknell and Harris et al., 1992). The transforming growth factor receptor β (endoglin) on the endothelial cells is upregulated in the dividing cells, providing another target. One of the present inventors found that the endoglin is up-regulated in activated HUVEC and split in culture, and is strongly expressed in human tissues on endothelial cells at sites of neovascularization, including a wide range of solid tumors and fetal placenta. In contrast, endothelial cells in most non-malignant adult tissues, including preneoplastic lesions, contain little or no endoglin. Importantly, it is believed that endoglin expression correlates with neoplastic progression in the breast, as shown by benign fibroadenomas and early carcinomas that bind to low levels of TEC-4 and TEC-11 antibodies, and intraductal carcinomas of late stage and invasive carcinomas that bind to high levels of these antibodies. Other markers of vascular endothelial cells associated with natural diseases include TIE, VCAM-1, P-selectin, E-selectin (ELAM-1), oiyß ^ integrin, pleitropin and endocyanin, each of which can be attacked using the invention. (b) Cytokine-Induced Vascular Endothelial Markers Due to the nature of disease processes, which frequently result in localized dysfunction within the body, there are methods available to manipulate the site of the disease while leaving other tissues relatively unaffected. . This is particularly true in malignant and benign tumors, which exist as distinct entities within the body of an animal. For example, the tumor environment can be manipulated to create additional markers that are specific for the vascular endothelial cells of the tumor. These methods generally mimic those that occur naturally in solid tumors, and also involve the local production of signaling substances, such as growth factors or cytokines, that induce the specific expression of certain molecules on the surface of nearby vascular endothelial cells. . The group of molecules that can be artificially induced to be expressed on the surface of vascular endothelial cells in a disease or tumor environment are referred to herein as "inducible endothelial cell markers," or specifically, "tumor endothelial cell markers. -inducibles ". This term is used - to refer to markers that are artificially induced, that is, induced as a result of manipulation by the hand of man, rather than those that are induced as part of a disease or process of tumor development in an animal. The term "inducible marker", as defined above, is chosen by simple reference in the context of the present application, independently of the fact that "natural markers" are also induced, eg, by substances derived from the tumor. Thus, although not required to practice the invention, there are techniques available for the selective targeting of vascular endothelial antigens on the surface of the vasculature associated with disease which, if desired, can be used in conjunction with the invention. These techniques involve manipulation of antigenic expression, or presentation on the cell surface, so that an objective antigen is expressed or becomes available on the surface of the vasculature associated with the disease and is not expressed or otherwise becomes accessible or available for link, or at least to a lesser degree, on the surface of the normal endothelium. Tumor endothelial markers can be induced by tumor-derived cytokines (Burrows et al., 1991; Ruco et al., 1990) and angiogenic factors (Mignatti et al., 1991).

Examples of cell surface markers that can be specifically induced in the tumor endothelium and then attacked using a bispecific coagulant ligand, as provided by the invention, include those listed in Table III (Bevilacqua et al., 1987; Dustin et al., 1986; Osborn et al., 1989; Collins et al., 1984). Table III also presents the mechanisms for the induction of the proposed markers, - the "inducente cytokine" or "intermediate cytokine", such as IL-1 and IFN-α; and the type of leukocyte cell and the associated cytokine activation molecule, the objective of which will result in the release of the cytokine. In the induction of a specific marker, an antibody "that induces cytokines" or "that induces antigen" is generally required. The antibody will selectively induce the release of the appropriate cytokine at the tumor site, thereby selectively inducing the expression of the desired target antigen by vascular endothelial cells. This bispecific antibody binds the cells of the tumor mass and the leukocytes that produce cytokine, thereby activating the leukocytes to release the cytokine. The preparation and use of bispecific antibodies such as these is based in part on the fact that the cross-linking of antibodies recognizing CD3, CD14, CD16 and CD28 has previously been shown to obtain cytokine production selectively after cross-linking with the second antigen. (Qian and collaborators, 1991). In the context of the present invention, since only leukocytes cross-linked with tumor cells will successfully be activated to release the cytokine, the release of cytokine will be restricted to the tumor site. Thus, expression of the desired marker, such as E-selectin, will be similarly limited to the endothelium of the tumor vasculature.

TABLE III POSSIBLE INDUCIBLE VASCULAR OBJECTIVES It is important to note that, of the possible inducible markers listed in Table III, E-selectin, and MHC Class II antigens, such as HLA-DR, HLA-DP and HLA-DQ (Collins et al., 1984), are by far the most preferred objectives for their use in connection with clinical modalities. The other adhesion molecules of Table III appear to be expressed in varying degrees in normal tissues, generally lymphoid organs or endothelium, making their target perhaps only suitable in animal models or in cases where their expression on normal tissues can be inhibited without significant side effects. It is preferred to direct E-selectin or a MHC Class II antigen since the expression of these antigens will probably be the most direct to selectively promote tumor-associated endothelium. E-selectin The target of an antigen that is not expressed on normal endothelial surfaces is the most direct form of induction methods. E-selectin is an adhesion molecule that is not expressed in normal endothelial vasculature or in other types of human cells. (Cotran et al., 1986), but it can be induced on the surface of endothelial cells through the action of cytokines such as IL-1, TNF, lymphotoxin and bacterial endotoxin (Bevilacqua et al., 1987). It is not induced by IFN-7 (Wu et al., 1990). The expression of E-selectin can thus be selectively induced in tumor endothelium through the selective administration of such a cytokine, or via the use of a composition that causes the selective release of these cytokines in the tumor environment. Bispecific antibodies are an example of a composition capable of causing the selective release of one or more of the above or other suitable cytokines at the site of the tumor, but no other part in the body. These bispecific antibodies are referred to herein as "antigen-inducing antibodies" and are, of course, distinct from any bispecific antibodies of the invention having white components and coagulants. Antigen-inducing antibodies are designed to cross-link cytokine effector cells, such as monocyte / macrophage lineage cells, T cells and / or NK cells or mast cells, with tumor cells of the target solid tumor mass. This cross-linking would effect a release of cytokine that is localized at the site of cross-linking, i.e., the tumor. The antibodies effective to induce antigen recognize a tumor cell surface antigen on the one hand, and on the other hand, recognize a "cytokine activating" antigen on the surface of a selected leukocyte cell type. The term "cytokine-activating" antigen is used to refer to any of the known molecules on the surfaces of leukocytes that, when linked by an effector molecule, such as an antibody or fragment thereof or a substance that is presented naturally or a synthetic analogue thereof, whether a soluble factor or a counter receptor bound to the membrane or another cell, promotes the release of a cytokine by the leukocyte cell. Examples of molecules that activate the cytokine include CD14 (the LPS receptor) and FcR for IgE, which will activate the release of IL-1 and TNFa; and CD16, CD2 or CD3 or CD28, which activate the release of IFN? and TNFβ, respectively. As soon as it is introduced into the bloodstream of an animal that has a tumor, this bispecific antibody that induces antigen will bind to the tumor cells within the tumor, will crosslink those tumor cells with effector cells, e.g., monocytes / macrophages , that have infiltrated the tumor, and after that they will effect the selective release of cytokine inside the tumor. However, importantly without the cross-linking of the tumor and the leukocyte, the antibody that induces the antigen will not effect the release of the cytokine. Thus, no release of cytokine will occur in parts of the body removed from the tumor and, therefore, expression of cytokine-induced molecules, e.g., E-selectin, will occur only within the tumor endothelium. Many useful "cytokine activating" antigens are known, which, when cross-linked with suitable bispecific antibody, will result in the release of cytokines by the cross-linked leukocyte. The generally preferred objective for this purpose is CD14, which is found on the surface of monocytes and macrophages. When CD14 is reticulated it stimulates monocytes / macrophages to release IL-1 (Schutt et al., 1988; Chen et al., 1990), and possibly other cytokines, which, in turn, stimulate the appearance of E-selectin on the near vasculature. Other possible targets for crosslinking in connection with induction of E-selectin and targeting include FcR for IgE, found in mast cells; FcR for IgG (CD16), found in NK cells; as well as CD2, CD3 or CD28, found on the surfaces of T cells. Of these, it is preferred to target CD14 due to the relative prevalence of monocyte / macrophage infiltration of solid tumors as opposed to the other types of leukocyte cells. In an exemplary induction modality, an animal having a solid tumor is injected with bispecific antibody (Fab'-Fab ') anti-CD14 / antitumor (such as the anti-CEA antibody, 9.2.27 against Mr high melanoma antigens, OV-TL3 or MOvld antibodies against antigens associated with the ovaries). The antibody is localized in the tumor, by virtue of its activity of binding to the tumor, and then activates monocytes and macrophages in the tumor by cross-linking its CD14 antigens (Schutt et al., 1988; Chen et al., 1990). Monocytes / macrophages have tumoricidal activity (Palleroni et al., 1991) and release IL-1 and TNF that rapidly induce E-selectin antigens on tumor vascular endothelial cells (Bevilacqua et al., 1987; Pober et al., 1991). MHC class II antiane The second preferred group of inducible markers contemplated for use in the present invention are the MHC Class II antigens (Collins et al., 1984), which include HLA-DR, HLA-DP and HLA-DQ. Class II antigens are expressed on vascular endothelial cells in most normal tissues in several species, including man. In vitro studies (Collins et al., 1984; Daar et al., 1984; O'Connell et al., 1990) and in vivo (Groenewegen et al., 1985) have shown that the expression of Class II antigens by vascular endothelial cells requires the continuous presence of IFN-? which is elaborated by TH1 cells and, to a lesser extent, by NK cells and CD8 + T cells. Class II MHC antigens are not unique to vascular endothelial cells, and are also constitutively expressed in B cells, activated T cells, monocyte / macrophage lineage cells and in certain epithelial cells, both in mice (Ham erling, .1976) as in man (Darr et al., 1984). Due to the expression of MHC antigens Class II on "normal" endothelium, its objective is not as direct as E-selectin. However, the induction and direction of MHC Class II antigens are made possible by using them together with an immunosuppressant, such as cyclosporin A (CsA), which has the ability to effectively inhibit the expression of Class II molecules in normal tissues (Groenewegen et al., 1985). Cyclosporin A acts by preventing the activation of T cells and NK cells (Groenewegen et al., 1985; DeFranco, 1991), thereby reducing the basal levels of IFN-α. below those necessary to maintain Class II expression in the endothelium. There are several other cyclosporin A related cyclosporins, including cyclosporins A, B, C, D, G, and the like, which also have immunosuppressive action and probably demonstrate an ability to suppress Class II expression. Other substances that may be similarly useful include FK506 and rapamycin. Thus, the practice of MHC Class II induction and attack mode requires pretreatment of the tumor-bearing animal with a dose of cyclosporin or another Class II immunosuppressant substance that is effective in suppressing Class II expression. . In case of cyclosporin A, this typically is in the range of about 10 to about 30 mg / kg of body weight. As soon as it is deleted in normal tissues, Class II antigens can also be selectively induced in the tumor endothelium, again through the use of a bispecific antibody. In this case, the bispecific antibody that induces the antigen will have specificity for a marker of tumor cells and for an activation antigen found on the surface of an effector cell that is capable of inducing the production of IFN-α. These effector cells will usually be helper T cells (TH) or natural killer cells (NK) In these modalities, it is necessary that T cells, or NK cells if CD16 is used, are present in the tumor to produce the intermediate cytokine in which the expression of Class II antigen is achieved using IFN-α, but is not achieved with other cytokines. Thus, for the practice of this aspect of the invention, it will be desired to select CD2, CD3, CD28, or more preferably CD28, as the cytokine activating antigen for targeting the bispecific antibody that induces antigen. The T cells that should be activated in the tumor are those adjacent to the vasculature since this is the most accessible region to the cells and it is also where the bispecific antibody will be most concentrated. Activated T cells should secrete IFN-? which induces Class II antigens on the vasculature of the adjacent tumor.

The use of a bispecific antibody (Fab'-Fab ') having one branch directed against a tumor antigen and the other branch directed against CD28 is currently preferred. This antibody will cross-link CD28 antigen on T cells in the tumor which, when combined with a second signal (provided, for example, by IL-1 which is commonly secreted by tumor cells (Burrows et al., 1-991; et al., 1990), has been shown to activate T cells through an independent pathway of inhibitory CsA CA (Hess et al., 1991; June et al., 1987; Bjorndahl et al., 1989). Cytokine activation molecules are also well known in the art For example, the preparation and use of anti-CD14 and anti-CD28 monoclonal antibodies that have the ability to induce cytokine production by leukocytes has now been described by several laboratories. (reviewed in Schutt et al, 1988, Chen et al 1990, and June et al, 1990, respectively) Furthermore, the preparation of monoclonal antibodies is also known. that stimulate the release of leukocyte from cytokines through other mechanisms and other activating antigens (Clark et al., 1986; Geppert et al., 1990). In still other embodiments, the inventors contemplate an alternative approach to suppress the expression of class II molecules, and selectively obtain expression of Class II molecule at the tumor site. This approach, which avoids the use of both cyclosporin A and a bispecific activation antibody, takes advantage of the fact that the expression of Class II molecules can be effectively inhibited by suppressing the production of IFN-α. by T cells, e.g., through the use of an anti-CD4 antibody (Street et al., 1989). Using this modality, the production of IFN-? it is inhibited by administering anti-CD4, resulting in the general suppression of Class II expression. Class II is then induced only at the site of the tumor, e.g., using tumor-specific T cells that are only activatable within the tumor. In this mode of treatment, a human animal or patient will generally be pretreated with an anti-CD4 dose that is effective to suppress IFN-α production. and by this suppress the expression of Class II molecules. It is contemplated that effective doses will be, for example, in the range of about 4 to about 10 mg / kg of body weight. After Class II expression is suppressed, T cell clones producing IFN-α will be prepared and introduced into the blood stream. (e.g., Thl or cytotoxic T lymphocyte, CTL) specific for an antigen expressed on the surface of tumor cells. These T cells are located in the tumor mass, due to their antigen recognition capacity and, after this recognition, they then release IFN-α. In this way, the release of cytokine to the tumor is again restricted, thus limiting the expression of Class II molecules to the vasculature of the tumor. The T cell clones that IFN-α produces it can be obtained from peripheral blood (Mazzocchi et al. 1990), however, a preferred source is from within the mass of the tumor (Fox et al., 1990). The presently preferred element for preparing this T cell clone is to remove a portion of the tumor mass from a patient; isolate cells, using collagenase digestion, when necessary; enrich the leukocytes that infiltrate the tumor using density gradient centrifugation, followed by deletion of other subsets of leukocytes by, eg, treatment with specific antibodies and complement; and then expand the leukocytes from tumor infiltration in vitro to provide the clone that produces IFN- ?. This clone will necessarily be immunologically compatible with the patient, and therefore will be well tolerated by the patient. It is proposed that particular benefits will be achieved by also selecting a T cell clone that produces IFN-α. high from the expanded leukocytes determining the pattern of cytokine secretion of each individual clone every 14 days. To this end, the remaining clones will be mitogenically or antigenically stimulated for approximately 24 hours and supernatants of their culture will be tested, e.g., using a sandwich-specific enzyme linked immunosorbent assay technique (Cherwinski et al., 1989), to determine the presence of IL-2, IFN- ?, IL-4, IL-5 and IL-10. Clones that secrete high levels of IL-2 and IFN-γ will be selected, the pattern of cytokine secretion characteristic of TH1 clones. The specificity of the tumor will be confirmed using proliferation assays. Further, it will be preferred to use an anti-CD4 Fab as the anti-CD4 antibody, because it will be removed from the body within 24 hours after the injection and thus will not cause suppression of tumor recognition T-cell clones that are subsequently administered. . The preparation of T cell clones having tumor specificity is generally known in the art, as exemplified by the production and characterization of T cell clones from the infiltration of solid melanoma tumors (Maeda et al., 1991). Using any of the MHC Class II induction suppression methods, there will likely be additional benefit from the fact that anti-Class II antibodies injected intravenously do not appear to reach epithelial cells or monocytes / macrophages in normal organs other than the liver and spleen. Presumably this is because the vascular endothelium in most normal organs is narrow, it is not open as it is in the liver and spleen, and thus the antibodies must be diffused through the basement membranes to reach the positive Class II cells. Also, any elimination of B cells that may result, e.g., after cross-linking, is unlikely to pose a significant problem since these cells are refilled from negative Class II progenitors (Lowe et al., 1986 ). Even the elimination of B cells, as occurs in patients with B-lymphoma, does not cause obvious harm (Vitetta et al., 1991). In summary, although the tumor coagulant compositions and antibodies of the present invention are elegantly simple, and do not require the induction of antigens for operability, the combined use of a bispecific antibody that induces antigen with this invention is also contemplated. These antibodies will generally be administered before the bispecific coagulation ligands of this invention. In general, the more "immunogenic" tumors are more convenient for the MHC Class II approach involving, eg, the cross-linking of T cells in the tumor through the bispecific anti-CD28 / antitumor antibody, because these tumors are more likely to be infiltrated by T cells, a prerequisite for this method to be effective. Examples of solid immunogenic tumors include renal carcinomas, melanomas, a minority of cancers of the breast and colon, as well as possibly cancers with pancreatic, gastric, liver, lung and glial tumors. These tumors are known as "immunogenic" because there is evidence that they obtain immune responses in the host and they have been found to be docile to cellular immunotherapy (Yamaue et al., 1990). In the case of melanomas and cancers of the large intestine, the most preferred antibodies for their use in these cases would be B72.3 (anti-TAG-72) and PRSC5 / PR4C2 (anti-Lewis antigen) or 9.2.27 (antigen antigen). melanoma Mr high). For most solid tumors of any origin, an anti-CD14 approach employing a macrophage / monocyte intermediate would be more convenient. This is because most tumors are rich in macrophages. Examples of tumors rich in macrophages include the majority of carcinomas of the breast, colon and lung. Examples of preferred antitumor antibodies for use in these cases would be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA11 (Smith et al., 1989). (c) Inducible Coagulant Markers Coagulants, such as tro Bina, Factor IX / IXa, X / Xa Factor, plasmin and metalloproteinases, such as interstitial collagenases, stromelysins and gelatinases, also act to induce certain markers. In particular, E-selectin, P-selectin, PDGF and ICAM-1 are induced by thrombin (Sugama et al., 1992; Shankar et al., 1994). Therefore for this induction, a bispecific anticoagulant / antitumor antibody will be used. The antibody will be localized in the tumor via its activity of binding to the tumor. The bispecific will then concentrate the coagulant, e.g., thrombin, in the tumor, resulting in the induction of E-selectin and P-selectin in the vascular endothelial cells of the tumor (Sugama et al., 1991; Shankar et al., 1994). ). Alternatively, targeting of truncated tissue factor to tumor cells or endothelium will induce thrombin deposition within the tumor. As thrombin is deposited, E-selectin and P-selectin can be induced on the vascular endothelial cells of the tumor. (d) Vascular Endothelial Cell Marker Antibodies A direct means of recognizing a vasculature target associated with disease, whether induced in the natural environment or by artificial means, is through the use of an antibody that has binding affinity for the particular cell surface receptor, molecule or antigen. These include antibodies directed against all cell surface components that are known to be present on, eg, vascular endothelial tumor cells, which are induced or overexpressed in response to tumor-derived factors, and to which they are induced by the manipulation of the hand of man. Anti-vWF recognizes the antigen VII R Ag and stains 100% of the types of tumors presented and stains 100% of the vessels in the tumor and presents a strong staining pattern in normal vessels. FB5 recognizes the endosialin antigen and stains 50% of the tumor types presented and stains 10 to 30% of the vessels in the tumor and presents a staining pattern in normal vessels in the lymphoid organs. TP3 recognizes the antigen 80 kDa osteosarcoma-related antigen protein and stains 50% of the tumor types presented and stains 10 to 30% of the vessels in the tumor and exhibits a strong staining pattern in normal vessels over the blood vessels little ones. BC-1 recognizes the antigen fibronectin isoforms and stains 60% of the tumor types presented and stains 10 to 30% of the vessels in the tumor and does not present staining pattern in normal vessels. TV-1 recognizes the fibronectin antigen and stains 100% of the tumor types presented and stains 100% of the vessels in the tumor and presents a strong staining pattern in all normal vessels. LM 609 recognizes the vitronectin receptor av ße and stains 85% of the tumor types presented and stains 70 to -80% of the vessels in the tumor and - presents a pattern of medium staining in normal vessels. TEC-11 recognizes endoglin and stains 100% of the tumor types presented and stains 100% of the vessels in the tumor and presents a weak staining pattern in most of the normal vessels. TECHO recognizes VEGF antigens and stains 100% of the tumor types presented and stains 100% of the vessels in the tumor and presents a weak staining pattern in most normal vessels. In a comparative study of anti-EC monoclonal antibodies in human tumors, TEC 110, TV-1 and TECH were found to be positive in gastrointestinal, parotid, breast, ovarian, lung and Hodgkin tumors. While FB-5 had a slight staining in gastrointestinal and lung tumors and was negative in other tumors listed. TP-3 was positive in gastrointestinal tumors and less in types of parotid tumor, ovaries and Hodgkins tumors. BC-1 was positive for gastrointestinal tumors as well as reproductive and respiratory tumors. IM 609 was positive in gastrointestinal, ovarian, uterine, lung and Hodgkin tumors as well as tumors of the reproductive and respiratory system. Two other antibodies that can be used in this invention are those described by Rettig et al., (1992) and Wang et al. (1993) which are directed against unrelated antigens of unknown function expressed in the vasculature of human tumors., but not in most normal tissues. The antibody described by Kimm et al. (1993) can also be used in this invention, particularly since this antibody inhibited angiogenesis and suppressed tumor growth in vivo. Antibodies that have not been shown above that are specific for human tumors can also be used. For example, Venkateswaran et al. (1992) described the production of anti-FGF monoclonal antibodies. Xu et al. (1992) developed and characterized a panel of 16 domain-specific monoclonal and polyclonal antibodies and isoforms against FGF receptor (flg). Massoglia et al. (1987) also reported monoclonal antibodies against the FGF receptor. (e) Generation of antibodies to the Vasculature of the Disease In addition to using a known antibody, such as that described above and others known and published in the scientific literature, a novel antibody can also be generated using standard immunization procedures, as described in more detail later in the present. To generate an antibody against a known vascular marker antigen associated with a disease, an animal with an immunogenic composition comprising the antigen would be immunized.

This may be a membrane preparation that includes, or is enriched for, the antigen; a relatively purified form of the antigen, a cell or membrane isolate; a highly purified form of the antigen, as obtained by a variety of purification steps using, e.g. , an extract of the original antigen or a recombinant form of the antigen obtained from a recombinant host cell. The present invention also further provides other methods for generating an antibody against an antigen present in vascular endothelial cells associated with disease, these methods being convenient for use even when the biochemical identity of the antigen remains unknown. These methods are exemplified through the generation of an antibody against the endothelial cells of the tumor vasculature. A first element to achieve antibody generation in this manner uses a preparation of vascular endothelial cells obtained from the tumor site of an animal or human patient. An experimental animal is simply immunized with a preparation of these cells and the antibodies thus produced are collected. The most useful form of this method is when specific antibodies are subsequently selected, as can be achieved using conventional hybridization technology and screening against tumor vascular endothelial cells. A discovery of the above method is that it mimics the tumor vasculature phenomenon in vi tro, and where purification of the cell is not necessary. Using this method, endothelial cells undergo tumor-derived products, which could be obtained from conditional or tumor media, in cell culture rather than in animal culture. This method generally involves stimulating endothelial cells with tumor conditioned medium and employing stimulated endothelial cells as immunogens to prepare a collection of antibodies. Again, specific antibodies should be selected, e.g., using conventional monoclonal antibody technology, or other techniques such as combinatorial immunoglobulin phagemid libraries prepared from RNA isolated from the spleen of the immunized animal. A specific antibody would be selected that preferentially recognizes the vascular endothelium stimulated from the tumor and reacts more strongly with endothelial cells associated with the tumor than with normal adult human tissues. (£) Anti-anglin antibodies One of the inventors has prepared and isolated antibodies that have relative specificity for tumor vascular endothelium. The monoclonal antibody called tumor-4 endothelial cell antibody and tumor endothelial cell antibody 11 (TEC4 and TECH) were obtained using the same method (U.S. patent application serial number 08 / 457,229 and 08 / 457.031, each incorporated herein by reference). The antigen recognized by TEC4 and TECH was finally determined to be the endoglin molecule. Epitopes on endoglin recognized by TEC4 and TECH are present on the cell surface of stimulated HUVE cells, and only minimally present (or immunologically accessible) on the surface of unstimulated cells. The monoclonal antibodies have been previously cultured against endoglin. However, analyzing the reactivity with HUVEC or surface determinants of HUVEC cell activated by TCM by means of FACS or indirect immunofluorescence shows that the epitopes recognized by TEC-4 and TECH are different from those of a previous antibody called 44G4 (Gougos and Letarte, 1988). ). (g) Use of Vascular Endothelial cell binding ligands. Biological ligands known to bind or interact with endothelial cell surface molecules, such as growth factor receptors, can also be used as the target component. Growth factors or ligands contemplated as useful as targets in this regard include VEGF / VPF, FGF, TGF / 3, ligands that bind to a TAR, fibronectin isoforms associated with tumor, scatter factor, hepatocyte growth factor (HGF) ), platelet factor 4 (PF4), PDGF and TIMP.

Particularly preferred targets are VEGF / VPF, the FGF family of proteins and TGFjS. Abraham and colleagues (1986) cloned FGF, which is therefore available as a recombinant protein. As reported by Ferrara et al. (1991) four VEGF species having 121, 165, 189, and 206 amino acids have been cloned. (h) Target of Linked Ligands Antibodies or specific target ligands can also be targeted to any component that binds to the surface of vascular endothelial cells at a disease site, such as a tumor. These components are exemplified by ligands and tumor-derived antigens, such as growth factors, which bind to specific cell surface receptors already present on endothelial cells, or to receptors that have been induced, or overexpressed, on these cells in response to to the environment of the tumor. The targets associated with the tumor vasculature can also be called endothelial cell-binding factors derived from the tumor. A level of specificity required to successfully target a disease target will be partially achieved because local endothelial cells will be induced to express, or reveal, receptors that are not present, or are under-expressed or masked, in normal endothelial cells. With tumors, greater specificity will result due to the fact that the endothelial cells in the tumor will capture the factors derived from the tumor, and bind them to the cell surface, reducing the amount of ligand available to other tissues. When combined with further dilution of the factor or ligand by distribution in the blood and in tissue fluid culture, endothelial cells in normal tissues will be expected to link relatively few of these factors. Thus, operationally, ligands or binding factors on the cell surface will be able to be used as markers of tumor endothelial cells. In addition to manufacturing by the same tumor cells, the binding factors of tumor endothelial cells can also originate from other cell types, such as macrophages and mast cells, which have infiltrated tumors, or can be made by platelets that are activate inside the tumor. Other growth factors or ligands contemplated to be useful as target substances associated with tumor vasculature include EGF, FGF, VEGF, TGFjS, HGF (NaKamura, 1991), angiotropin, TGF-α, TNF-α, PD-ECGF and ligands of TIE link (Bicknell and Harris, 1992). The currently preferred target substances are VEGF / VPF, and the FGF family of proteins, transforming the growth factor β (TGF-β); TGF-a; tumor necrosis factor-a (TNF-a); angiotropin; endothelial cell growth factor derived from platelet (PD-ECGF); TIE link ligands; pleiotropin. In addition, non-antibody target components, such as annexins and peptides comprising the tripeptide sequence RGD, which specifically target the tumor vasculature (Pasqualini et al., 1997), are also contemplated for use in certain aspects of the invention. . Another aspect of the present invention is the use of objective antibodies, or binding regions thereof, which are specific for epitopes present only in ligand-receptor complexes, these epitopes are absent of both the individual (free) ligand and the receptor in its unlinked form. These antibodies recognize and bind to the unique conformation that results when a ligand, such as a growth factor, binds to its receptor, such as a growth factor receptor, to form a specifically linked complex. These epitopes are not present in the uncomplexed forms of the ligands or receptors. The inventors contemplate that the ligand-receptor complexes to which these antibodies bind are present in a significantly higher number in endothelial cells associated with tumors than in endothelial cells not associated with tumors. These antibodies will therefore be useful as target substances and will serve to further increase the specificity of the bispecific coagulants of the invention. (i) Receptor Constructs Soluble endothelial cell surface receptor domains are also contemplated for use as target ligands in the present invention. This concept is generally based on the well-known sandwich binding phenomenon that has been exploited in a variety of in vitro and in vivo binding protocols. BasicallyAs the endothelial cells express specific receptors, the cells bind to and adsorb the corresponding ligands, the ligands are then available to bind to other receptor constructs if they are to be introduced into the system. A range of useful endothelial cell receptors has been identified in the previous sections, with particularly preferred targets being VEGF / VPF, FGF, TGF3, TIE-1 and TIE-2. Each of these receptors could be manipulated to form a soluble binding domain for use as a target ligand. iv. Objectives of Stromal Cells Associated with the Disease (a) Matrix / Extracellular Stromal Objectives The utility of basement membrane markers in tumor pathology was described by Birembaut et al. (1985). These studies showed that the distributors of basement membrane (BM) collagen type IV, laminin (LM), heparan sulfate proteoglycan (HSP) and fibronectin (FN) were interrupted in the tumor pathology. Burtin et al. (1983) also described alterations of the basement membrane and connective tissue antigen in human metastatic lymph nodes. A preferred objective for use with the invention is RIBS. Ugarova et al. (1993) reported that conformational changes occur in fibrinogen and are produced by their interaction with the glycoprotein of the GPIIb-IIIa platelet membrane. The binding of fibrinogen to the glycoprotein GPIIb-IIIa membrane in activated platelets leads to platelet aggregation. This interaction results in conformational changes in fibrinogen as evidenced by the expression of receptor-induced binding sites, RIBS, epitopes that are expressed by binding but not by free ligand. Two RIBS epitopes have been located by Ugarova et al. (1993). A sequence resides in? Ll2-119 and is recognized by Mab 9F9; the second is the sequence RGDF in Aa 95-98 and is recognized by Ab 155B16. These epitopes are also exposed by adsorption of fibrinogen on plastic surface and digestion of the molecule by plas ina. The proteolytic exposure of the epitopes coincides with the dissociation of carboxyl terminal aspects of the Aa chains formed by the fragment X - The inaccessibility of the sequence RGDF of the sequence Aa 95-98 in fibrinogen suggests that this sequence does not participate in the initial binding of the molecule to GPIIb-IIIa. The binding of fibrinogen to its receptor alters the conformation of the carboxyl terminal aspects of the Aa chains, exposing the sequences residing in the spiral linker segments between the D and E domains of the molecule, generating RIBS epitopes. In practical terms, RIBS sequences are proposed as epitopes for use in directing them with a coaguligand. The monoclonal antibodies 9F9 and 155B16 can be used in this manner advantageously, as well as the antibodies described by Zamarron et al. (1991). (b) Additional Cellular Targets Combinations for the use of the present invention have the additional advantage that they can be used to direct coagulants to the vasculature associated with the disease by directing them to cell types found within the region of the disease. Platelets participate in hemostasis and thrombosis by adhering to the walls of injured blood vessels and accumulating at the site of injury. Although the deposition of platelets at sites of blood vessel injury is responsible for the primary arrest of bleeding under physiological conditions, it can lead to vascular occlusion with damage to the resulting ischemic tissue and thrombus embolization under pathological conditions. The interactions of platelets with their environment and with each other represent complex processes and begin at the cell surface. Therefore, the surface membrane provides a reactive interface between the external medium, which includes components of the wall of the blood vessels, and the plasma and the interior of the platelet. p-155, a multimeric platelet protein that is expressed on activated platelets (Hayward et al. 1991), can be attacked using the invention. Platelets respond to a large number of stimuli suffering complex biochemical and morphological changes. These changes are involved in physiological processes that include adhesion, aggregation, and coagulation. The activation of platelets produces alterations of the membranes that can be recognized by monoclonal antibodies. Monoclonal antibody JS-1 (Hayward et al., 1991) is one of these antibodies contemplated for use as part of a coaguligand. Ligand-induced binding sites (LIBS) are sites expressed on cell surface receptors only after the binding of the ligand causes the receptor to change shape, through subsequent biological events. These can be seen as a counterpart to the RIBS and are also preferred objectives for use in the present invention. Frelinger et al. (1990; 1991) developed 13 anti-LIBS antibodies, any of which can be used to deliver a coagulant to a disease site or tumor according to the present invention. Murine monoclonal anti-platelet antibodies MA-TSPI-1 (directed against human thrombospondin) and MA-PMI-2, MA-PMI-1, and MALIBS-1 (directed against LIBS on the human platelet glycoprotein Hb / IIIa) from Dewerchin and collaborators (1991) can also be used, as well as RUU 2.41 and LIBS-1 of Heynen et al. (1994), - OP-G2 of Tomiyama et al. (1992); and Ab-15. Many other targets, such as antigens or smooth muscle cells, pericytes, fibroblasts, macrophages and infiltrating lymphocytes and leukocytes can also be used. v. Toxins For certain applications, it is considered that the second therapeutic substance will be pharmacological substances linked to antibodies or growth factors, particularly cytotoxic or otherwise anti-cellular substances that have the ability to kill or suppress the growth of endothelial cell cell division. In general, secondary aspects of the invention contemplate the use of any drug substance that can be conjugated to a target substance, preferably an antibody, and administered in active form to the target endothelium or stroma. Exemplary anti-cell substances include chemotherapeutic substances, radioisotopes as well as cytotoxins. In the case of chemotherapeutic substances, the inventors propose that substances such as a hormone, a steroid, an antimetabolite such as arabinoside cytosine, fluorouracil, methotrexate or aminopterin, an anthracycline, mitomycin C; a vinca alkaloid; demecolcin, - etoposide; mithramycin, - or an antitumor alkylating substance such as chlorambucil or melphalan, will be particularly preferred. Other embodiments may include substances such as cytokine, growth factor, bacterial endotoxin or the lipid A fraction of bacterial endotoxin. In any case, it is proposed that substances such as these, if desired, can be successfully conjugated to a target substance, preferably an antibody, in a manner that allows their targeting, internalization, release or presentation to the blood components in the blood. target endothelial cell site as required using known conjugation technology (see, e.g., Ghose et al., 1983 and Ghose et al. 1987). In certain preferred embodiments, the immunotoxins will generally include a toxin derived from plant, fungus or bacteria, such as the A chain toxins, a ribosome inactivation protein, a-sarcin, aspergillin, restrictocin, a ribonuclease, diphtheria toxin or exotoxin pseudomonas to mention only a few examples. The use of toxin antibody constructs is well known in the immunotoxin technique, as is its binding to antibodies. Of these, a particularly preferred toxin for binding with antibodies will be a deglycosylated ricin A chain. The deglycosylated ricin A chain is preferred because of its extreme potency, longer half-life, and because it is economically feasible to manufacture it in clinical and scale grade. (a) Preparation of Conjugates Target Substance-Toxin While the preparation of immunotoxins is, in general, well known in the art (see, e.g., U.S. Patent Nos. 4,340,535, and European Patent Number 44167 , both incorporated herein by reference), the inventors are aware that certain advantages can be achieved through the application of a certain preferred technology, both in the preparation of the immunotoxins and in their purification for subsequent clinical administration. For example, although IgG-based immunotoxins will typically exhibit better binding capacity and slower elimination in the blood than their Fab1 counterparts, immunotoxins based on Fab 'fragments will generally exhibit better tissue penetration capacity compared to immunotoxins based in IgG. Additionally, although numerous types of linkers containing disulfide linkers are known which can be successfully used to conjugate the toxin moiety with the target substance, certain ligands will generally be preferred over other ligands, based on different pharmacological characteristics and capabilities. For example, ligands containing a disulfide bond that is sterically "hindered" will be preferred, due to its greater stability in vivo, thus preventing release of the toxin fraction prior to binding at the site of action. In addition, although certain advantages according to the invention will be realized through the use of any of several toxin fractions, the inventors have found that the use of the ricin A chain, and even more preferably the deglycosylated A chain, will provide benefits particular. A wide variety of cytotoxic substances are known that can be conjugated with endothelial cell antibodies. Examples include numerous toxins derived from plant, fungus, or even bacteria, which, by way of example, include several A chain toxins, particularly ricin A chain, ribosome inactivating proteins such as saporin or gelonin, a-sarcin, aspergillin, restrictocin, ribonucleases such as placental ribonuclease, angiogenic, diphtheria toxin, and pseudomonas exotoxin. to name just a few. The most preferred toxin moiety for use in connection with the invention is the A chain toxin that has been treated to modify or remove carbohydrate residues, so called chain A deglycosylated. The inventors have had the best success through the use of deglycosylated ricin A chain (dgA) which is now commercially available from Inland Laboratories, Austin, TX. However, it may be desirable from a pharmacological point of view to employ the smallest possible molecule that nevertheless provides an adequate biological response. It may then be desired to employ smaller A chain peptides which will provide an adequate anti-cell response. To this end, it has been discovered by others that the A chain of ricin can be "truncated" by removing 30 N-terminal amino acids by Nagarase (Sigma), and still retain adequate toxin activity. It is proposed that when desired, this truncated A chain can be used in conjugates according to the invention. Alternatively, it can be found that the application of recombinant DNA technology to the toxin A chain fraction will provide additional significant benefits according to the invention. Since the cloning and expression of the A chain of biologically active ricin has been enabled through the publication of others (O 'Hare et al., 1987; Lamb et al., 1985; Halling et al., 1985), it is now possible to identify and prepare smaller peptides or otherwise variants that nevertheless exhibit adequate toxin activity. Moreover, the fact that the A chain of ricin has now been cloned allows for the application of site-directed mutagenesis, through which it can be easily prepared and screened for chain A-derived peptides and obtain additional useful fractions for its use in connection with the present invention. The crosslinking of the toxin A chain region of the conjugate with the region of the target substance is an important aspect of the invention. In certain cases, a crosslinker which exhibits disulfide function is required to be used by the conjugate to have biological activity. The reason for this is not clear, but is probably due to a need for certain toxin fractions to be easily liberable from the target substance once the substance has "delivered" the toxin to the target cells. Each type of regulator, as well as how cross-linking is carried out, will tend to vary the pharmacodynamics of the resulting conjugate. Finally, in cases where a releasable toxin is contemplated, it is desired to have a conjugate that remains intact under conditions found everywhere in the body except at the intended site of action, at this point it is desirable that the conjugate has good " release". Therefore, the particular crosslinking scheme, including in particular the particular crosslinking reagent used and the structures that are crosslinked, will have some importance. Depending on the specific toxin compound used as part of the fusion protein, it may be necessary to provide a peptide spacer operably linked to the target substance and the toxin compound that is capable of being bent into a disulfide linked hairpin structure. The proteolytic dissociation within the fork will then produce a heterodimeric polypeptide wherein the target substance and the toxin compound are linked by only a single disulfide bond. (See, for example, Lord et al., 1992). An example of this toxin is the ricin A chain toxin. When other toxin compounds are used, a non-dissociable peptide spacer can be provided to operably link the target substance and the toxin compound of the fusion protein. The toxins that can be used together with the non-dissociable peptide spacers are those that can, by themselves, be converted by proteolytic cleavage in a cytotoxic disulfide linked form (see for example, Ogata et al., 1990). An example of a toxin compound is a Pseudomonas exotoxin compound. Nucleic acids can be used herein which comprise nucleic acid sequences encoding a target substance of interest and nucleic acid sequences encoding a toxin substance of interest. These nucleic acid sequences encoding target substance and encoding toxin substance are linked in such a way that the translation of the nucleic acid yields the target substance / toxin compounds of the invention. (b) Binding of other substances to the target substances It is contemplated that most of the therapeutic applications of the additional immunotoxin aspects of the present invention will involve the targeting of a toxin moiety to the endothelium of the tumor or stroma. This is due to the larger capacity of most toxins to deliver a cell-killing effect compared to other potential substances. However, there may be circumstances, such as when the target antigen is not internalized by a route consistent with efficient intoxication by target substance / toxin compounds, such as immunotoxins, where it will be desirable to target chemotherapeutic substances such as antitumor drugs., other cytokines, antimetabolites, alkylating substances, hormones, and the like. The advantages of these substances over their counterpart conjugated non-target substances is the added selectivity that the target substance has, such as an antibody. Examples such as steroids, cytosine arabinoside, methotrexate, aminopterin, anthracyclines, mitomycin C, vinca alkaloids, demecolin, etoposide, mithramycin, and the like could be mentioned by way of example. The list, of course, is exemplary only because the technology for linking pharmaceutical substances to target substances, such as antibodies, for tissue-specific administration is well established (see, e.g., Ghose and Blair, 1987). A variety of chemotherapeutic substances and other pharmacological substances have now been successfully conjugated to antibodies and shown to function pharmacologically (see, e.g., Vaickus et al. 1991). Exemplary antineoplastic substances that have been investigated include dexorubicin, daunomycin, methotrexate, vinblastine, and several others (Dillman et al., 1988; Pietersz et al., 1988). Moreover, the binding of other substances such as neocarzinostatin (Kimura et al., 1983), macromycin (Manabe et al., 1984), trenimon (Ghose, 1982) and a-amanitin (Davis and Preston, 1981) has been described. saw. Coaguligands The second objective substance for optional use with the invention may also comprise an objective component that is capable of promoting coagulation. These "substances that promote coagulation" or "coaguligands" include any of the above objective substances that are operatively associated with one or more coagulation factors. The target substance may be linked directly to a factor that directly or indirectly stimulates coagulation, or the target substance may be linked to a second binding region that is capable of binding and releasing a clotting factor that directly or indirectly stimulates coagulation. (a) Clotting factors Exemplary coagulation factors are the types of truncated, dimeric, ultimeric and mutant tissue factor molecules of the present invention, as described herein in detail. A variety of other coagulation factors can be used in connection with the present invention, as exemplified by the substances presented below. When a coagulation factor is covalently linked to a first binding substance or target, a site other than its functional coagulation site is used to join the molecules. Suitable binding regions other than the active sites, or functional regions, of the coagulation factors are also described in each of the following sections. Coagulation factors Thrombin, Factor V / Va and derivatives, Factor VIII / HIVa and derivatives, Factor IX / IXa and derivatives, Factor X / Xa and derivatives, Factor Xl / XIa and derivatives, Factor XII can also be used in the present invention. / XHa and derivatives, Factor XIII / XIHa and derivatives, activator of Factor X and activator of Factor V. Venom coagulants Williams and Snouf, in 1962, demonstrated that Russell's viper venom contains a coagulant protein. Kisiel (1979) isolated a venom glycoprotein that activates the Factor V; and Di Scipio et al. (1977) showed that a venom protease activates human Factor X. The activator of Factor X is the component contemplated for use in this invention. The specific monoclonal antibodies for the activator of Factor X present in the viper venom of Russell have also been produced (e.g., MPl from Pukrittayakamee et al., 1983), and could be used to administer the substance at a specific target site within the body. Prostaqlandins and synthetic enzymes Thromboxane A2 is formed from endoperoxides by the sequential actions of the enzymes cyclooxygenase and thromboxane synthetase in platelet microsomes. The thromboxane A2 is easily generated by platelets and is a potent vasoconstrictor, by virtue of its ability to - produce platelet aggregation (Whittle et al., 1981). Both thromboxane A2 and active analogues thereof are contemplated for use in the present invention. A synthetic protocol for generating thromboxane A2 is described by Bhagwat et al. (1985). The thromboxane A2 analogs described by Ohuchida et al. (1981) (especially compound 2) are particularly contemplated for use herein. It is possible that thromboxane synthase, and other enzymes that synthesize prostaglandins that activate platelets, can also be used as "coagulants" in the present context. Shen and Tai (1986) describe monoclonal antibodies, and immunoaffinity purification of thromboxane synthase; and Wang et al (1991) reports the CDNA for human thromboxane synthase. Inhibitors of fibrinolysis The a2-antiplasmin, or a2-plasmin inhibitor, is a proteinase inhibitor naturally present in human plasma that functions to efficiently inhibit the lysis of fibrin clots induced by the plasminogen activator (Moroi and Aoki, 1976). . A2-antiplasmin is a particularly potent inhibitor, and is contemplated for use in the present invention. The a2-antiplasmin can be purified as first described by Moroi and Aoki (1976). Other purification schemes are also available, such as using affinity chromatography on plasminogen-sepharose, ion exchange chromatography on DEAE-sephadex and chromatography on concavalin-A-Sepharose; or using affinity chromatography on a sepharose column having an elastase-digested plasminogen formulation containing three N-terminal triple cycle structures on the plasmin A chain (LBSI), followed by gel filtration (Wiman and Collen, 1977 Wiman, 1980, respectively). As the a2-antiplasmin cDNA sequence is available (Tone et al., 1977), a preferred method for the production of a2-antiplasmin will be via recombinant expression. Also available are monoclonal antibodies against a2-antiplasmin that can be used in the bispecific binding ligand embodiments of the invention. For example, Hattey et al. (1987) describes two monoclonal antibodies against a2-antiplasmin, MPW2AP and MPW3AP. Since each of these monoclonal antibodies was reported to react equally well with the original a2-antiplasmin, both could be used to administer exogenous a2-antiplasmin to a target site or accumulate endogenous a2-antiplasmin and concentrate it within the target region. Other antibodies such as JTPI-2, described by Mimuro et al. Could also be used. (b) Substances that bind coagulation factors Another group of target coagulation ligands for use with the tissue factors of this invention are those in which the target region is not directly linked to a coagulation factor, but is linked to a coagulation factor. second binding region that binds to a coagulation factor. When a second binding region is used to ligate and administer a clotting factor, the binding region is chosen so as to recognize a site in the coagulation factor that does not significantly impair its ability to induce coagulation. The regions of the coagulation factors suitable for binding in this manner will generally be the same as the regions that are convenient for the covalent bond to the target region., as described in the previous sections. However, since bispecific ligands of this class can be expected to release the coagulation factor after administering it to the tumor site or region, there is more flexibility allowed in the coagulation factor regions suitable for binding to a second substance or antibody. of link. The second convenient link regions for use in this manner will generally be antigen-combining sites of antibodies having binding specificity for the coagulation factor, including the functional portions of the antibodies, such as the scFv, Fv, Fab fragments. ', Fab and F (a') 2 • Bispecific binding ligands containing antibodies, or fragments thereof, directed against Tissue Factor, Thrombin, Precalinein, Factor V / Va, Factor VIII / VHIa, Factor IX / IXa , Factor X / Xa, Factor Xl / XIa, Factor XII / XHa, Factor XIII / XIHa, venom of Russell's viper. Thromboxane A2 or a2-antiplasmin are exemplary embodiments of this aspect of the invention. (c) Tissue Factor Prodrugs The exemplary truncated Tissue Factor prodrugs have the following structures: tTF | .2i9 (X) n? (Y) n2 Z ligand, where tTF? .219 represents the Tissue Factor minus the cytosolic and transmembrane domains; X represents a hydrophobic transmembrane domain with no amino acids (AA) in length (n = l-20 AA), - Y represents a hydrophilic protease recognition sequence of n2 amino acids in length (sufficient amino acids to ensure adequate recognition of protease); Z represents a disulfide thioester or other linking group such as (Cys) 1.2; Ligand represents an antibody or other objective fraction of recognition of tumor cells, EC tumor, connective tissue (stroma) or basal lamina markers.

The truncated Tissue Factor prodrug is contemplated for intravenous injection allowing it to locate diseased tissue (e.g., tumor). As soon as it is located in diseased tissue, the endogenous proteases (eg, metalloproteinases, thrombin, Factor Xa, Factor Vlla, Factor IXa, plasmin) will dissociate the hydrophilic protease recognition sequence of the prodrug which will allow the sequence of Hydrophobic transmembrane is inserted into a local cell membrane. Once the tail has been inserted into the membrane, the truncated tissue factor will regain its coagulation induction properties resulting in the formation of clot in the desired tissue vasculature. (d) Bispecific Antibodies In general, the preparation of bispecific antibodies is also well known in the art, as exemplified by Glennie et al. (1987). Bispecific antibodies have been used clinically, for example, to treat patients with cancer (Bauer et al., 1991). A method for the preparation of bispecific antibodies involves the separate preparation of antibodies having specificity for the target tumor cell antigen, on the one hand, and the coagulating substance (or other desired target, such as an activating antigen) on the other. Bispecific antibodies have also been developed particularly for use as immunotherapeutic substances. As mentioned above in conjunction with the induction of antigen, certain of these antibodies were developed to cross-link lymphocytes and tumor antigens (Nelson, 1991; Segal et al., 1992). Examples include chimeric molecules that bind T cells, e.g., in CD3, and tumor antigens, and trigger lymphocyte activation by physical cross-linking of the TCR / CD3 complex in close proximity to the target cell (Staerz et al. 1985, Pérez et al., 1985; 1986a, 1986b; Ting et al., 1988). Undoubtedly, tumor cells from carcinomas, lymphomas, leukemias and melanomas have been reported to be susceptible to elimination mediated by bispecific antibody using T cells (Nelson, 1991, Segal et al., 1992, de Leij et al., 1991). These types of bispecific antibodies have also been used in several phase I clinical trials against various tumor targets. The bispecific crosslinking antibodies can be administered as described in references such as DeLeij et al. (1991); Clark et al. (1991); Rivoltini et al. (1992), - Bolhuis et al. (1992), - and Nitta et al. (1990). Although numerous methods are known in the art for the preparation of bispecific antibodies, the method of Glennie et al. (1987) involves the preparation of peptide F (ab '?) 2 fragments of the two chosen antibodies, followed by the reduction of each one to provide Fab '? SH fragments separately. The SH groups in one of the two coupling partners are alkylated with a cross-linking reagent such as o-phenylenedimaleimide to provide free maleimide groups in a partner. This partner can be conjugated with the other by means of a thioether bond, to give the desired heteroconjugate. Due to ease of preparation, high yield and reproducibility, the method of Glennie et al. (1987) is often preferred for the preparation of bispecific antibodies, however, there are numerous distinct approaches that can be employed and are considered by the inventors. For example, other techniques are known in which cross-linking with SPDP or protein A is carried out, or a trispecific construction is prepared (Titus et al 1987; Tutt et al., 1991). Another method for producing bispecific antibodies is by fusion of two hybridomas to form a quadroma (Flavell et al., 1991, 1992; Pimm et al., 1992, - French et al., 1991).; Embleton et al., 1991). As used herein, the term "quadroma" is used to describe the productive fusion of two B-cell hybridomas. Using standard techniques now, two hybridomas that produce antibody to produce daughter cells are fused, and those cells that have been selected are selected. maintained the expression of both sets of immunoglobulin clonotype genes. A preferred method for generating a quadroma involves the selection of an enzyme-deficient mutant from at least one of the parent hybridomas. This first mutant hybridoma cell line is then fused with cells from a second hybridoma that has been exposed lethally, e.g., to iodoacetamide, preventing its continued survival. The cell fusion allows the rescue of the first hybridoma by acquiring the gene for its enzyme deficiency from the lethally treated hybridoma, and the rescue of the second hybridoma through the fusion with the first hybridoma. It is preferred, but not required, the fusion of immunoglobulins of the same isotype, but of a different subclass. A mixed subclass antibody allows use if there is an alternative assay for the isolation of a preferred quadroma. In greater detail, a method of developing a quadroma and selection involves obtaining a hybridoma line that secretes the first monoclonal antibody chosen and makes it deficient for the essential metabolic enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtain deficient mutants of the hybridoma, cells are grown in? presence of increasing concentrations of 8-azaguanin (1x10 M to 1 x 10"5 M) The mutants are subcloned by limiting the dilution and testing their sensitivity to hypoxanthine / aminopterin / thymidine (HAT). in, for example, DMEM supplemented with 10% fetal calf serum (FCS), 2mM L-glutamine and 1 M penicillin-streptomycin A complementary hybridoma cell line that produces the second desired monoclonal antibody is used to generate the quadromas using standard cell fusion techniques (Galfre et al., 1981), or using the protocol described by Clark et al. (1988) In summary, 4.5 x 107 first hypoxanthine / aminopterin / thymidine sensitive cells are mixed with 2.8 x 107 seconds. hypoxanthine / aminopterin / thymidine-resistant cells that have been previously treated with a lethal dose of the irreversible biochemical inhibitor iodoacetamide (5mM in phosphate buffered saline) du 30 minutes on ice before melting. Cell fusion is induced using polyethylene glycol (PEG) and the cells are plated in 96 well microculture plates. The quadromas are selected using medium containing hypoxanthine / aminopterin / thymidine. Cultures containing bispecific antibody are identified using, for example, isotype-specific, solid-phase isotype-specific ELISA and isoform-specific immunofluorescence. In an identification modality for identifying the bispecific antibody, microtiter plate wells (Falcon, Becton Dickinson Labware) are coated with a reagent that specifically interacts with one of the parent hybridoma antibodies and that lacks cross-reactivity with both antibodies. The plates are washed, blocked, and the supernatants (SNs) to be tested are added to each well. The plates are incubated at room temperature for two hours, the supernatants are discarded, the plates are washed, and alkaline phosphatase-anti-antibody conjugate added for 2 hours at room temperature. The plates are washed and a phosphatase substrate is added, e.g., P-Nitrophenyl phosphate (Sigma, St. Louis) is added to each well. Plates are incubated, 3N NaOH is added to each well to stop the reaction, and OD410 values are determined using an enzyme-linked immunosorbent assay (ELISA) reader. In another identification mode, microtiter plates pretreated with poly-L-lysine are used to join one of the target cells to each well, the cells are then fixed, v.gr. using 1% glutaraldehyde and bispecific antibodies are tested for their ability to bind the intact cell. In addition, FACS, immunofluorescence staining, idiotype-specific antibodies, antigen binding competition assays, and other methods common in the art of antibody characterization can be used in conjunction with the present invention to identify preferred quadromas. After the isolation of the quadroma, the bispecific antibodies are purified away from other cellular products. This can be accomplished by a variety of protein isolation procedures, known to those skilled in the art of immunoglobulin purification. The means for preparing and characterizing antibodies are well known in the art (see, v, Gr Antibodies: A Laboraty Manual, 1988). For example, supernatants from selected quadromas are passed over sepharose protein A or protein G columns to bind IgG (depending on the isotype). Then the antibodies are eluted with, e.g. a citrate regulator with PH 5.0. The eluted fractions containing the bispecific antibodies are dialyzed against an isotonic regulator. Alternatively, the eluate is also passed over an anti-immunoglobulin sepharose column. Bispecific antibodies are eluted with 3.5 M magnesium chloride. Bispecific antibodies purified in this manner are tested for their binding activity by, eg, an isotype-specific linked enzyme-linked immunosorbent assay and an immunofluorescence staining assay of the target cells, as described above. Bispecific antibodies and parent antibodies can also be characterized and isolated by SDS-PAGE electrophoresis, followed by silver or Coomassie staining. This is possible when one of the parent antibodies has a higher molecular weight than the other, where the band of bispecific antibodies migrates halfway between that of the two parent antibodies. The reduction of the samples verifies the presence of heavy chains with two different apparent molecular weights. In addition, recombinant technology is now available for the preparation of antibodies in general, allowing the preparation of recombinant antibody genes that encode an antibody having the desired dual specificity (Van Duk et al., 1989). Thus, after selecting the monoclonal antibodies having the most preferred binding characteristics, the respective genes for these antibodies can be isolated, eg, by immunological selection of a phage display library (Oi and Morrison, 1986).; Winter and Milstein, 1991). Then, through the rearrangement of the Fab coding domains, the appropriate chimaeric construction can be easily obtained. vi i. Combination Treatment Tissue Factor compositions in combination with either immunotoxins or coaguligands are contemplated for use in the clinical treatment of various human cancers and even other disorders, such as benign prostatic hyperplasia and rheumatoid arthritis, in which term arrest medium or longer blood flow would be advantageous.

The combination of the tissue factor compositions described in the present application with immunotoxins and coaguligands are considered to be particularly useful tools in antitumor therapy. From the data presented herein, including animal studies, and knowledge in the art regarding the treatment of lymphoma (Glennie et al., 1988), appropriate doses and treatment regimens can be directly developed targeting the cells T (Nolan and Kennedy, 1990) and directing drugs (Paulus, 1985). It is currently proposed that the effective doses of immunotoxins and coaguligands for use with the tissue factor constructs described above in the treatment of cancer will be between about 0.1 mg / kg and about 2 mg / kg, and preferably, between about 0.8. mg / kg and approximately 1.2 mg / kg, when administered via route IV at a frequency of approximately once a week. There will necessarily be some variation in dosage depending on the condition of the subject being treated. The person responsible for the administration, in any case, will determine the appropriate dose for the individual subject. This optimization and adjustment is carried out routinely in the art and in no way reflects an undue amount of experimentation. Naturally, before its widespread use, animal studies and additional clinical trials will be carried out. The various elements for conducting a clinical trial, including patient treatment and supervision, will be known to those skilled in the art in the light of the present disclosure. The following information is being presented as a general line for use in establishing these trials. It is contemplated that patients chosen for combined studies would have failed to respond to at least one course of conventional therapy and had to have objectively measurable disease as determined by physical examination, laboratory techniques, or radiographic procedures. When portions of murine monoclonal antibody are used in immunotoxins or coaguligands, patients should have no history of allergy to mouse immunoglobulin. Any chemotherapy should be stopped at least two weeks before entering the study. With respect to the administration of tissue factor constructs with either immunotoxins or coaguligands, it is considered that certain advantages will be found in the use of a resident central venous catheter with a triple lumen gate. The therapeutic mixtures should be filtered, for example, using a 0.22 μ filter, and appropriately diluted, such as with saline, to a final volume of 100 ml. Before use, the test sample should also be filtered in a similar manner, and its concentration assessed before and after filtration by determining the A28o-The expected recovery should be within the range of 87 to 99%, and will be considered adjustments for the loss of protein. These combinations of tissue factor and immunotoxin or coaguligand can be administered for a period of about 4-24 hours, with each patient receiving 2 to 4 infusions at intervals of 2 to 7 days. The administration can also be carried out by a stable infusion regimen for a period of 7 days. The infusion given at any dose level should depend on any observed toxicity. Therefore, if grade II toxicity is reached after any single infusion, or at a particular time period for a steady-state infusion, other doses should be retained or the infusion stopped at a steady state until the toxicity improves. Increasing doses of tissue factor with either immunotoxins or coaguligands should be administered to groups of patients until approximately 60% of patients show unacceptable grade III or IV toxicity in any category. Doses that are 2/3 of this value should be defined as the safe dose. Physical exam, tumor measurements, and laboratory tests, of course, should be done before treatment and at intervals-up to a month later. Laboratory tests should include complete blood counts, serum creatinine, creatinine kinase, electrolytes, urea, nitrogen, SGOT, bilirubin, albumin, and total serum protein. Serum samples taken up to 60 days after treatment should be evaluated by radioimmunoassay to determine the presence of intact tissue factor, immunotoxin and / or coaguligating or components thereof and antibodies against any portion thereof. Immunological analysis of serum, using any standard assay such as, for example, a linked enzyme immunosorbent assay or radioimmunoassay, will allow the pharmacokinetics and elimination of the therapeutic substance to be evaluated. To evaluate antitumor responses, it is contemplated that patients should be examined at 48 hours a week and again 30 days after the last infusion. When a palpable disease is present, perpendicular diameters of all masses should be measured daily during treatment, within one week after completing therapy, and at 30 days. To measure non-palpable disease, serial CT scans will be performed at one-centimeter intervals throughout the chest, abdomen, and pelvis at 48 hours to a week and again at 30 days. Tissue samples should also be evaluated histologically, and / or by flow cytometry, using biopsies from disease sites or even blood or fluid samples if appropriate. Clinical responses can be defined by acceptable measurement. For example, a complete response can be defined by the disappearance of all measurable tumors one month after treatment. While a partial response can be defined by a reduction of 50% or more of the sum of the products of the perpendicular diameters of all tumor modules evaluable one month after treatment, without tumor sites showing enlargement. Similarly, a mixed response can be defined by a reduction of the product of perpendicular diameters of all measurable lesions by 50% or more one month after treatment, with advancement in one or more sites. F. Prolonged Average Life of Tissue Factor It is demonstrated herein that the antitumor activity of the truncated tissue factor is increased by conjugating the truncated tissue factor with carrier molecules, such as immunoglobulins, which delay the removal of the truncated tissue factor from the body. . For example, linking the truncated tissue factor to immunoglobulin increases the antitumor activity by prolonging the in-vivo half-life of the truncated tissue factor so that the truncated tissue factor persists for longer and has more time to induce thrombotic events in the vessels of tumor. The prolongation in the half-life is the result either of the increase in size of the truncated tissue factor above the threshold for glomerular filtration, or of the active readsorption of the conjugate within the kidney, a property of the immunoglobulin Fc piece (Spiegelberg and Weigle, 1965). It is also possible that the immunoglobulin component changes the conformation of the truncated tissue factor to make it more active or stable. Other carrier molecules in addition to immunoglobulin are contemplated to produce similar effects and are then included within the present invention. Fl. Modifications Since a first interpretation of the prolonged half-life observed after binding of the truncated tissue factor to the immunoglobulin is simply that the resulting increase in size leads to prolonged plasma half-life, the inventors contemplate that other modifications that increase the Size of the tissue factor constructions can be advantageously used in connection with the present invention, as long as the elongation modification does not substantially restore the membrane binding functionality with the modified tissue factor construct. In the absence of such a possibility, which can easily be proved, virtually any generally biologically acceptable inert molecule can be conjugated with a Tissue Factor construct in order to prepare a modified Tissue Factor with increased half-life in vivo. The modification can also be made to the structure of the tissue factor itself to make it more stable, or perhaps to reduce the catabolism regime in the body. One mechanism for these modifications is the use of D-amino acids instead of L-amino acids in the Tissue Factor molecule. Technicians with ordinary experience in the field will understand that the introduction of these modifications needs to be followed by rigorous testing of the resulting molecule to ensure that it still retains the desired biological properties. Other stabilizing modifications include the use of the addition of stabilizing fractions to either the N-terminus or the C-terminus or both, which is generally used to prolong the half-life of the biological molecules. By way of example only, one may wish to modify the terms of the Tissue Factor constructions by acylation or amination. The variety of these modifications can also be used together, and portions of the Tissue Factor molecule can also be replaced by peptidomimetic chemical structures that result in the maintenance of biological function and still improve the stability of the molecule. F2 Conjugates i. Proteins Useful techniques in connection with conjugation proteins of interest for carrier proteins are widely used in the scientific community. It will generally be understood that in the preparation of these Tissue Factor conjugates for use in the present invention, the protein chosen as a carrier molecule should have certain defined properties. For example, it must of course be biologically compatible and not result in any significant adverse effect after administration to a patient. In addition, it is generally required that the carrier protein be relatively inert, and not immunogenic, both properties will result in maintenance of tissue factor function and allow the resulting construct to prevent excretion through the kidney. Exemplary proteins are albumins and globulins. ii. Non-Proteins In common with the protein conjugates described above, the Tissue Factor molecules of the present invention may also be conjugated to non-protein elements in order to improve their half-life in vivo. Again, the choice of non-protein molecules for use in these conjugates will readily be apparent to those skilled in the art. For example, one or more of a variety of natural or synthetic polymers, including polysaccharides and polyethylene glycol, can be used. In the context of preparing conjugates, either - proteinaceous or non-proteinaceous, care must be taken that the introduced conjugate does not substantially re-associate the modified Tissue Factor molecule with the plasma membrane in such a way as to increase its coagulation capacity and give resulting in a molecule that exert harmful side effects after its administration. As a general rule, it is believed that hydrophobic or conjugated additions should be largely avoided in relation to these modalities. iii. Immunoconjugates When antibodies are used to conjugate the truncated Tissue Factor compositions of the present invention, the choice of antibody will generally depend on the intended use of the Tissue Factor-antibody conjugate. For example, when Tissue Factor immunoconjugates are contemplated. its use in addition to the Tissue Factor molecules alone, the type of tumor should be considered, e.g., whether it is preferable to target the tumor cells, or more preferably, the tumor vasculature or the tumor stroma. When the Tissue Factor immunoconjugates are themselves the primary therapeutic substances, the immunoconjugates will in no way be an "objective immunoconjugate". In these aspects, the conjugation of the Tissue Factor molecule to an antibody or portion thereof is simply carried out in order to generate a construct having improved half-life and / or bioavailability as compared to the Tissue Factor molecule. original. In any case, certain advantages can be achieved through the application of particular types of antibodies. For example, although IgG-based antibodies are expected to exhibit better binding and elimination capacities in the blood slower than their Fab 'counterparts, compositions based on Fab' fragments gener exhibit better penetration capacity in the tissue. (a) Monoclonal Antibodies Elements for preparing and characterizing antibodies are well known in the art (See, e.g., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988). Methods for generating monoclonal antibody (MAbs) generally begin along the same lines as those for preparing polyclonal antibodies. Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogenic composition according to the present invention, either with or without prior immunotolerance, depending on the antigen composition and the protocol being used (e.g. tolerate a population of normal cells and then immunize them with a population of tumor cells), and collect the antiserum from the immunized animal. A wide range of animal species can be used for the production of antiserum. Typically the animal used for the production of antiserum is a rabbit, a mouse, a rat, a hamster, a guinea pig or a goat. Due to the relatively large volume of blood in rabbits, a rabbit is a preferred choice for the production of polyclonal antibodies. As is well known in the art, a given composition can vary in its immunogenicity. Frequently it is therefore necessary to elevate the host immune system, as can be achieved by coupling a peptide or immunogen polypeptide to a carrier. Preferred and exemplary carriers are U-shaped limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as egg albumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. The elements for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobenzoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine. As is well known in the art, the immunogenicity of a particular in uniogenic composition can be increased by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include Freund's adjuvant (a non-specific stimulator of the immune response containing killed Micobacterium tuberculosis), incomplete Freund's adjuvants and aluminum hydroxide adjuvant. The amount of immunogen composition used in the production of polyclonal antibodies varies according to the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies that can be monitored by sampling the immunized animal's blood at several points after immunization. A second boost of reinforcement can also be given. The reinforcement and titling process is repeated until a suitable degree is achieved. When a desired level of titration is obtained, the immunized animal can be bled and the serum isolated and stored, and / or the animal can be used to generate monoclonal antibodies. Monoclonal antibodies can be readily prepared by the use of well known techniques, such as those exemplified in U.S. Patent No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunization of a suitable animal with a selected immunogen composition, e.g., a purified or partially purified tumor cell by vascular endothelial cell protein, polypeptide, peptide, or intact cell composition.

The immunizing composition is administered in an effective manner to stimulate the cells that produce antibodies. Rodents such as mice and rats are the preferred animals, however, the use of rabbit, sheep, frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986, page 60-61), but mice are preferred, with BALB / c being the most preferred mice since it is used more routinely and generally gives a higher percentage of fusions. stable After immunization, somatic cells are selected with the potential to produce antibodies, specifically B lymphocytes (B cells), for use in the monoclonal antibody generation protocol. These cells can be obtained from spleens, tonsils or lymph nodes of biopsies, or from a peripheral blood sample. Spleen cells and peripheral blood cells are preferred, the former because they are a rich source of cells that produce antibodies that are in the plasmablast division stage, and the latter because the peripheral blood is readily accessible. Frequently, a set of animals will have been immunized and the spleen of the animal with the highest antibody titre will be removed and the lymphocytes of the spleen will be obtained by homogenizing the spleen with a syringe. Typically, the spleen of an immunized mouse contains approximately 5 X 10 7 up to 2 X 108 ° lmfocytes.

The B lymphocytes that produce antibodies from the immunized animal are then fused with cells from an immortal myeloma cell, generally one of the same species as the animal that was immunized. Suitable myeloma cell lines for use in fusion processes that produce hybridoma are preferably non-producers of antibodies, which have high fusion efficiency, and enzyme deficiencies that render them incapable of growing in a certain selective medium that supports the growth of only the desired fused cells (hybridomas). Any of several myeloma cells can be used, as those skilled in the art know (Goding, pages 65-66, 1986; Campbell, pages 75-83, 1984). For example, when the immunized animal is a mouse, one can use P3-X63 / Ag8, X63-Ag8.653, NSI / l.Ag 4 1, Sp210-Agl4, FO, NSO / U, MPC-11, MPCH- X45-GTG 1.7 and S194 / 5XX0 Bul; for rats R210.RCY3, Y3-Ag 1.2.3, IR983F, 4B210 or some of the mouse cell lines listed above can be used; and U-266, GM1500-GRG2, LICR-L0N-HMy2 and UC729-6, are useful in connection with human cell fusions. Methods for generating hybrids producing antibody-producing spleen or lymph node cells and myeloma cells usually comprise mixing somatic cells with myeloma cells in a ratio of 4: 1, although the ratio can vary from about 20: 1 to about 1: 1, respectively, in the presence of a substance or substances (chemical or electrical) that promote the fusion of cell membranes. Fusion methods using Sendai viruses have been described by Kohler and Milstein (1975; 1976), and those using polyethylene glycol (PEG), such as 37% (volume / volume) of polyethylene glycol, by Gefter et al., (1977). . The use of electrically induced fusion methods is also adequate (Goding pages 71-74, 1986). Fusion procedures usually produce viable hybrids at low frequencies, approximately 1 X 10"6 up to 1 x 10. However, this does not pose a problem, since the fused, viable hybrids differ from the non-fused cells, parents, (particularly unfused myeloma cells that would normally continue to divide indefinitely) by culturing them in a selective medium.The selective medium is generally one containing a substance that blocks the de novo synthesis of nucleotides in the tissue culture medium. Preferred are aminopterin, methotrexate, and azaserin.Aminopterin and methotrexate block de novo synthesis of both purines and pyrimidines, while azaserin blocks only purine synthesis.When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (hypoxanthine / aminopterin / thymidine medium). When azaserin is used, the medium is supplemented with hypoxanthine. The preferred selection medium is hypoxanthine / aminopterin / thymidine. Only cells capable of operating these nucleotide restoration pathways are able to survive in the hypoxanthine / aminopterin / thymidine medium. The myeloma cells are defective in key enzymes of the restoration pathway, e.g., hypoxanthine phosphoribosyl transferase (HPRT), and can not survive. B cells can operate this route, but they have a limited life extension in culture and usually die within approximately two weeks. Therefore, the only cells that can survive in the selective medium are hybrids formed from myeloma and B cells. This culture provides a population of hybridomas from which specific hybridomas are selected. Typically, the selection of hybridomas is carried out by culturing the cells by a dilution of a single clone in microtiter plates, followed by tests of the individual clonal supernatants (after approximately two to three weeks) to determine the desired reactivity. The assay could be sensitive, simple and rapid, such as radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, point-in-point assays, and the like.

The selected hybridomas would be serially diluted and cloned into cell lines that produce individual antibodies, whose clones can then be propagated indefinitely to provide monoclonal antibodies. The cell lines can be exploited for the production of monoclonal antibodies in two basic ways. A hybridoma sample can be injected (often into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors that secrete the specific monoclonal antibody produced by the hybrid of the fused cell. The body fluids of the animal, such as serum or ascites fluid, can be drained to provide monoclonal antibodies in high concentration. Individual cell lines could also be grown in vitro, where the monoclonal antibodies are secreted naturally in the culture medium from which they can be easily obtained in high concentrations. The monoclonal antibodies produced by either of the two media can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as high pressure liquid chromatography or affinity chromatography. The inventors also contemplate the use of a molecular cloning approach to generate monoclonal. For this, libraries of RNA immunoglobulin phagemids isolated from the spleen of an immunized animal are prepared, and phagemids expressing the appropriate antibodies are selected by panning using cells expressing the antigen and control cells v.gr. , normal cells against tumor. The advantages of this approach over conventional hybridoma techniques are that approximately 104 times more antibodies can be produced and selected in a single round, and that new specificities are generated by the combination of H and L chain that also increases the chance of finding antibodies adequate. When monoclonal antibodies are employed in the present invention, they may be of human, murine, monkey, rat, hamster, chicken or even rabbit origin. The invention contemplates the use of human antibodies, "humanized" or chimeric antibodies of mouse, rat or other species, having human constant and / or variable domain regions, and other recombinant antibodies and fragments thereof. Of course, due to the ease of preparation and immediate availability of the reagents, murine monoclonal antibodies are typically preferred. (b) Fab functional antibody binding regions Functional antibody fragments can be obtained by proteolysis of the whole immunoglobulin by the non-specific thiolprotease, papain. Papain must first be activated by reducing the sulfhydryl group at the active site with cysteine, 2-mercaptoethanol or dithiothreitol. The heavy metals in the enzymatic material must be removed by chelation with EDTA (2 mM) to ensure the maximum enzymatic activity. The enzyme and the substrate are usually mixed together at a ratio of 1: 100 by weight. After incubation, the reaction can be stopped by irreversible alkylation of the thiol group with iodoacetamide or simply by dialysis. The completion of digestion should be monitored by SDS-PAGE and the different fractions separated by protein-sepharose or ion exchange chromatography. P (ab «) 2 The usual procedure for the preparation of F (ab *) 2 fragments of rabbit immunoglobulin G and of human origin is the proteolysis limited by the enzyme pepsin (Protocol 7.3.2.). The conditions, 100% excess weight / weight antibody in acetate buffer at pH 4.5, at 37 aC, suggest that the antibody dissociates at the C-terminal side of the inter-heavy chain disulfide bond. The rates of digestion of mouse immunoglobulin G may vary with the subclasses and it may be difficult to obtain high yields of F (ab ') 2 fragments without some undigested or completely degraded immunoglobulin G. In particular, IgG2b is very susceptible to complete degradation. The other subclasses require different incubation conditions to produce optimal results. The digestion of rat IgG by pepsin requires conditions that include dialysis in 0.1 M acetate buffer, pH 4.5, and then incubation for 4 hours with 1% w / w pepsin; digestion of IgG, and IgG2a improves if dialysis is first performed against regulatory 0.1 M, pH 2.8, at 49C, for 16 hours followed by acetate regulator. IgG2b gives more consistent results with incubation in staphylococcal V8 protease (3% w / w) in 0.1 M sodium phosphate buffer, pH 7.8, for 4 hours at 37 aC. iv. Second generation of tissue factor immunoconjugates The inventors contemplate that the Fc portion of the immunoglobulin in the truncated tissue-immunoglobulin Factor construct employed in the advantageous studies described herein may actually be the relevant portion of the antibody molecule, giving as a result, an increased live life. It is reasonable to assume that conjugation of the Fc region results in an active readsorption of a TF-Fc conjugate into the kidney, restoring the conjugate to the systemic circulation. As such, any of the deficient coagulation tissue factor constructs or variants of the invention can be conjugated to an Fc region in order to increase the in vivo half-life of the resulting conjugate. Several methods are available to produce Fc regions of sufficient purity to allow their conjugation to the Tissue Factor constructs. By way of example only, the chemical dissociation of antibodies to provide the defined domains or portions is well known and practiced easily, and recombinant technology can also be employed to prepare either substantial amounts of Fc regions or, undoubtedly, prepare the conjugate of whole TF-Fc after generation of a recombinant vector expressing the desired fusion protein. Other manipulations of the general immunoglobulin structure can also be carried out in order to provide the second generation of tissue factor constructions with increased half-life. By way of example only, one can consider replacing the CH3 domain of an IgG molecule with a truncated Tissue Factor or a variant thereof. In general, the most effective mechanism to produce this hybrid molecule will be to use molecular cloning techniques and recombinant expression. All of these techniques are generally known to those skilled in the art, and are described in detail herein. F3 Linking Media The above compositions can be linked to the Roof Factor compositions in any operational manner - allowing each region to perform its intended function without significant impairment of the functions of the Tissue Factor. In this way, the binding components will be able to prolong the half-life of the construction, and the Tissue Factor is able to promote the coagulation of the blood. i. Biochemical crosslinkers The binding of any of the above components to a Tissue Factor composition will generally employ the same technology as that developed for the preparation of immunotoxins. It is considered as a general line that any biochemical cross-linker that is suitable for use in an immunotoxin will also be useful in the present context, and can also be considered additional linkers. Crosslinking reagents are used to form molecular bridges that bind functional groups of two different molecules together, e.g., a stabilizing and coagulating substance. To link two different proteins in a staggered manner, heterobifunctional crosslinkers can be used which eliminate the formation of unwanted homopolymers.

TABLE IV HETERO-FUNCTIONAL RETICULATORS An exemplary hetero-bifunctional crosslinker contains two reactive groups *, one which reacts with the primary amine group (e.g., N-hydroxysuccinimide) and the other reacts with a thiol group (e.g. pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the crosslinker can react with the lysine residues of a protein (e.g., the selected antibody or fragment) and through the thiol reactive group, the crosslinker, already bound to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the other protein (e.g., the coagulant). Therefore it can be seen that the preferred composition of Tissue Factor will generally have, or will be derived to have, a functional group available for cross-linking purposes. This requirement is not considered to be limiting since a wide variety of groups can be used in this way. For example, primary or secondary amine groups, hydrazide or idrazine groups, carboxylic alcohol, phosphate, or alkylating groups can be used for linking or crosslinking. For an overview of link technology, one may wish to refer to Ghose and Blair (1987). The spacer arm between the two reactive groups of a crosslinker can have different lengths and chemical compositions. A longer spacer arm allows for better flexibility of the conjugate components while some particular components in the bridge (eg, benzene group) can lend extra stability to the reactive group or an increased resistance of the chemical bond to the action of several. aspects (e.g., disulfide bond resistant to reducing substances). The use of peptide separators, such as L-Leu-L-Ala-L-Leu-L-Ala is also contemplated. It is preferable to employ a crosslinker that has reasonable stability in the blood. Numerous types of disulfide bonds containing linkers are known that can be used successfully to conjugate target substances and coagulants. Linkers containing a disulfide bond that is sterically hindered may show greater stability in vivo, preventing release of the Tissue Factor before binding to the site of action. These linkers are then a preferred group of linking substances. One of the most preferred crosslinking reagents for use in immunotoxins is SMPT, which is a bifunctional crosslinker containing a disulfide bond that is "sterically hindered" by an adjacent benzene ring and methyl groups. It is believed that steric hindrance of the disulfide bond serves a protective function of binding the attack of thiolate anions such as glutathione which may be present in tissues and blood, and by which it is helped to prevent uncoupling of the conjugate prior to administration of the substance attached to the tumor site. It is contemplated that the SMPT substance can also be used in connection with the bispecific coagulant ligands of this invention. The crosslinking reagent SMPT, together with many other known crosslinking reagents, provides the ability to crosslink functional groups such as the SH of cysteine or primary amines (e.g., the epsilon amino group of lysine). Another possible type of crosslinker includes hetero-bifunctional photoreactive phenylazides containing a dissociable disulfide bond such as sulfosuccinimidyl-2- (p- • azido salicylamido) ethyl-1,3 '-dithiopropionate. The N-hydroxy-succinimidyl group reacts with the primary amino groups and the phenylazide (after photolysis) reacts non-selectively with any amino acid residue. In addition to the hindered crosslinkers, unimpeded linkers may also be used according to the present invention. Other useful crosslinkers do not consider containing or generating a protected disulfide, include SATA, SPDP and 2- i inothiolane (Wawrzyncza and Thorpe, 1987). The use of these crosslinkers is very well understood in the art. As soon as it is conjugated, the truncated Tissue Factor is generally purified to separate the conjugate from nonconjugated or coagulant target substances or other contaminants. A large number of purification techniques are available for use to provide conjugates of a sufficient degree of purity to render them clinically useful. Purification methods based on size separation, such as gel filtration, gel permeation or high performance liquid chromatography, will generally be of much use. Other chromatographic techniques can also be used, such as separation by Blue-Sepharose. ii. Recombinant fusion proteins The truncated tissue factor compositions of the invention can also be fusion proteins prepared by molecular biology techniques. The use of recombinant DNA techniques to achieve these ends is now a common practice for those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and in vivo recombination / genetic recombination. Additionally, DNA and RNA synthesis can be performed using an automated synthesizer (see, for example, the techniques described in Sambrook et al., 1989).; and Ausubel et al., 1989). The preparation of this fusion protein generally involves the preparation of a first and a second coding region of DNA and the functional ligand or the joining of said regions, in frame, to prepare a single coding region encoding the desired fusion protein. . In the present context, the truncated Tissue Factor or Tissue Factor mutant DNA sequence will generally be linked in frame with the DNA sequence encoding an inert protein carrier, immunoglobulin, Fc region, or the like. It is generally not thought to be particularly relevant if the Tissue Factor portion of the fusion protein or the inert portion is prepared as the N-terminal region or as the C-terminal region. In connection with the second generation of Tissue Factor immunoglobulin molecules, the Tissue Factor coding sequences can also be inserted into the immunoglobulin coding regions, so that the Tissue Factor sequences functionally interrupt the sequences of immunoglobulin and the encoded protein can be considered a "tribrido". As soon as the coding of the desired region has occurred, an expression vector is created. Expression vectors contain one or more promoters upstream of the inserted DNA regions that act to promote DNA transcription and thereby promote the expression of the encoded recombinant protein. This is the meaning of "recombinant expression" and has been discussed elsewhere in the specification. F4 Assays As with other aspects of the present invention, as soon as a candidate Tissue Factor construct has been generated with the intention of providing a construction with increased live half-life, the construct will generally have to be tested to ensure that the desired properties have been imparted. to the resulting compound. The various tests for use to determine these changes in function are routine and are readily practiced by those skilled in the art. In Tissue Factor conjugates designed simply in order to increase their size, the confirmation of their increased size is completely routine. For example, the candidate composition will simply be separated using any methodology that is designed to separate biological components based on size and the separated products will be analyzed in order to determine that a Tissue Factor construction of increased size has been generated. By way of example only, separation gel and separation columns, such as gel filtration columns, can be mentioned. The use of gel filtration columns in the separation of mixtures of conjugated and unconjugated components may also be useful in other aspects of the present invention, such as in the generation of relatively high levels of conjugates, immunotoxins or coaguligands. As the objective of the present class of conjugates is to provide a deficient coagulation tissue factor molecule having an increased live half life, the modified variants of the tissue factor candidates or the conjugates should generally be tested in order to confirm that it is present this property. Again, these tests are routine in the art. A first test would be to determine the half-life of the modified Tissue Factor or candidate conjugate in an in vitro assay. These assays generally comprise mixing the candidate molecule in serum and determining whether or not the molecule persists in a relatively intact form for a longer period of time, compared to the initial sample of deficient coagulation tissue factor. Again aliquots of the mixture will be sampled and their size and, preferably, their biological function determined. Live assays of the biological half-life or "elimination" can easily be carried out. In these systems, it is generally preferred to label the test factor Tissue Factor constructs with a detectable marker and to follow the presence of the marker after administration to the animal, preferably via the intended route in the final therapeutic treatment strategy. As part of this process, samples of body fluids, particularly serum and / or urine samples, would be taken and the samples analyzed to determine the presence of the marker associated with the construction of the tissue factor, which would indicate the longevity of the construction in the natural environment in the body. One or more of a combination of Tissue Factor molecules with increased half-life can thus be used in conjunction with the therapeutic methods described herein. The doses proposed for administration will generally be between about 0.2 milligrams and about 200 milligrams per patient, as with the original tissue factor constructs described above. However, since these Tissue Factor molecules have been modified, it is possible that effective doses may be even lower, such as on the order of about 0.1 milligrams. If therapeutic treatment regimens are more likely to be altered when Tissue Factors with increased half-life are used in the number of times the drugs are administered, rather than a change in the dose given. For example, when an original Tissue Factor construction is proposed for use on days 1, 3, and 7 within the treatment period, the improved counterparty Tissue Factor with longer half-life would be administered only on day 1 and on day 7. In any case, all these optimizations in terms of dosage and administration times will be easily determined by technicians with experience in the field. G. Combinations of Tissue Factor and Vlla Factor The inventors have further demonstrated that the activity that induces coagulation of the truncated Tissue Factor bound to A20 cells markedly increased in the presence of Factor Vlla. In common with previous studies, these results in vi tro are also transferred to the live environment. The studies are presented herein to demonstrate that the antitumor activity of several constructs of deficient coagulation tissue factor increases after co-administration with Factor Vlla. Still using an experimental animal model of the HT29 tumor, which is remarkably difficult to coagulate, the co-administration of the coagulation tissue Factor constructs of Eicient and the exogenous Factor Vlla resulted in considerable necrosis of the tumor tissue. These data can be explained since the truncated tissue factor binds to the Factor VII but does not efficiently mediate its activation to the Factor Vlla by the Xa and the adjacent Factor Vlla molecules. Providing a source of preformed (exogenous) Vlla Factor overcomes this blockade, allowing a more efficient coagulation. The success of the combined treatment of deficient coagulation tissue factor and Factor Vlla is generally based on the surprising location of the construction of the tissue factor within the tumor vasculature. In the absence of this surprising localization and specific functional effects, the co-administration of Factor Vlla would have no meaning in the context of the treatment of the tumor, and may even be harmful as it promotes unwanted thrombosis in several healthy tissues. The combined use of truncated Tissue Factor and Vlla Factor in a non-targeted manner has been proposed previously in connection with the treatment of hemophiliacs and patients with other blood disorders, in which there is a fundamental deterioration of the coagulation cascade. In the present invention, the coagulation cascade is generally fully operative, and the therapeutic invention concentrates this activity within a defined region of the body. It is therefore another object of the present invention to increase the antitumor effects of any of the Weapon Factor constructs of the invention by combining the use of the Tissue Factor with the additional administration of Factor Vlla. Since the truncated tissue factor binds to the vascular endothelium of the tumor, it is possible to inject truncated Tissue Factor into animals that have tumor, wait a period of time for the excess Truncated Tissue Factor to be removed, and then inject Factor Vlla to magnify the thrombotic action of the truncated Tissue Factor inside the vessels of the tumor. In this way, the truncated tissue factor or other deficient coagulation tissue factor construct can be seen to form a reservoir within the tumor, which allows subsequent administration of the Vlla Factor to increase and perpetuate the antitumor effect. Another observation of the present invention is that the thrombotic activity of the factor VII activation mutants of the truncated Tissue Factor (G164A) and truncated Tissue Factor (W158R) was restored to a large extent by the Vlla Factor. These mutations are within a region of the truncated Tissue Factor that is important for the conversion of Factor VII into Factor Vlla. As with the same truncated Tissue Factor, studies in the present show that the preformed Vlla Factor often overcomes this block in the formation of coagulation complex. The present invention exploits this in the aforementioned observations with the aim of providing in vivo cancer therapy. Undoubtedly, the studies presented herein confirm that coadministration of an activating mutant variant of Factor VII Factor Tissue with preformed Vlla Factor results in considerable necrotic damage to tumors, even in small tumor models that do not they are the most docile for the treatment with the present invention. This aspect of the invention is particularly surprising, since it was not previously believed that these mutants would have any therapeutic activity in any form other than, perhaps, competitive inhibition of the Tissue Factor as it can be used to inhibit or reduce coagulation. Apart from these hypotheses, the generation of these mutants has been motivated by scientific interest and could perhaps be used as controls in certain in vitro studies. Only the studies of the present inventors make these mutants clinically useful, either in the context of targeted administration (WO 96/01653), or in the still more surprising combined uses of the present invention. In particular embodiments, this application of the present invention therefore first involves injecting tTF (G164A), tTF (W158R) or an equivalent thereof into animals that have tumors. The truncated Tissue Factor mutant is then allowed to localize in the tumor vessels and the residue is removed. Then, this is followed by the injection of Factor Vlla, which allows localized Truncated Tissue Factor mutants to express thrombotic activity. This strategy offers the advantage that it is very safe. Truncated Tissue Factor mutants are practically non-toxic, as is Vlla Factor. Thus, administration of the truncated Tissue Factor mutant followed by Factor Vlla will be harmless to the host, although it efficiently induces thrombosis in the tumor vessels. Gl. Factor Vlla Factor VII can be prepared as described by Fair (1983), and as shown in U.S. Patent Nos. 5,374,617, 5,504,064 and 5,504,067, each of which is incorporated herein by reference. The coding portion of the human Factor VII cDNA sequence was reported by Hagen et al., (1986). The amino acid sequence from 1 to 60 corresponds to the pre-pro / front sequence that is removed by the cell before secretion. The mature chain of Factor VII polypeptides consists of amino acids 61 to 466. Factor VII is converted into its active form, Factor Vlla, by dissociating a single peptide bond between arginine-212 and isoleucine-213. Factor VII can be converted in vi tro to Factor Vlla by incubating the purified protein with Factor Xa immobilized in Affi-Gel ™ 15 beads (Bio-Rad).

• The conversion can be monitored by SDS-polyacrylamide gel electrophoresis of reduced samples. The free Factor Xa in the preparation of Factor Vlla can be detected with the chromogenic substrate methoxycarbonyl-D-cyclohexylglycyl-glycyl-arginine-p-nitroanilide (Spectrozyme Factor Xa, American Diagnostica, Greenwich, CT) at a final concentration of 0.2 mM in the presence of 50 mM EDTA. The recombinant Vlla Factor can also be purchased from Novo Biolabs (Danbury, CT). G2 Treatment The use of Factor Vlla in connection with the present invention is not confined to its ability to significantly improve the usefulness of Factor VII activation mutants described herein. It is also contemplated that the Vlla Factor will be used together with the coagulation tissue factor molecules deficient in activity equivalent to the truncated Tissue Factor employed first. In these treatment modalities, the dose of the tissue factor construct will generally be between about 0.2 milligrams and about 200 milligrams per patient. The appropriate doses for the Factor Vlla can be better determined in light of this information. For example, one may wish to create a 1: 1 ratio of the Tissue Factor and Vlla Factor construction in a precomplex and administer the precomplex composition to the animal. If this is desired, a quantity of Tissue Factor and a sufficient amount of Factor Vlla are generally mixed to allow the formation of an equimolar complex. To achieve this, it should be preferable to use a molar excess of 2-3 Vlla Factor in order to ensure that each of the Tissue Factor molecules is appropriately complexed. Then simply the non-complexed Fabric Factor and the Vlla Factor of the complexed mixture would be separated using any convenient technique, such as gel filtration. After formation of the TF: VIIa complex, the complex can simply be administered to a patient in need of treatment in a dose of approximately 0.2 milligrams and approximately 200 milligrams per patient. As noted above, it may generally be preferred to administer in advance a deficient coagulation tissue factor construct to a patient, leaving sufficient time for the tissue factor to be specifically located within the tumor. After this pre-administration, a suitable dose of Vlla Factor would then be designed to coordinate and complex with the tissue factor located within the tumor vasculature. Again, the Vlla Factor dose could be designed in order to allow a 1: 1 molar ratio of Tissue Factor and Vlla Factor to be formed in the tumor environment. Given the differences in the molecular weight of these two molecules, it will be seen that it would be advisable to add approximately double the amount in milligrams of the Vlla Factor in comparison with the milligrams of the Tissue Factor. However, the above analysis is only exemplary, and any dose of Factor Vlla that generally results in an improvement in coagulation would obviously be of clinical importance. About this, it is noteworthy that the studies presented herein actually use a 16: 1 tissue factor surplus compared to the Vlla Factor, which is generally about a 32-fold molar surplus of the tissue factor construct. However, coagulation and impressive tumor necrosis were specifically observed. Therefore, it will be evident that the effective doses of Factor Vlla are quite broad. By way of example only, a dose of Vlla Factor between about 0.01 milligrams and about 500 milligrams per patient may be considered to be administered to a patient. Each of the above analyzes can equally be applied to the use of Tissue Factor constructs that have been mutated to impair their ability to activate Factor VII. Since the above calculations are based on a binding ratio, it is not thought necessary to use particularly increased levels of Factor Vlla in combination with the activation mutants described. However, given that the administration of Factor Vlla is not believed to be particularly harmful in itself, the potential for using increased doses of Factor Vlla is certainly evident. Although the detailed guidance provided above is believed to be sufficient to enable a person of ordinary skill in the art to know how to practice these aspects of the invention, reference may also be made to other quantitative analyzes to assist in the optimization of Factor doses. of Fabric and Factor Vlla for its administration. By way of example only, reference may be made to U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067, which describe a range of therapeutically active doses and plasma levels of Factor Vlla. Morrissey and Comp have reported that, in the context of bleeding disorders, the deficient coagulation tissue factor can be administered in an effective dose to produce in the plasma an effective level of between 100 nanograms / milliliter and 50 micrograms / milliliter, or a preferred level of between 1 microgram / milliliter and 10 microgram / milliliter or from 60 to 600 microgram / kilogram of body weight, when administered systemically; or an effective level of between 10 micrograms / milliliter and 50 micrograms / milliliter or a preferred level of between 10 micrograms / milliliter and 50 micrograms / milliliter, when administered topically (U.S. Patent No. 5,504,064). Factor Vlla is administered in an effective dose to produce in the plasma an effective level of between 20 nanograms / milliliter and 10 micrograms / milliliter, (1.2 to 600 micrograms / kilogram), or a preferred level of between 40 nanograms / milliliter and 700 micrograms / milliliter (2.4 to 240 micrograms / kilogram) or a level of between 1 microgram of Factor VHa / milliliter and 10 micrograms of Factor Vlla / milliliter when administered topically. In general, deficient coagulation tissue factor and Factor VII activator would be administered to produce levels of up to 10 micrograms of deficient coagulation tissue factor / milliliter of plasma and between 40 nanograms and 700 micrograms of Factor Vlla / milliliter of plasma. Although these studies were conducted in the context of bleeding disorders, they also have relevance in the context of the present invention, since the levels must be effective but adequately supervised to avoid systemic toxicity due to high levels of coagulation tissue factor. deficient and Factor Vlla activated. Therefore, the Factor VII activator is administered in an effective dose to produce in the plasma an effective level of Factor Vlla, as defined above. G3 Factor VII Activators As described in U.S. Patent No. 5,504,064, incorporated herein by reference, activators of endogenous Factor VII may also be administered in place of the same Factor Vlla. As described in the above patent, Vlla Factor can also be formed in vivo, shortly before, at the time of, or preferably shortly after, the administration of the deficient coagulation tissue factors. In these embodiments, endogenous Factor VII is converted to Factor Vlla by infusing an activator of Factor Vlla, such as Factor Xa (FXa) in combination with phospholipids (phosphatidyl choline / phosphatidylserine (PCPS)). Factor VII activators in vivo include Factor Xa / PCPS, Factor IXa / PCPS, thrombin, Factor XHa and the Factor VII activator for the venom of Oxyuranus s c u t t l l t t i n c o m b i n a c i n n o n Phosphatidylcholine / Phosphatidylserine. These have been shown to activate Factor VII in Factor Vlla in vi tro. The activation of Factor VII to Factor Vlla for Xa / PCPS in vivo has also been directly measured. In general, the Factor VII activator is administered in a dose between 1 and 10 micrograms / milliliter of carrier (U.S. Patent No. 5,504,064). The phospholipid can be provided in various forms such as phosphatidylcholine / phosphatidylserine (PCPS) vesicles. The phosphatidylcholine / phosphatidylserine vesicle preparations and the Xa / PCPS administration method are described in Giles et al., (1988), the teachings of which are specifically incorporated herein. Other preparations of phospholipids can be replaced by PCPS, as long as they accelerate the activation of Factor VII by Factor Xa. The effectiveness, and therefore the determination of the optimal composition and dosage, can be monitored as described below. A very effective dose of Xa / PCPS, which elevates Vlla factor levels in vivo in the chimpanzee, has been reported to be 26 pmoles FXa + 40 pmoles of phosphatidylcholine / phosphatidyl serine per kilogram of body weight. That dose produced an 18-fold increase in the endogenous levels of Factor Vlla (up to 146 nanograms / milliliter). A marginally detectable effect was observed using a smaller dose in dogs, where the infusion of 12 pmoles of Factor Xa + 19 pmoles of phosphatidylcholine / phosphatidylserine per kilogram of body weight produced a threefold increase in the levels of the endogenous Vlla Factor. Accordingly, the doses of Factor Xa which are at least 12 pmoles of Factor Xa per kilogram of body weight, and preferably 26 pmoles of Factor Xa per kilogram of body weight, should be useful. Doses of phosphatidylcholine / phosphatidylserine which are at least 19 phosphatidyl choline / phosphatidylserine per kilogram of body weight, and preferably 40 pmol of phosphatidylcholine / phosphatidylserine per kilogram of body weight, are equally useful (U.S. Patent No. 5,504,064 ). The effectiveness of any Factor VII activator in infusion can be monitored after intravenous administration, extracting samples of citrated blood at variable times (at 2, 5, 10, 20, 30, 60, 90 and 120 minutes) after a bolus infusion of the activator, and prepare plasma with few platelets for blood samples. The amount of endogenous Factor Vlla can then be measured in the samples of citrated plasma by performing a Factor Vlla coagulation assay based on Tissue Factor. The desired levels of endogenous Factor Vlla would be the same as the target levels of Factor Vlla in plasma indicated for the infusion of purified Factor VII and the deficient Coagulation Factor Factor. Therefore, other Factor VII activators could be tested in vivo for the generation of Vlla Factor with proper experimentation and to adjust the dose to generate the desired levels of Vlla Factor using the deficient coagulation tissue factor assay based on Factor Vlla. of the plasma samples. The appropriate dose of the Factor VII activator (which produces the desired level of the endogenous Vlla Factor) can then be used in combination with the recommended amounts of deficient coagulation tissue factor. Doses can be timed to provide prolonged elevation in Factor Vlla levels. Doses would preferably be administered until the desired antitumor effect is achieved, and then repeated as necessary to control bleeding. The half-life of the Factor Vlla in vivo has been reported to be approximately two hours, although this could vary with different therapeutic modalities and individual patients. Therefore, the half-life of the Vlla Factor in the plasma in a given treatment modality should be determined with the coagulation assay based on deficient coagulation tissue factor. H. Examples The following examples are included to demonstrate the preferred embodiments of the invention. It should be appreciated by those skilled in the art that the techniques described in the examples that follow represent techniques discovered by the inventor that function well in the practice of the invention, and thus can be considered to constitute preferred modes for their practice. However, experienced technicians should, in light of the present disclosure, appreciate that many changes can be made in the specific embodiments described and still obtain a similar or similar result without departing from the spirit and scope of the invention. EXAMPLE 1 SYNTHESIS OF TRUNCATED TISSUE FACTOR The truncated Tissue Factor herein is designated as the extracellular domain of the mature Tissue Factor protein (amino acids 1-219 of the mature protein, - SEQ ID N0: 1). Identification Sequence No. 1 is coded by, e.g., 'Identification Sequence No. 10. A. H6 [tTF] H6 Ala Met Ala [tTF]. The complementary DNA of Truncated Tissue Factor (cDNA) was prepared as follows: RNA from J-82 cells (from human bladder carcinoma) was used for the cloning of truncated Tissue Factor. Total RNA was isolated using the GlassMax ™ RNA micro-isolation reagent (Gibco BRL). The RNA was reverse transcribed into cDNA using the GeneAmp RNA polymerase chain reaction set (Perkin Elmer). The cDNA of the truncated Tissue Factor was amplified using the same set with the following two primers: 5 'primer: 5' GTC ATG CCA TGG CCT CAG GCA CTA CAA (SEQ ID NO: 15) 3 'primer: 5' TGA CA CA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 16) The underlined sequences encode the N term of the truncated Tissue Factor. The remainder of the sequence in the 5 'primer is the restriction site for Ncol allowing cloning of truncated Tissue Factor in the expression vector. The sequence in the 3 'primer is the HindIII site for cloning the truncated Tissue Factor in the expression vector. Amplification was performed by polymerase chain reaction as suggested by the manufacturer. In brief, 75 μM dNTP was used; 0.6 μM primer, 1.5 mM MgCl2 and 3.0 cycles of 30 seconds at 95QC, 30 seconds at 55SC and 30 seconds at 729C were performed. The truncated Tissue Factor was expressed as a fusion protein in an unnatural state in E. coli inclusion bodies using the H6 expression vector pQE-60 (Qiagen). The expression vector of E. cóli H6 pQE-60 was used for the expression of the truncated tissue factor (Lee et al., 1994). The cDNA of the truncated Tissue Factor amplified by polymerase chain reaction was inserted between the Ncol and HindIII site. The H6 pQE-60 has an integrated coding sequence (His) 6 so that the expressed protein has the sequence of (His) 6 at the N-terminus, which can be purified on a Ni-NTA column. In addition, the fusion protein has a thrombin dissociation site and residues 1-219 of the Tissue Factor. To purify the truncated Tissue Factor, the truncated Tissue Factor containing H6 DNA pQE-60 was transformed into E. coli TG-1 cells. Cells were cultured at OD600 = 0.5 and IPTG was added at 30 μM to induce the production of Tea Factor truncated. Cells were harvested after shaking for 18 hours at 30SC. The cell side was denatured in 6 M Gu-HCl and the lysate was loaded onto a Ni-NTA column (Qiagen). The bound truncated Tissue Factor was washed with 6 M urea and the truncated Tissue Factor was doubled with a gradient of 6 M-1 M urea at room temperature for 16 hours. The column was washed with washing buffer (0.05 Na H2 P04, 0.3 M NaCl, 10% glycerol) and the truncated Tissue Factor was eluted with 0.2 M Imidozole in wash buffer. Eluted truncated tissue factor was concentrated and loaded onto a G-75 column. Truncated Tissue Factor monomers were collected. B. Truncated Tissue Factor Gly [tTF]. The complementary DNA of GlytTF (cDNA) was prepared in the same manner as described in the previous section except that the 5 'primer was replaced by the next primer in the polymerase chain reaction. 5 »primer: 5 'GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT CTG GCA CTA CAA ATA CT (SEQ ID NO: 17) The underlined sequence encodes the N term of the truncated Tissue Factor. The remaining sequence encodes a restriction site for Ncol and a dissociation site for thrombin.

The H6 expression vector pQE60 and the method for protein purification is identical to that described above except that the final protein product was treated with thrombin to remove the H6 peptide. This was done by adding a portion of thrombin (Sigma) to 500 parts of truncated tissue factor (weight / weight), and dissociation was carried out at room temperature for 18 hours. Thrombin was removed from the truncated Tissue Factor by passage of the mixture through a thrombin affinity column Benzamidine Sepharose 6B (Pharmacia). The resulting truncated tissue factor, designated tTF219, consisted of residues 1 to 219 of the Tissue Factor plus one additional glycine in the N-terminus. This migrated as a single 26 KDa molecular weight band when analyzed by SDS-PAGE, and the N-terminal sequence was confirmed by degradation of Edman Had the Sequence of identification sequence No. 1.

C. Truncated tissue factors modified by cysteine (His) 6-N'-cys 'tTF219-tTF, abbreviated hereafter as H6-N' -cys-tTF219, was prepared by mutating tTF219 by polymerase chain reaction with a 5 * primer encoding a Cys in front of the term N1 of mature truncated tissue factor. H6-tTF-, 19-cys-C 'was also prepared using a primer 31 encoding a Cys after amino acid 219 of the truncated Tissue Factor. Expression and purification were as for tTF219, except that the Ellman reagent (5 '5'-dithio-bis-2-nitrobenzoic acid) was applied after redoubling it to convert the Cys N' or C-terminal into a stable activated disulfide group. The products had the sequences shown in Identification Sequence No. 2 and Identification sequence No. 3. The thrombin dissociation removed the tag (His) 6 and converted the proteins into N'-cys-tTF219 and tTF219-cys-C having the sequences shown in Identification sequence No. 4 and Identification sequence No. 5. The products were > 95% pure judged by SDS-polyacrylamide gel electrophoresis. H6-tTF220-cys-C 'and H6-tTF221-cys-C' were prepared by mutating tTF219 by polymerase chain reaction with 3 'primers encoding Ile-Cys and Ile-Phe-Cys after amino acid 219 of the truncated Tissue Factor . The expression, beating and purification were as for H6-tTF219-cys-C. The proteins have the sequences shown in Identification Sequence No. 6 and Identification Sequence No. 7 EXAMPLE II SYNTHESIS OF THE DIMERIC TISSUE FACTOR The inventors reasoned that the Tissue Factor dimers may be more potent than the monomers for initiating coagulation. . It is possible that the natural Tissue Factor on the surface of J82 bladder carcinoma cells may exist as a dimer (Fair et al., 1987). The binding of a Factor VII or Factor Vlla molecule to a Tissue Factor molecule can also facilitate the binding of another Factor VII or another Factor Vlla to another Tissue Factor (Fair and collaborators, 1987, Bach et al., 1986). In addition, the Tissue Factor shows structural homology to the members of the cytokine receptor family (Edgington et al., 1991) some of which are dimerized to form active receptors (Davies and Wlodawer, 1995). The inventors therefore synthesized Tissue Factor dimers, as follows. Although the synthesis of the dimers hereinafter are described in terms of chemical conjugation, the inventors also contemplated the recombinants and other means for producing the dimers of the present invention. A. [tTF] Linker [tTF] The Gly [tTF] Linker [tTF] was made with the structure Gly [tTF] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Ser [tTF]. Two pieces of DNA were amplified with polymerase chain reaction separately and ligated and inserted into the vector as follows: Polymerase chain reaction 1: Preparation of truncated tissue factor and the 5 'half of the linker DNA. The primer sequences in the polymerase chain reaction were as follows: 5 'primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ ID NO: 18) 3 'primer: 51 CGC GGA TCC ACC GCC ACC AGA TCC ACC GCC TCC TTC TCT GAA TTC CCC TTT CT (SEQ ID NO: 19) The Gly DNA [tTF] was used as the DNA standard. Other conditions of the polymerase chain reaction were as described in the section on the truncated tissue factor. Polymerase chain reaction 2: The preparation of the 3 'half of the linker DNA and the truncated Tissue Factor DNA. The primer sequences in the. Polymerase chain reaction were as follows: 5 'primer: 5' CGC GGA TCC GGC GGT GGA GGC TCT TCA GGC ACT ACA AAT ACT GT (SEQ ID NO: 20) 3 'primer: 5' TGA CA CA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 21) Truncated Tissue Factor DNA was used as the pattern in the polymerase chain reaction. The product of the polymerase chain reaction 1 was digested with Ncol and BamH. The polymerase chain reaction product 2 was digested with HindIII and BamHI. The DNA of the polymerase chain reaction 1 and the digested polymerase chain reaction 2 was ligated with DNA H6 pQE 60 digested by Ncol and HindIII. For the vector constructs and the protein purification the procedures were the same as described in section Gly [tTF] B. Cys [tTF] Linker [tTF] The Cys [tTF] Linker [tTF] was also constructed with the structure Ser Gly Cys [tTF 2-219] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Ser [tTF] was made DNA by polymerase chain reaction using the following primers: 5 'primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ ID NO: 22) 3 'primer: 5' TGA CA CA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 23) DNA was used [tTF ] linker [tTF] as the pattern. The remaining polymerase chain reaction conditions were the same as described in the section on Truncated Tissue Factor. The vector constructs and the protein purification were all described in the purification of H6 C [tTF]. C. [tTF] linker [tTF] cys We also made the dimer [tTF] linker [tTF] cys with the protein structure [tTF] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Ser [tTF] Cys . DNA was made by polymerase chain reaction using the following primers: 5 'primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT GCA CTA CAA ATA CT (SEQ ID NO: 24) 3 'primer: 5' TGA CAÁ GCT TAG CAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 25) The [tTF] linker [tTF] DNA was used as the standard. The remaining conditions of the polymerase chain reaction were the same as described in the section on Truncated Tissue Factor. The vector constructs and the protein purification were performed again as described in the purification section of [tTF] cys. D. Chemically conjugated dimers Monomer [tTF] Cys was reduced, which had been treated with Ellman's reagent to convert free Cys into activated disulfide group, with half a molar equivalent of dithiothreitol. This generated free Cys residues in half of the molecules. The monomers were chemically conjugated to form [tTF] Cys-Cys [tTF] dimers. This was done by adding a molar amount equal to the DTT for the protected [tTF] Cys at room temperature for one hour to deprotect and expose the cysteine at the C-terminus of [tTF] Cys. An equal molar amount of protected [tTF] Cys was added to the DTT / [tTF] Cys mixture and incubation continued for 18 hours at room temperature. The dimers were purified on a G-75 gel filtration column. H6-tTF220-cys-C and H6-tTF221-cys-C and H6-N'-cys-tTF219 dimers were also prepared. The Cys monomer [tTF] was chemically conjugated to form dimers using the same method. EXAMPLE III SYNTHESIS OF TISSUE FACTOR MUTANTS Three truncated Tissue Factor mutants are described that lack the ability to convert the Factor VII in Factor Vlla linked with truncated tissue factor. There are 300 times less Factor Vlla in the plasma compared to Factor VII (Morrissey et al., 1993). Therefore, the Circulating mutant truncated tissue factor should be less able to initiate coagulation and therefore exhibit very little toxicity. However, as soon as the truncated Tissue Factor has been localized to the tumor site, as surprisingly demonstrated herein, the Vlla Factor can be injected to interchange with the Factor VII bound to the Factor of Tumor.

Truncated fabric. The mutated proteins have the sequences shown in Identification Sequence No. 8 and Identification Sequence No. 9 and are active in the presence of Factor Vlla. A. [tTF] G164A The "[tTF] G164A" has the mutant protein structure with amino acid 164 (Gly) of tTF219 replaced by Ala. The Chameleon double-stranded site mutagenesis game (Stratagene) was used to generate the mutant. The DNA standard is Gly DNA [tTF] and the sequence of the mutagenizing primer is: 5 'CAA GTT CAG CCA AGA AAAC (SEQ ID NO: 26) The mutant G164A is represented by Sequence Identification No. 9. The vector constructions and the protein purification methods described above were used in the purification of Gly [tTF]. B. [tTF] 158R The tryptophan at amino acid 158 of tTF219 was mutated into arginine by polymerase chain reaction with a primer encoding this change. The expression, beating and purification was as for tTF219. The mutated protein has the sequences shown in Identification Sequence No. 8 C. [tTF] W158R S162A [tTF] W158R S162A is a double mutant in which amino acid 158 (Trp) of tTF219 is replaced by Arg and amino acid 162 (Ser) is replaced by Ala. The same method of mutagenization as described for [tTF] G164A and [tTF] W158R is used. The mutagenization primer is: 5 'ACÁ CTT TAT TAT CGG AAA TCT TCA GCT TCA GGA AAG (SEQ ID NO: 27) The above vector constructions and protein purification are the same as those used to purify Gly [tTF]. EXAMPLE IV PREPARATION OF TTY BISPECIFIC ANTIBODY ADUTS AND SYNTHESIS OF TISSUE FACTOR CONJUGATES A. Preparation of bispecific antibody adducts for tTF. Bispecific antibodies were constructed that had a Fab 'arm of the 10H10 antibody that is specific for a non-inhibitory epitope of truncated Tissue Factor linked to a Fab' arm of antibodies (0X7, Mac51, CAMPATH-2) of irrelevant specificity. When mixed with truncated Tissue Factor, the bispecific antibody binds to the truncated Tissue Factor via the 10H10 arm, forming a non-covalent adduct. Bispecific antibodies were synthesized according to the method of Brennan et al. (1985, incorporated herein by reference) with minor modifications. Briefly, fragments of F (ab ') 2 were obtained from IgG antibodies by digestion with pepsin (type A, EC 3.4.23.1) and purified to homogeneity by chromatography on Sephadex G100. F (ab ') 2 fragments were reduced for 16 hours at 20 ° C with 5 M 2-mercaptoethanol in 0.1 M sodium phosphate buffer, pH 6.8, containing 1 mM EDTA (PBSE regulator) and 9 mM NaAs02. The Ellman reagent (ER) was added to give a final concentration of 25 mM and, after 3 hours at 205 ° C, Ellman-derived Fab 'fragments (Fab'-ER) were separated from Ellman reagents on Sephadex columns. G25 in PBSE. To form the bispecific antibody, Fab'-ER derived from an antibody was concentrated at approximately 2.5 mg / ml in an Amicon ultrafiltration cell and reduced with 10 mM 2-mercaptoethanol for one hour at 20 ° C. The resulting Fab '-SH was filtered through a Sephadex G25 column in PBSE and mixed with a 1: 1 molar excess of Fab'-ER prepared from the second antibody. The mixtures were concentrated by ultrafiltration at about 3 mg / ml and stirred for 16 hours at 20 ° C. The products of the reaction were fractionated on Sephadex G100 columns in phosphate buffered saline. Fractions containing the bispecific antibody (110 kDa) were concentrated at 1 mg / ml, and stored at 4SC in 0.02% sodium azide. To form the adducts of tTF bispecific antibodies, the bispecific antibody was mixed with one molar equivalent of tTF or derivatives thereof for 1 hour at 49C. The eluted adduct with a molecular weight of approximately 130 kDa in gel filtration columns, corresponding to a bispecific antibody molecule was linked to a tTF molecule. l. Preparation of IgG-H6-N'-cys-tTF219 and lgG-H6-tTF219-cys-C To 26 mg of immunoglobulin G at a concentration of 10 mg / ml in phosphate-buffered saline solution with nitrogen were added 250 micrograms of SMPT (Pharmacia) in 0.1 ml of dry DMF. After stirring for 30 minutes at room temperature, the solution was applied to a column (1.6 cm in diameter X 30 cm) of Sephadex G25 (F) equilibrated in the same regulator. The derived immunoglobulin G was collected in a volume of 10 to 12 ml and concentrated to approximately 3.5 ml by ultrafiltration (Amicon, membrane YM2). The H6-N '-cys-tTF219 or H6-tTF219-cys-C (15 mg) was reduced by incubation at room temperature in the presence of 0.2 mM DTT until the Ellman substance was released (ie OD at 412 nm reached a maximum). It was then applied to the Sephadex G25 column (F) (1.6 cm in diameter x 30 cm) equilibrated with a nitrogen-washed regulator. Cys-tTF (-15ml) was added directly to the derivatized IgG solution. The mixture was concentrated to approximately 5 ml by ultrafiltration and incubated at room temperature for 18 hours before resolution by gel filtration chromatography on Sephacryl S200. The peak containing the material that had a molecular weight of 175,000-200,000 was collected. This component consisted of an IgG molecule linked to one or two molecules of tTF. The conjugates have the structure: CH-, 2. Preparation of Fab »-H6-N'-cys-tTF219 Fab 'fragments were produced by reducing F (ab') 2 fragments of IgG with 10 mM mercaptoethylamine. The resulting Fab 'fragments were separated from the reducing substance by gel filtration on Sephadex G25. The freshly reduced Fab 'fragment and the modified H6-N'-cys-tTF219 from Ellman were mixed in equimolar amounts at a concentration of 20 μM. The progress of the coupling reaction was followed by the increase in absorbance at 412 nm due to the 3-carboxylate-4-nitrothiophenolate anion released as a result of conjugation. The conjugate had the structure: Fab'-SS-tTF B. Synthesis of Tissue Factor Conjugates 1. Chemical Derivation and Antibody Conjugation The tTF antibody conjugates were synthesized by binding the antibody chemically derived to the chemically derived truncated tissue factor via a disulfide bond. The antibody was reacted with a 5-fold molar excess of succinimidyl oxycarbonyl-α-methyl-α- (2-pyridyldithio) toluene (SMPT) for one hour at room temperature to produce an antibody derived with an average of 2 pyridyl disulfide groups per antibody molecule. The derived antibody was purified by gel permeation chromatography. A 2.5-fold molar excess of truncated Tissue Factor was reacted with a 45-fold molar excess of 2-iminothiolane (2IT) for one hour at room temperature to produce truncated Tissue Factor with an average of 1.5 sulfhydryl groups per molecule of Truncated Fabric Factor. The derived truncated tissue factor was also purified by gel permeation chromatography and immediately mixed with the derived antibody. The mixture was allowed to react for 72 hours at room temperature and then a Sephacryl S-300 column was applied to remove the conjugate from the antibody-Truncated Tissue Factor from the free truncated Tissue Factor and released pyridine-2-thione. The conjugate was separated from the free antibody by affinity chromatography on a column of anti-truncated tissue factor. The predominant molecular species of the final conjugate was the conjugate of only substituted antibody-truncated Tissue Factor (Mr about 176,000) with smaller amounts of multiply substituted conjugates (Mr> about 202,000) titrated by SDS-PAGE. 2. Conjugation of Truncated Tissue Factor Modified by Cysteine to Derived Antibody C-antibody conjugates [tTF] and [tTF] C were synthesized by direct coupling of truncated Tissue Factor modified with cysteine to chemically derived antibody via a disulfide bond. The antibody was reacted with a 12-fold molar excess of 2IT for one hour at room temperature to produce antibody derived with an average of 1.5 sulfhydryl groups per antibody molecule. The derivatized antibody was purified by gel permeation chromatography and immediately mixed with a 2-fold molar excess of cysteine-modified truncated tissue factor. The mixture was allowed to react for 24 hours at room temperature and then the conjugate was purified by gel-permeation and affinity chromatography as described above. The predominant molecular species of the final conjugate • was the only substituted conjugate (Mr about 176,000) with lower amounts of multiple substituted conjugates (Mr> about 202,000) assessed by SDS-PAGE. 3. Conjugation of truncated tissue factor modified with cysteine to Fab 'fragments Fab'-C [tTF] and [tTF] C antibody conjugates were prepared. These conjugates may be more potent in vivo because they must remain on the cell surface for longer than the bivalent conjugates due to their limited internalization capacity. The Fab 'fragments are mixed with a 2-fold molar excess of cysteine-modified truncated tissue factor for 24 hours and then the conjugate is purified by gel permeation and affinity chromatography as described above. EXAMPLE V TUMOR INFARCTION THROUGH THE TISSUE FACTOR A. Methods 1. In Vitro Coagulation Assay This test was used to verify that the truncated Tissue Factor, several derivatives and mutants thereof, and immunoglobulin-tTF conjugates acquire activity that induces the coagulation as soon as it is located on a cell surface. A20 lymphoma cells (positive I-Ad) (2 X 10 cells / ml, 50 μl) were incubated for one hour at room temperature with a bispecific antibody (50 micrograms / ml, 25 microliters) consisting of a Fab 'arm of the B21-2 antibody directed against I-Ad bound to a Fab 'arm of the 10H10 antibody directed against a non-inhibitory epitope on truncated tissue factor. The cells were washed at room temperature and variable concentrations of truncated Tissue Factor, derivatives or mutants thereof, or immunoglobulin-Truncated Tissue Factor conjugates were added for one hour at room temperature. The bispecific antibody captures truncated Tissue Factor or truncated Tissue Factor bound to immunoglobulin, bringing it into close approximation with the cell surface, where coagulation can proceed. The cells were washed again at room temperature, resuspended in 75 microliters of phosphate buffered saline and heated to 37 ° C. Calcium (12.5 M) and citrated human or mouse plasma (30 microliters) were added. Time was recorded for the first fibrin strands to form. The coagulation time against the truncated tissue factor concentration was plotted and the curves were compared with standard curves prepared using standard tTF219 preparations. In some studies, varying concentrations of recombinant human Factor Vlla were added together with tTF219 and mutants thereof, to determine if the coagulation capacity was increased by the presence of Factor Vlla. 2. Factor Xa Production Assays This assay is useful in addition to or as an alternative to the in vitro coagulation assay to demonstrate that the truncated Tissue Factor and the immunoglobulin-tTF conjugates acquire activity that induces coagulation as soon as they are localized. a cell surface. The assay measures the conversion rate of Factor X into Factor Xa by a substrate that generates chromophore (S-2765) for Factor Xa. A20 cells (2 X 10 7 cells) were suspended in 10 milliliters of medium containing 0.2 weight / volume sodium azide. To 2.5 ml of cell suspension were added 6.8 micrograms of the "capture" bispecific antibody B21-2 / 10H10 for 50 minutes at room temperature. The cells were washed and resuspended in 2.5 ml of medium containing 0.2 weight percent / volume of sodium azide. Truncated Tissue Factor and immunoglobulin-tTF conjugates dissolved in the same medium were distributed in 100 microliter volumes at a range of concentrations in 96-well microtiter plates. 100 microliters of the bispecific antibody / cell suspension was then added to the wells. The plates were incubated for 50 minutes at room temperature. The plates were centrifuged, the supernatants were discarded and the cell pellets were resuspended in 250 microliters of washing buffer (150 mM NaCl, 59 mM Tris-HCl, pH 8, 0.2 weight / volume percent of bovine serum albumin) . The cells were washed again and the cells were resuspended in 100 microliters of a 12.5 fold dilution of Proplex T (Baxter, Inc.) containing factors II, VII, IX and X in dilution buffer (Supplemental washing regulator with 12.5 mM calcium chloride). The plates were incubated at 37 aC for 30 minutes. To each well was added detention solution (12.5 mM of ethylenediamine-tetraacetic acid (EDTA) of sodium) in washing buffer. The plates were centrifuged. One hundred microliters of supernatant from each well was added to 11 microliters of S-2765 (N-cv-benzyloxycarbonyl-D-Arg-L-Gly-L-Arg-p-nitroanilide dihydrochloride, Chromogenix AB, Sweden). The optical density of each solution was measured at 409 nm. The results were compared with standard curves generated from standard tTF2i9. 3. Tumor thrombosis in vivo This model was used to demonstrate that truncated Tissue Factor and immunoglobulin-tTF conjugates induced thrombosis of the tumor blood vessels and caused tumor infarction in vivo. The tumor test systems were of four types: (i) 3LL mouse lung carcinoma growing subcutaneously in C57BL / 6 mice; (ii) C1300 mouse neuroblastoma growing subcutaneously in BALB / c nu / nu mice; (iii) HT29 human colorectal carcinoma growing subcutaneously in BALB / c mice nu / nu, - and (iv) mouse neuroblastoma C1300 Mu? growing subcutaneously in BALB / c nu / nu mice. The tumor C1300 Mu? is a transfectant that secretes interferon-? derived from the C1300 tumor (Watanabe et al., 1989). In addition, the tumor model C1300 (Mu?) From (Burrows et al., 1992; incorporated herein by reference) was used and modified as follows: (i) the B21-2 antibody was used to target IA "; ii) C1300 tumor cells (Mu?), a subline of C1300 tumor cells (Mu?) 12, growing continuously in BALB / c nu / nu mice, and (iii) tetracycline was omitted from drinking water of the mice to prevent the bacteria of the intestines from inducing I-Ad in the gastrointestinal epithelium.Unlike the immunotoxins, the coaguligands and the tissue factor constructions do not damage the intestinal epithelium that expresses I-Aú. Tumor To establish tumors, 10 to 1.5 x 10 tumor cells were injected at 20 on the right anterior flank of the mice.When tumors grew to different sizes, the mice were randomly assigned to different study groups. an injection intravenous 0.5 mg / kg Truncated Tissue Factor alone or bound with IgG, Fab ', or bispecific antibody. Other mice received equivalent amounts of IgG, Fab 'or only bispecific antibody. Injections were performed slowly in the tail veins for approximately 45 seconds, usually followed by 200 microliters of saline. In some studies, the effect of administering chemotherapeutic drugs against cancer on the thrombotic action of truncated tissue factor on tumor blood vessels was investigated. Mice bearing HT29 human-colored subcutaneous tumors of 1.0 cm in diameter were given intraperitoneal injections of doxorubicin (1 mg / kg / day), camptothecin (1 mg / kg / day), etoposide (20 mg / kg / day) or interferon-? (2 x 105 units / kg / day) for two days before the injection of truncated tissue factor and again on the day of injection of truncated tissue factor. Twenty-four hours after being injected with truncated Tissue Factor or immunoglobulin-Truncated Tissue Factor conjugates, the mice were anesthetized with methane and exsanguinated by perfusion with heparinized saline. Tumors and normal tissues were cut and immediately fixed at 3 percent (volume / volume) formalin. Paraffin sections were cut and stained with hematoxylin and eosin. Blood vessels that had open lumens containing erythrocytes and blood vessels containing thrombi were counted. The paraffin sections were cut and stained with hematoxylin and eosin or with Martius Scarlet Blue (MSB) trichrome for the detection of fibrin. 5. Antitumor effects Accepted animal models were used to determine whether administration of truncated tissue factor from immunoglobulin-truncated tissue factor conjugates suppressed the growth of solid tumors in mice. The tumor test systems were: (i) L540 human Hodgkin disease tumors growing in SCID mice; (ii) neuroblastoma C1300 Mu? (which secretes interferon) growing in nu / nu mice, - (iii) H460 human non-small cell lung carcinoma growing in nu / nu mice. To establish solid tumors, 1.5 × 10 tumor cells were injected subcutaneously into the right anterior flank of SCID or BALB / c nu / nu mice (Charles et al.

River Labs., Wilmingham, MA). When the tumors grew at different diameters, the mice were assigned to different experimental groups, each containing from 4 to 9 mice. The mice then received an intravenous injection of 0.5 mg / kg truncated Tissue Factor alone or bound with bispecific antibody. Other mice received equivalent amounts of bispecific antibody only.

The injections were carried out for approximately 45 seconds in one of the tail veins, followed by 200 microliters of saline. The infusions were repeated six days later. The perpendicular diameters of the tumors were measured at regular intervals and the volumes of the tumors were calculated. B. Results 1. In vitro Coagulation by Truncated Tissue Factor and Variants To direct truncated Tissue Factor to I-Ad in tumor vascular endothelium, the inventors prepared a Bieepecific antibody with Fab 'arm of antibody B21-2, specific for IA, linked to the Fab 'arm of the 10H10 antibody, specific for a non-inhibitory epitope in module C of the truncated tissue factor. This bispecific antibody, B21-2 / 10H10, mediated truncated Tissue Factor binding in a manner specific for antigen to I -A "in B lymphoma cells of A20 in vi tro mice When mouse plasma was added to cells A20 at which the truncated Tissue Factor had been bound by B21-2 / 10H10, coagulated rapidly.Fibrin strands were visible 36 seconds after the addition of plasma to the antibody treated cells, compared to 164 seconds when plasma was added to untreated cells (Figure 4A) Only when the truncated Tissue Factor was bound to the cells did this increase the coagulation observed: no effect was seen on the coagulation time when the cells were incubated with Tissue Factor truncated only, with F (ab ') 2, homodimeric, with Fab' fragments, or with truncated Tissue Factor plus bispecific antibodies that only had one or two specificities needed to bind the Tissue Factor truncated to A20 cells. The tTf2j9 prepared as in Example 1 had identical capacity to a "standard" tTF2i9 preparation obtained from Dr. Thomas Edgington (The Scripps Research Institute, La Jolla, CA) to induce the coagulation of mouse or human plasma after its binding via bispecific antibody B21-2 / 10H10 to A20 lymphoma cells (Figure 5).

The mouse plasma was coagulated in 50 seconds when both the tTF219 preparation of Example I and the standard truncated Tissue Factor were applied to the cells at 3 X 10 M. Thus, the tTF21g prepared as described herein appears be correctly redoubled and fully active. There was a linear relationship between the logarithm of the number of truncated Tissue Factor molecules bound to the cells and the rate of plasma coagulation by the cell (Figure 4B). In the presence of the cells alone, the plasma coagulated in 190 seconds, while at 300,000 molecules of Tissue Factor truncated per cell the clotting time was 40 seconds. Even with only 20,000 molecules per cell, coagulation was faster (140 seconds) than with untreated cells. These in vitro studies showed that the thrombogenic potency of truncated tissue factor is increased by the proximity of the cell surface mediated through antibody-directed binding to Class II antigens on the cell surface. H ^ -N '-cys-tTF219 and H6-tTF219-cys-C' were as active as the truncated Tissue Factor in inducing plasma coagulation as soon as they were linked via the bispecific antibody to A20 cells. The plasma coagulated in 50 seconds when H6-N '-cys-tTF2? 9 and H6-tTF-219-cys-C' were applied at 3 x 10"9 M, the same concentration as for the truncated tissue factor ( Figure 5) Thus, the mutation of the truncated Tissue Factor to induce a sequence (His) 6 and a Cys residue at the N 'or C terminus does not reduce its coagulation-inducing activity H6-tTF220-cys-C, tTF220-cys-C, H6-tTF221-cys-C and tTF221-cys-C were as active as tTF219 in inducing plasma coagulation as soon as they were located on the surface of A20 cells via the bispecific antibody, B21-2 / 10H10. With all the samples at 5 x 10"10 M, the plasma coagulated in 50 seconds (Figure 6 and Figure 7) 2. In vitro coagulation by truncated tissue factor dimers The dimer H6-N'cys-tTF219 was so active as the same tTF219 in inducing plasma coagulation as soon as it was located on the surface of A20 cells by the bispecific antibody, B21-2 / 10H10 At a concentration of 1-2 x 10"10 M, both samples induced coagulation in 50 seconds (Figure 8), however, the dimer H6-tTF221-cys-C was 4 times less active than the monomer H6-tTF221-cys-C of tTF219 itself. At a concentration of 4 x 10"9 M, the dimer H6-tTF221-cys-C induced plasma coagulation in 50 seconds, whereas the corresponding monomer needed to be applied at 1 x 10" 9 M for the same effect in the coagulation. 3. Tumor thrombosis in vivo A histological study was performed to determine whether intravenous administration of coaguligand B2l-2 / l0H10-tTF induced selective thrombosis of the tumor vasculature in mice having subcutaneous neuroblastomas C1300 (Mu?) From 0.8 to 1.0 cm in diameter (Figure 9). Within 30 minutes, all vessels throughout the tumor were thrombosed, containing aggregates of occlusive platelets, packed erythrocytes, and fibrin. At this time, the tumor cells were histologically indistinguishable from the tumor cells of untreated mice. After four hours, however, there were signs of tumor cell damage. Most of the tumor cells separated from each other and had pyknotic nuclei, and the tumor interstitium commonly contained erythrocytes. Around 24 hours, the tumor showed advanced necrosis, and at 72 hours, the entire central region of the tumor had condensed into an amorphous residue. These studies indicated that the predominantly occlusive effect of the coaguligand of B21-2 / 10H10-tTF on the tumor vessels is mediated through the binding of Class II antigens on the vascular endothelium of the tumor. Surprisingly it was observed that there was a non-specific thrombotic action of the truncated Tissue Factor discernible in the tumor vessels at later times: in tumors of mice that had been injected 24 hours previously with truncated Tissue Factor only or truncated Tissue Factor mixed with the Tumor Factor. control bispecific antibody, OX7 / 10H10, the tumors assumed a blackened, bruised appearance beginning in 30 minutes and being progressively more marked until 24 hours. A histological study revealed that 24 hours after the injection of tTF2j practically all the vessels in all regions of the tumor were thrombosed (Figure 9). The vessels contained aggregates of platelets, packed red cells and fibrin. Most of the tumor cells had separated from each other and developed pyknotic nuclei and many regions of the tumors were necrotic. This was more pronounced in the tumor nucleus. Erythrocytes were commonly observed in the tumor interstitium. It is possible that the resident thrombogenic activity of the tumor vasculature (Zacharski, et al., 1993) makes these veins more susceptible to thrombosis even by non-directed truncated tissue factor. Alternatively, increased procoagulant changes could have been induced by interferon-? derived from the tumor. Similar results were obtained when tTF219 was administered to mice that had large C1300 tumors (greater than 1000 mm3). Again, virtually all vessels were thrombosed 24 hours after the injection (Figure 10). In this way, the effects observed in tumors C1300 Mu? no - were related to the secretion of interferon? by the tumor cells. Other studies were performed on C57BL / 6 mice that had large 3LL tumors (greater than 800 mm3). Again, thrombosis of the tumor vessels was observed, although somewhat less pronounced than with the C1300 tumor and the C1300 Mu tumor. On average 62 percent of the 3LL tumor vessels were thrombosed (Figure 11). The vessels in tumors C1300 and C1300 Mu? small (less than 500 mitr) were largely unaffected by the administration of tTF2 | 9. Thus, as tumors grow, their susceptibility to thrombosis by tTF219 increases. This is possibly because the 'cytokines released by the tumor cells or by the host cells that infiltrate the tumor activate the vascular endothelium of the tumor, inducing procoagulant changes in the vessels. The coaguligand treatment was well tolerated, the mice did not lose weight and retained normal appearance and normal activity levels. At the treatment dose of 0.6 mg / kg B21-2 / 10H10 plus 0.5 mg / kg Truncated Tissue Factor, toxicity was observed in only two out of forty mice (tail vein thrombosis). It is important to note that neither thrombi nor histological or morphological abnormalities were visible in paraffin sections of liver, kidney, lung, intestine, heart, brain, adrenal, pancreas, or spleen of mice that had tumors 30 minutes or 24 hours after the administration of coaguligand or free truncated tissue factor. In addition, no signs of toxicity (behavioral changes, physical signs, weight changes) were observed in the treated animals. 4. Antitumor Effects The inventors then investigated whether intravenous administration of the coaguligand B21-2 / 10H10-tTF could inhibit the growth of large tumors (0.8 to 1.0 cm in diameter) in mice. The combined results of three separate studies indicate that mice that received coaguligando B21-2 / 10H10-tTF had complete tumor regressions that lasted four months or longer. These antitumor effects were significantly greater than for other treatment groups (Figure 12A). Surprisingly, the inventors found that the antitumor effect of cuaguligand B21-2 / 10H10-tTF was attributable, in part, to an unmanaged effect of the truncated tissue factor. Tumors in mice that received truncated Tissue Factor alone or mixed with control bispecific antibodies (CAMPATH II / 10H10 or B21-2 / OX7) grew significantly more slowly than tumors in mice that received antibodies or only saline (Figure 12A; Figure 12B). Mice that had C1300 mu tumors? small (300 mm3) were injected intravenously with 16-20 micrograms of tTF219. The treatment was repeated a week later. The first treatment with tTF219 had a slight inhibitory effect on tumor growth, consistent with the lack of marked thrombosis observed with small tumors previously (Figure 12B). The second treatment had a substantially greater, statistically significant effect (P less than 0.01), probably because the tumors had increased size. One week after the second treatment with tTF219, the tumors were 60 percent the size of the tumors in the mice that received only diluent. The greater effectiveness of the second injection probably derives from the greater thrombotic action of tTF219 on the vessels in large tumors, observed previously. Similar antitumor effects were observed in mice that had human lung carcinomas H460 (Figure 13). The first treatment with tTF219 occurred when the tumors were small (250 mm3) and had little effect on the growth rate. The second treatment with tTF219 occurred when the tumors were older (900 mm3) and caused the tumors to return to 550 mm3 before regrowing. Antitumor effects were also observed in mice that had HT29 human colorectal carcinomas (Figure 14). Nu / nu mice that had large tumors (1200 mm-) on their flanks were injected intravenously with tTF2l9 or phosphate buffered saline (control), and growth of tumors was monitored every day for ten days. Tumors in mice treated with tTF2j discontinued growth for approximately 7 days after treatment, while tumors in mice treated with phosphate buffered solution continued to grow unimpeded. In animals that did not show complete regression of the tumor after coaguligand treatment B21-2 / 10H10-tTF, the tumors grew again from a surviving microscopic ring of cells at the periphery of the tumor. Immunohistochemical examination of these tumors revealed that the vascular endothelium in the invading border of the tumors lacked detectable Class II antigens consistent with a lack of thrombosis of these vessels allowing coaguligating the cellular survival of the local tumor. Thus, coadministration of a drug acting on the same tumor cells would probably improve efficacy, as has been observed with other anti-sparing therapy (Burrows and Thorpe, 1992; Burrows and Thorpe 1993; Burrows and Thorpe 1994; Patent Application of the United States of America numbers 07 / 846,349; 08 / 205,330; 08 / 295,868; and 08 / 350,212). The inventors previously demonstrated that a powerful cytotoxic ricin A chain immunotoxin directed against the same tumor cells virtually lacked antitumor activity when administered to mice with large tumors C1300 (Mu?) (Burrows and Thorpe, 1993; United States of America numbers 07 / 846,349; 08 / 205,330; 08 / 295,868; and 08 / 350,212). The lack of activity was due to the inability of the immunotoxin to have access to tumor cells in large tumor masses, serving as a witness to the comparative effectiveness of coaguligand therapy. Studies using coaguligands confirmed the potential therapeutic of the selective initiation of the blood coagulation cascade in the tumor vasculature (U.S. Patent Applications Nos. 08 / 273,567; 08 / 482,369; 08 / 485,482; 08 / 487,427; 08 / 479,733; 08 / 472,631; 08 / 479,727; and 08 / 481,904). Induction or tumor infarction by directing a thrombogen to tumor endothelial cell markers is therefore an effective anticancer strategy and can still result in the eradication of primary solid tumors and vascularized metastases. Successful truncation of tissue factor alone or immunoconjugates of truncated tissue factor with an antibody of irrelevant specificity was initially a surprising result of the targeted studies. Although mice that received only truncated Tissue Factor did not have complete tumor regressions, it is clear that the surprising antitumor activity of truncated Tissue Factor returns to this and to its functionally related Tissue Factor derivatives useful in the treatment of solid tumors. The benefits of these compositions as detailed herein are more far-reaching and include the lack of side effects of the use of these Tissue Factors. Furthermore, it is well within the experience of the technicians in the field to produce the type of truncated Tissue Factors compositions presented in the present invention. These compositions can then be used in the treatment of solid tumors alone or in combination with other solid anti-cancer substances. EXAMPLE VI COAGULATION OF MOUSE PLASMA THROUGH CONJUGATES IMMUNOGLOBULIN-TISSUE FACTOR The IgG-Hg-N '-cys-tTF219 conjugate was active to induce coagulation of mouse plasma when it was located on the surface of A20 cells by means of the bispecific antibody, B21-2 / 10H10. It induced coagulation in 50 seconds when it was applied at concentrations of Truncated Te Factor of 5 x 10 ~ 9 M compared to 1 x 10 M for TF2 [and unconjugated H6-N '-cys-tTF19 (Figure 15). The activity that induces the coagulation of the IgG-H6-N 'conjugate was therefore reduced 5-fold in relation to the H ^ -N' -cys-tTF2i9 or the tTF219 itself. The slight reduction of the immunoglobulin G conjugation could be due to the immunoglobulin G fraction of IgG-H6-N '-cys-tTF219 prevents access of the bispecific antibody B21-2 / 10H10 to the truncated tissue factor fraction (ie say, an aberrant reduction related to the test method). It is probably not because the immunoglobulin G fraction of IgG-H6 ~ N '-cys-tTF219 interferes with the formation of coagulation initiation complexes because, in previous work, the inventors have found that the fraction of Truncated tissue in an analogous construction, B21-2 IgG-H6-N '-cys-tTF219, is active as the Truncated Te Factor is linked via B21-2 / 10H10 to I-Ad antigens in A20 cells (Figure 16) . Similarly, B21-2 IgG-H6-tTF219-cys-C 'was as active to induce coagulation as the N'-linked conjugation (Figure 16). The ability of IgG-H6-N '-cys-tTF219 and Fab * -H6-N'-cys-tTF219 to convert Factor X to Xa in the presence of Factors II was tested, VII and IX, once located on the surface of A20 lymphoma cells by means of the bispecific antibody B21-2 / 10H10. The Fab '-tTF construct was as active as the H6-N' -cys-tTF219 itself in inducing Xa formation. The IgG-tTF construct was slightly (2 times) less active than the same H6-N '-cys-tTF219 (Figure 17). EXAMPLE VII INHIBITION OF TUMOR GROWTH C1300 VERY THROUGH IMMUNOGLOBULIN CONJUGATE-TISSUE FACTOR Mice with small tumors (300 mm3) subcutaneous C1300 Mu? were treated with tTF219 or with a complex of tTF219 and a bispecific antibody, 0X7 Fab '/ IOHIO Fab', not targeted to a component of the tumor environment. The treatment was repeated six days later (Figure 18). The bispecific antibody was designed simply to increase the mass of tTF219 from 25 kDa to 135 kDa, and thus prolong its circulatory half-life, and it was not intentional to impart an objective function to the truncated Tissue Factor. Tumors in mice treated with immunoglobulin-tTF conjugate grew more slowly than those mice that received only tTF219. Fourteen days after the first injection, the tumors were 55 percent the size of those in the controls that received only diluent. In mice that received tTF219 alone, tumors were 75 percent of size in controls that received only diluent (Figure 18). EXAMPLE VIII INCREASE OF IMMUNOGLOBULIN CONJUGATE ANTITUMOR ACTIVITY-TRUNCATED TISSUE FACTOR THROUGH ETOPOSIDE Mice treated with human Hodgkin's disease L540 were treated with a complex of tTF219 and a bispecific antibody together with the conventional anticancer drug, etoposide. The etoposide greatly increased the action of the immunoglobulin-tTF conjugate. In this tumor model alone, mice that received the antibody-tTF complex only showed little reduction in tumor growth relative to tumors in mice that received only diluent (Figure 19). In contrast, tumors in mice that received both the etoposide and the immunoglobulin-tTF conjugate decreased in size and did not resume growth for seventeen days. At the end of the study (day 20), the tumors in the mice that received etoposide plus immunoglobulin-tTF had an average of 900 mm3 in volume compared to 2300 mm3 in the mice treated with diluent and 2000 mm3 in mice treated with immunoglobulin alone. tTF. In mice that received only etoposide tumors averaged 1400 mm3 on day 14 (Figure 19). These results indicate that the etoposide can predispose the tumor vessels to thrombosis by means of tTF or immunoglobulin-tTF conjugates. Regardless of the mechanism, the results clearly show that the advantageous combination of Tissue Factor, or a Tissue Factor conjugate with a classical chemotherapeutic substance.

EXAMPLE IX INCREASE OF PLASMA COAGULATION BY Vlla The ability of the tTF2j9 associated with the cell to induce the coagulation of mouse or human plasma increased greatly with the presence of the free factor Vlla (Figure 20). In the absence of Factor Vlla, A20 cells treated with bispecific antibody B21-2 / 10H10 and 10"10 M of tTF219 coagulated the plasma in 60 seconds, whereas in the presence of 13.5 nM Factor Vlla, the plasma coagulated in 20 seconds ( Figure 20) This represents approximately a 100-fold increase in the induction potency of coagulation of tTF in the presence of Factor Vlla. Even in the presence of 0.1 nM Factor Vlla, a 2-5 fold increase in induction power was observed Coagulation of Truncated Tissue Factor This finding leads to aspects of the invention that relate to the co-administration of Factor Vlla together with truncated Tejdobond Factor or derivatives thereof, or with immunoglobulin conjugates-Truncated Tissue Factor, with in order to increase thrombosis of tumor vessels in vivo EXAMPLE X REDUCED COAGULATION OF MOUSE PLASMA THROUGH TISSUE FACTOR ACTIVATION MACTORS FACTOR VII Mutations in W158 and G164 of tTF219 have been reported to markedly reduce the capacity of the Tissue Factor to induce recalcified plasma coagulation (Ruf et al., 1992; Martin; and collaborators, 1995). The residues of 157-167 of the Tissue Factor seem to be important to accelerate the activation of Factor VII in Factor Vlla, but not the binding of Factor VII to the Tissue Factor. The inventors mutated W158 to R and G164 to A and determined s :. the mutants acquired the ability to coagulate plasma as soon as they were localized by means of a bispecific antibody, B21 / 2-10H10, on the surface of A20 cells. It was found that the mutants were 30 to 50 times less effective than what was tTf2J9 in inducing plasma coagulation (Figure 21). EXAMPLE XI RESTORATION OF THE COAGULATION CAPACITY OF THE MUTANTS ACTIVATION OF FACTOR VII THROUGH THE FACTOR Vlla The mutant TF2 [Q (G164A) is a very weakly coagulant mutant of TF219 (Ruf, et al., 1992). The mutation is present in a region of the Tissue Factor (amino acid 157-167) thought to be important for the conversion of Factor VII into Factor Vlla. Thus, the addition of Factor Vlla to cells coated with bispecific antibody and TF2i9 (G164A) would be reasoned to induce plasma coagulation. In support of this, A20 cells coated with B21-2 / 10H10 followed by tTF2j (G164A) had increased ability to induce plasma coagulation in the presence of Factor Vlla (Figure 22). The addition of the Vlla Factor to 1 nM or more produced only coagulation times marginally slower than that observed with tTF219 and the Vlla Factor in the same concentrations. The mutant tTF219 (W158R) gave results similar to tTF219 (G164A). Again, in addition to Vlla Factor at 1 nM or more at A20 cells coated with B21-2 / 10H10 followed by tTF219 only gave coagulation times marginally slower than tTF219 and Factor Vlla at the same concentrations. These results support the aspects of the invention which provide that tTF219 (G164A) or tTF219 (W158R), when coadministered with Factor Vlla to animals that have tumor, will induce thrombosis of the tumor vessels. This approach is considered to be advantageous because tTF (G164A), tTF (W158R) or the given Vlla Factor separately are practically non-toxic to mice, and they are reasonably expected in humans. The co-administration of the mutant tTF and Factor Vlla is expected not to cause toxicity, and to cause efficient thrombosis of the tumor vessels. The administration of mutant truncated tissue factor together with Factor Vlla is contemplated so that it results in an improved therapeutic index in relation to tTF219 plus Vlla Factor. EXAMPLE XII INCREASED ANTITUMOR ACTIVITY OF ACTIVATION MACHINES AND FACTOR Vlla For these studies, the inventors chose the xenograft tumor model HT29 (human colorectal carcinoma). HT29 cells (10 7 cells / mouse) were injected subcutaneously into BALB / c nu / nu mice. The size of the tumor was measured and the tumors were treated when their size was 0.5 and 1.0 cm3. The animals were given an intravenous injection of one of the following tTF219 (16 μg), tTF219 (16 μg) + Vlla Factor (1 μg), tTF2.9 (G164A) (64 μg), tTF219 (G164A) (64 μg) + Factor Vlla (1 μg), Factor Vlla alone (1 μg), or saline. The animals were sacrificed 24 hours after treatment, flooded with saline and heparin and exsanguinated. Tumors and organs were collected, fixed with formalin and histological sections were prepared. The average area of necrosis in tumor sections was quantified and calculated as a percentage of the total area of the tumor in the section. In these small HT29 tumors, analysis of tumor sections from animals treated with saline, Factor Vlla, tTF-19 or tTF219 (G164A) showed some necrosis (Figure 23). The tumor necrosis induced by tTF was the most developed, although this was not surprising, on this occasion, as the results of previous studies using different tumor models and / or larger tumors. An analysis of the tumor sections of animals treated with tTF219 + Factor Vlla or tTF219 (G164A) + Factor Vlla revealed considerable necrosis (12.5% and 17.7% respectively, Figure 23) and a higher correlation between the blood vessels recently bosados and areas of necrosis. The combined use of Factor Vlla with Tissue Factor, even a tissue factor construct with particularly deficient in vitro coagulant activity, is therefore a particularly advantageous aspect of the present invention. Since the HT29 tumor model is difficult to troad in general and these tumors had small size, these results probably translate into more impactful results in other systems and in humans. All compositions and / or methods described and claimed herein may be made and executed with proper experimentation in light of the present disclosure. Although the compositions and methods of this invention have been described in terms of the preferred embodiments, it will be apparent to those skilled in the art that variations may be applied to the compositions and / or methods and in the steps or sequence of the steps of the invention. method described herein without departing from the concept, spirit and scope of the invention. More specifically, it will be apparent that certain substances that are both chemically and physiologically related can be substituted for the substances described herein and at the same time achieve similar or similar results. All of these substitutes and similar modifications apparent to those skilled in the art are considered to be within the spirit, scope and concept of this invention as defined by the appended claims.

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LIST OF SEQUENCES (1) GENERAL INFORMATION (i) APPLICANT: (A) NAME: Board of Regents, University of Texas System (B) STREET: 201 West 7th Street (C) CITY: Austin (D) STATE: Texas (E) COUNTRY: United States of America (F) POSTAL CODE (ZIP): 78701 (ii) TITLE OF THE INVENTION: METHODS AND COMPOSITIONS OF TISSUE FACTOR FOR COAGULATION AND TUMOR TREATMENT (iii) NUMBER OF SEQUENCES: 27 (iv) PREVIOUS APPLICATION DATA : (A) APPLICATION NUMBER: US 60 / 042,427 (B) SUBMISSION DATE: March 27, 1997 (iv) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 60 / 036,205 (B) SUBMISSION DATE: January 27, 1997 (vv) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 60 / 035,920 (B) SUBMISSION DATE: January 22, 1997 (2) INFORMATION FOR SEQ ID NO: 1: (i) ) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 620 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SECUENC IA: SEQ ID NO: 'l: Gly Leu Tyr Ser Glu Arg Gly Leu Tyr Thr His Arg Thr His Arg Ala 1 5 10 15 Ser Asn Thr His? Rg Val Ala Leu Ala Leu Ala Ala Leu Ala Thr Tyr 20 25 30 Arg Ala Ser AG? Leu Glu Thr His? Rg Thr Arg Pro Leu Tyr Ser Ser 35 40 45 Glu Arg Thr His Arg Ala Ser Asn Pro His Glu Leu Tyr Ser Thr'His 50 55 60 Arg He Leu Glu Leu Glu Gly Leu Thr Arg Pro Gly Leu Pro Arg Leu 65 70 75 80 Tyr Ser Pro Arg Val Wing Leu Wing Being Asn Gly Leu Asn Val Wing Leu 85 90 95 Thr Tyr Arg Thr His Arg Val Wing Leu Gly Leu Asn He Leu Glu Being 100 105 110 Glu Arg Thr His Arg Leu Tyr Being Ser Glu Arg Gly Leu Tyr Wing Being 115 120 125 Pro Thr Arg Pro Leu Tyr Being Ser Glu Arg Leu Tyr Ser Cys Tyr Ser 130 135 140 Pro His Glu Thr Tyr Arg Thr His Arg Thr His Arg Ala Ser Pro Thr 145 150 155 160 His Arg Gly Leu Cys Tyr Ser Ala Ser Pro Leu Glu Thr His Arg Ala 165 170 175 Ser Pro Gly Leu He Leu Glu Val Ala Leu Leu Tyr Ser Ala Ser Pro 180 185 190 Val Ala Leu Leu Tyr Ser Gly Leu Asn Thr His Arg Thr Tyr Arg Leu 195 200 205 Glu Ala Leu Ala Ala Arg Gly Val Ala Leu Pro His Glu Ser Glu Arg 210 215 220 Thr Tyr Arg Pro Arg Ala Leu Ala Gly Leu Tyr Ala Ser Asn Val Ala 225 230 235 240 Leu Gly Leu Ser Glu Arg Thr His Arg Gly Leu Tyr Ser Glu Arg Ala 245 250 255 Leu Wing Gly Leu Tyr Gly Leu Pro Arg Leu Glu Thr Tyr Arg Gly Leu 260 265 270 Wing Being Asn Being Glu Arg Pro Arg Gly Leu Pro His Glu Thr Kis Arg 275 280 285 Pro Arg Thr Tyr Arg Leu Glu Gly Leu Thr His Arg Ala Ser Asn Leu 290 295 300 Glu Gly Leu Tyr Gly Leu Asn Pro Arg Thr His Ars He Leu Glu Gly 305 310 315 320 Leu Asn Ser Glu Arg Pro His Glu Gly Leu Gly Leu Asn Val Ala Leu 325 330 335 Gly Leu Tyr Thr His Arg Leu Tyr Ser Val Ala Leu Ala Ser As Val 340 345 350 Wing Leu Thr His Arg Val Wing Leu Gly Leu Wing Pro Pro Gly Leu Wing 355 360 365 Arg Gly Thr His Arg Leu Glu Val Wing Leu Wing Arg Gly Wing Arg Gly 370 375 380 Wing Being Asn Wing Being Asn Thr His Arg Pro His Glu Leu Glu Ser Glu 385 390 395 400 Arg Leu Glu Wing Arg Gly Wing Ser Pro Val Wing Leu Pro His Glu Gly 405 410 415 Leu Tyr Leu Tyr Ser Wing Pro Pro Leu Glu He Leu Glu Thr Tyr Arg 420 425 430 Thr His Arg Leu Glu Thr Tyr Arg Thr Tyr Arg Thr Arg Pro Leu Jyr 435 440 445 Ser Ser Glu Arg Ser Glu Arg Ser Glu Arg Ser Glu Arg Gly Leu Tyr 450 455 460 Leu Tyr Ser Leu Tyr Ser Thr His Arg Ala Leu Ala Leu Tyr Ser Thr 465 470 475 480 His Arg Ala Ser Asn Thr Kis Arg Ala Ser Asn Gly Leu Pro Kis Glu 485 490 495 Leu Glu He Leu Glu Wing Being Pro Val Wing Leu Wing Being Pr Leu Tvr 500 505 510 Being Gly Leu Tyr Gly Leu Wing Being Asn Thr Tyr Arg Cys Tyr Ser Pro 515 520 525 Hie Glu Ser Glu Arg Val Wing Leu Gly Leu Asp Wing Leu Ala Val Ala 530 535 540 Leu He Leu Glu Pro Arg Ser Glu Arg Ala Arg Gly Thr K s Arg Val 545 550 555 560 Wing Leu Wing Being Asn Wing Arg Gly Leu Tyr Being Ser Glu Arg Thr Kis 565 570 575 Arg Ala Ser Pro Glu Arg Pro Arg Val Wing Leu Gly Leu Cys Tyr 580 585 590 Ser Met Glu Thr Gly Leu Tyr Gly Leu Asn Gly Leu Leu Tyr Ser Gly 595 600 605 Leu Tyr Gly Leu Pro His Glu Wing Arg Gly Gly Leu 610 615 620 (2) INFORMATION FOR SEQ ID NO: 2: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 234 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 2 His His His His His His Wing Met Wing Ala Leu Val Pro Arg Gly Ser Cys 1 5 10 15 Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser Thr 20 25 30 Asn Phe Lys Thr He Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val 35 40 45 Tyr Thr Val Gln He Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys 50 55 60 Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu He Val Lys 65 70 75 80 Asp Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing Gly 85 90 95 Asn Val Glu Be Thr Gly Be Wing Gly Glu Pro Leu Tyr Glu Asn Ser 100 105 110 Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr He 115 120 125 Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp 130 135 140 Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp 145 150 155 160 Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp lys Ser Ser 165 170 175 Be Ser Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu He 180 185? = D Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln A. to Val He 195 200 205 Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys 210 215 220 Ket Gly Gln Glu Lys Gly Glu Phe Arg Glu 225 230 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 234 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 3: His His His His His His His Wing Met Wing Ala Leu Val Pro Arg Gly Ser Gly 1 5 10 15 Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser Thr Asn 20 25 30 Phe Lys Thr He Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 35 40 45 Thr Val Gln He Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe 50 '55 60 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu He Val Lys Asp 65 70 75 80 Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing Gly Asn 85 90 95 Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro 100 105 110 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr He Gln 115 120 125 Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu 130 135 140 Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val 145 150 155 160 Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser 165 170 175 Be Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu He A.sp 180 185 190 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val He Pro 195 200 2C5 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys K t 210 215 220 Gly Gln Glu Lys Gly Glu Phe Arg Glu Cys 225 230) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 220 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: l ineal (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 4: Ser Cys Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys 1 5 10 15 Be Thr Asn Phe Lys Thr He Leu Glu Trp Glu Pro Lys Pro Val Asn 20 25 30 Gln Val Tyr Thr Val Gln He Ser Thr Lys Ser Gly Asp Trp Lys Ser 35 40 45 Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu He 50 55 60 Val Lys Asp Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro 65 70 75 80 Wing Gly Asn Val Glu Ser Thr Gly Ser Wing Gly Glu Pro Leu Tyr Glu 85 90 95 Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro 100 105 110 Thr He Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val 115 120 125 Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu 130 135 140 Arg Asp Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys 145 150 155 160 Be Being Be Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe 165 170 175 Leu He Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Wing 180 165 190 Val He Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val 195 200 205 Glu Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu 210 215 220 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 220 amino acids - (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 5: Ser Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Thr Asp Thr Asp Glu He Val 50 55 60 Lys Asp Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing 65 70 75 83 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Tnr 100 105 110 He Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Be Ser Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 He Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Wing Val 180 185 190 He Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu Cys 210 215 220 INFORMATION FOR SEQ ID NO: 6: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 235 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 6: His His His His His His His Wing Met Wing Leu Val Pro Arg Gly Ser Gly 1 5 10 15 Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser Thr Asn 20 25 30 Phe Lys Thr He Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 35 40 45 Thr Val Gln He Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe 50 55 60 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu He Val Lys Asp 65 70 75 80 Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing Gly Asn 85 90 95 Val Glu Ser Thr Gly Be Wing Gly Glu Pro Leu Tyr Glu Asn Ser Pro 100 105 110 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr He Gln 115 120 125 Be Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu 130 135 140 Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val 145 150 155 160 Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser 165 170 175 Be Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu He Asp 180 185 190 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val He Pro 195 200 205 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met 210 215 220 Gly Gln Glu Lys Gly Glu Phe Arg Glu lie Cys 225 230 235) INFORMATION FOR SEQ ID NO: 7: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 236 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 7: His His His His His His His Wing Met Wing Leu Val Pro Arg Gly Ser Gly 1 5 10 15 Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser Thr Asn 20 25 30 Phe Lys Thr He Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 35 40 45 Thr Val Gln He Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe 50 55 60 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu He Val Lys Asp 65 70 75 80 Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing Gly Asn 85 90 95 Val Glu Ser Thr Gly Be Wing Gly Glu Pro Leu Tyr Glu Asn Ser Pro 100 105 110 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr He Gln 115 120 125 Be Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu 130 135 140 Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val 145 150 155 160 Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser 165 170 175 Be Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu He Asp 180 165 190 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val He Pro 195 200 205 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met 210 215 220 Gly Gln Glu Lys Gly Glu Phe Arg Glu He Phe cys 225 230 235 (2) INFORMATION FOR SEQ ID NO: 8: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 219 amino acids ( B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 8: Ser Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser 1. 5 10 15 Thr Asn Phe Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Thr Asp Thr Asp Glu He Val 50 55 60 Lys Asp Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing 65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr 100 105 _ _ ... no He Gln Ser Phe Glu Gln-Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Arg Lys Ser 145 150 155 160 Be Ser Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 He Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Wing Val 180 185 190 He Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Ket Gly Gln Glu Lys Gly Glu Phe Arg Glu 210 215 INFORMATION FOR SEQ ID NO: 9: (1) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 219 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 9 Ser Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser 1 5 10 15 Thr Asn Phe Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Thr Asp Thr Asp Glu He Val 50 55 60 Lys Asp Val Lys Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing 65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Be Pro Glu Phe Thr Pí or Tyr Leu Glu Thr Asn Leu Gly Gln Pro Tnr 100 105 110 He Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Being Being Wing Lys Lys Tnr Wing Lys Thr Asn Thr Asn Glu Phe Leu 165 170 175 He Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Wing Val 180 185 190 He Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu 210 215 (2) INFORMATION FOR SEQ ID NO: 10: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 657 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 10: TCAGGCACTA CAAATACTGT GGCAGCATAT AATTTAACTT GGAAATCAAC TAATTTCAAG 60 ACAATTTTGG AGTGGGAACC CAAACCCGTC AATCAAGTCT ACACTGTTCA AATAAGCACT 120 AAGTCAGGAG ATTGGAAAAG CAAATGCTTT TACACAACAG ACACAGAGTG TGACCTCACC 180 GACGAGATTG TGAAGGATGT GAAGCAGACG TACTTGGCAC GGGTCTTCTC CTACCCGGCA "240 GGGAATGTGG AGAGCACCGG TTCTGCTGGG GAGCCTCTGT ATGAGAACTC CCCAGAGTTC 300 ACACCTTACC TGGAGACAA CCTCGGACAG CCAACAATTC AGAGTTTTGA ACAGGTGGGA 360 ACAAAAGTGA ATGTGACCGT AGAAGATGA CGGACTTTAG TCAGAAGGAA CAACACTTTC 420 CTAAGCCTCC GGGATGTTTT TGGCAAGGAC TTAATTTATA CACTTTATTA TTC-GAAATCT 480 TCAAGTTCAG GAAAGAAAAC. AGCCAAAACA AACACTAATG AGTTTTTGAT TGATGTGGAT 540 AAAGGAGAAA ACTACTGTTT CAGTGTTCAA GCA37GATTC CCTCCCGAAC AGTTAACCGG 630 AAGAGTACAG ACAGCCCGGT AGAGTGTATG GGCCAC-3AGA AAGGGGAATT CAGAGAA 6 57 (2) INFORMATION FOR SEQ ID NO: 11: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 13865 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 11: (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: GAATTCTCCC AGAGGCAAAC TGCCAGATGT GAGGCTGCTC TTCCTCAGTC ACTATCTCTG 60 GTCGTACCGG GCGATGCCTG AGCCAACTGA CCCTCAGACC TGTGAGCCGA GCCGGTCACA 120 CCGTGGCTGA CACCGGCATT CCCACCGCCT TTCTCCTGTG CGACCCGCTA AGGGCCCCGC 180 GAGGTGGGCA GGCCAAGTAT TCTTGACCTT CGTGGGGTAG AAGAAGCCAC CGTGGCTGGG 240 AGAGGGCCCT GCTCACAGCC ACACGTTTAC TTCGCTGCAG GTCCCGAGCT TCTGCCCCAG 300 GTGGGCAAAG CATCCGGGAA ATGCCCTCCG CTGCCCGAGG GG? GCCCAGA "GCCCGTGCTT 360 TCTATTAAAT GTTGTAAATG CCGCCTCTCC CACTTTATC? CCAAATGGAA GGGAAGAATT 420 CTTCCAAGGC GCCCTCCCTT TCCTGCCAT? GACCTGC ?? C CCACCTAAGC TGCACGTCGG 480 AGTCGCGGGC CTGGGTGAAT CCGGGGGCCT TGGGGGACCC GGGCAACTAG ACCCGCCTGC 540 GTCCTCCAGG GCAGCTCCGC GCTCGGTGGC GCGGTTGAAT CACTGGGGTG AGTCATCCCT 600 TGCAGGGTCC CGGAGTTTCC TACCGGGAGG AGGCGGGGCA GGGGTGTGGA CTCGCCGGGG 660 GCCGCCCACC GCGACGGCAA GTGACCCGGG CCGGGGGCGG GGAGTCGGGA GGAGCGGCGG 720 GGGCGGGCGC CGGGGGCGGG CAGAGGCGCG GGAGAGCGCG CCGCCGGCCC TTTATAGCGC 780 GCGGGGCACC GGCTCCCCAA GACTGCGAGC TCCCCGCACC CCCTCGCACT CCCTCTGGCC 840 GGCCCAGGGC GCCTTCAGCC CAACCTCCCC AGCCCCACGG GCGCCACGGA ACCCGCTCGA 900 TCTCGCCGCC AACTGGTAGA CATGGAGACC CCTGCCTGGC CCCGGGTCCC GCGCCCCGAG 960 ACCGCCGTCG CTCGGACGCT CCTGCTCGGC TGGGTCTTCG CCCAGGTGGC CGGCGCTTCA 1020 GGTGAGTGGC ACCAGCCCCT GGAAGCCCGG GGCGCGCCAC ACGCAGGAGG GAGGCGACAG 1080 TCCTGGCTGG CAGCGGGCTC GCCCTGGTTC CCCGGGGCGC CCATGTTGTC CCCCGCGCCT 1140 ACGGGACTCG GCTGCGCTCA CCCAGCCCGG CTTGAATGAA CCGAGTCCGT CGGGCGCCGG 1200 CGGGAGTTGC AGGGAGGGAG I GGCGCCCC AGACCCCGCT GCCCCTTCCG CTGGAGAGTT 1260 TTGCTCGGGG TGTCCGAGTA ATTGGACTGT TGTTGCATAA GCGGACTTTT AGCTCCCGCT 1320 TTAACTCTGG GGAAAGGGCT TCCCAGTGAG TTGCGACCTT CAATATGATA GGACTTGTGC 1380 CTGCGTCTGC ACGTGTTGGC GTGCAGAGGT TTGGATATTA TCTTTCATTA TATGTGCATC 1440 TTCCCTTAAT AAAGAGCGTC CCTGGTCTTT TCCTGGCCAT CTTTGTTCTA GGTTTGGGTA 1500 GAGGCAATCC AAAAGGGCTG GATTGCTGCT TAGATTGGAG CAGGTACAAC GTTGTGCATG 1560 CCCCGTATTT CTACGAGGTG TTCGGGACGG CGTAGAGACT GGGACCTGCT GCGTACTGGC 1620 AAAGCAGACC TTCATAAGAA ATAATCCTGA TCCAATACAG CCGACGGTGT GACAGGCCAC 1680 ACGTCCCCGT GGGTCTCTGT GGAAGTTTCA GTGTAGCGAC ATTTCAGATA AAAGTGGAAA 1740 AAGTGAAGTT TGGCTTTTTT CATTTGTATG CAGTCCTAAC TCTTGTCACA CGTGTGGGAT 1800 TTATCTTTTT CCATAACTTA CTGAAAACCC TTCCTGGCGG GCTGAACCTG ACTCTTCCTG 1860 AGCTGAGTCC TGGACTGGCA CACTGATGGC TCTGGGCTCT TCCCGGTCAA GTTATAACAA 1920 GGCTTTGCCC ATGAATAATT TCAAACGAAA ATGTCAAGAT CCTTGCCGGT GTCCTGGGAT 1980 TACAAGGTGA ATCTTGTCAT GAAGAAATTC TAGGTCTAGA AAAAATTTGA AGATTCTTTT 2040 TCTCTTGATA ATTCACTAAT GAAGCTTTTG TGGTTGAAAA ATAAAAAGTG AGGTTTATGG 2100TGATGTCAGG TGGGAAGGTG TTTTATACAT CAATACATTC GAGTGCTCTG AAGTGCATGT 2160 AATAATAGCT GTTTCTCTGT TGTTTAAAGG CACTACAAAT ACTGTGGCAG CATATAATTT 2220 AACTTGGAAA TCAACTAATT TCAAGACAAT TTTGGAGTGG GAACCCAAAC CCGTCAATCA 2280 AGTCTACACT GTTCAAATAA GGTAAGCTGG GTACAGAAAA AGAAAATTAA GGTCTTTGAT 2340 GTTTCTACTG TCCTATGCTG AACAAGAATG TCTTTAAAGC TGATTACTGG ATGAAATTAT 2400 TTAACAGATG ACGAAGAAGA AGGGATTCTT GGCAATTCGC TGGCCGGTGT CATACTCTAT 2460 TAGGCCTGCA ACATTTCCAG ACCTTAAACT GATAGAACAT TTTAATTGTT TTAATTGTTT 2520 TTGGAAATGA TGGGAGAGTT CCTAAGTGGA GTATAAACTG TGGAGAGATG AACCATCTTG 2580 AGTAGGCACT GAAGTGTGCT TTGGGTCATG ATAGATTAAT TAATCTCATC TAAACATTGA 2640 TGTCTTTTTC CGTTGCTGTC TAGACTGTGA ACAATGTCTA ACACCTTAGG GAAGAGGTGG 2700 GGAGGAATCC CAATGTATAC ATTGCCCTTA AGCAGTGTTT GATTCATTCA TCTTTGGACT 2760 CCATGAATCG AAATCTGGTA GAATACATGA TCTTAGTGGA GGAGGCCAAA TGCGTGACTC 2820 ACTGAGCCTG GCAGAGCAGA AATACTCTGC TGTCTGCACC CTCTGGGTCT GGTGTGGCTC 2880 TGCTTCTTGG TGCTTCAACT CTGACTGGCA GCTGTCCCCA GGAGGCGATA ATTCAGCATG 2940 TTCAATCTAA AGGTTATGAC TTCCTTGATG GTTTTCACCA TATTCTTGGC AAGTTTTTGG 3000 TTTTTGAAAT GTTCTAGGAG GCTTGGTAGA GATCTTATGA AATAGAGAAT AGCTGCTGTG 3060 GAAATTATTT TAATGCTAAT TACATAAAAG TACAAAAGTA GCACTAGCTA AAACAAAAGG 3120 TATTTTGCTG TTCTGTTTTG TTTTAGCTTG TGCCAGGCCT TTTACAGCAT TAGGAATGCA 3180 ACTTCTAGAT AACGATGCAT CTTTTAAGTG AATGTTCTTG TTTTTCAAAA TGAACTTCAT 3240 GACAGTAGTT GCCAAACCAG CAAGGAGAAC TTGCATGCAT ACGTGCATGC ATGTGTGGAT 3300 ATGTATGGGG GTGGGGGGAG AGAAAGATGA AGGAATTTCA TAACATGAAA TAATGATTAC 3360 AGTTCTGGTC AAACTTGTCA ATTCAGATTT CACCAATTGA GAATTAGTAA GTAATTTCTC 3420 TGATACAGGC CTGAAGTTTA CCTTAGTAAA CACTTTACTT CCATATGGTA AAAATTAGAT 3480 TTTGGGAGGA ATGCTTACCT CCTAAATATA TTCAATCTAA TATTTGAGGA CACATGGGAA 3540 TATATTTATG ATTCATCTGC TTTTTAAACA TAAGCCTTTG TTAACTGTAA GTTCTTGAAC 3600 TTTATAAGGC TGCTGTTATT TAAATGAGCA CAGCTCCTGA TCTGCAAACA GCAGAGCGCA 3660 GGGCTACAGC TTGGGGGATG CCAGCCGACT CAGGOTGGTC CTGTGGACTG AACAATCTCT 3720 TGCTGCTGTA CTGOAGGGCC TGGGAGCTTT TCCATCAGCC TCGGCCTGAG GTGTGCACTC 3780 TTCTCCTGCC CACCCCAGGA ATAAATGAGA TTCCTGGTTA AAAAGGACCA GAGCAGTCAT 3S40 TTTACAGTTG AGGAAACTGT TGCTCTGAGA AGTGAGGGAT TTATTCATGA CTACACTGAT 3900 G3TGAGTGCC CATGTCAGGT CTGGAACCAA AGTCTACCCA GTATCCACAC ACCACCATCC 3960 CTCAGGTGGC TCTGCCACAG TCTGATGGGA GGCTCCAAAG CGGGAGGAAG AAGGAAAGTC 4020 TTGCCCACTG CATCTCCTCA GTTGGCCTTC CTCTCTGCCT GTTTTCCCTC CCTACAGTTA 4080 GCATCTTAAG CAGCTGCCTC TCTTCCCTCC CGACTGCTCT CACTACTGCA GCCTGGCTCC 140 AGCCGCAGGA CACTACTGCT GTGCAGAAGC CCCTACTTGG AACTCCAACT GCATTTTTCA 4200 CCTTTTGCTAA CAGTTTTCAG TGGTGGTTGG GAAATGTTAT TGGCTTAAGC CTTAGCACAA 4260 ACCGTCACCG GTGATATTCA TTCCATGGAA ATGTTCTGAA TTCTAAAGCT GAATTTACAA 4320 AGCTTCTGGA AAACAACCTG CAACCAAATT AGTGACTGAA TTTTTTAGTT AACTCAAAAT 4380 TCCAAATCAG AGGGTTTTGC AATGCCTGGA GGAACCTTGG AGGCTTTTAA AGTGTTAATG 44 0 CTATTAATGG CATTCAGAGG GATTTTCTAC AGAATTGTCC CTTCATTACC TGTTTATACA 4500 GTTTTACTAC TTACCAGGGT ACTGTATAAA TCCTTGTGCT AAATTTTGCT ATAGAGTATG 4560 TGGTCCCTGC TGTGAGCTGG GAGGAACCAA ATACTGTATC TCTATGTTAC ATAGAAAGCC 4620 CTAGGAGACT TTCTCCTGTT ATCTGAACAA CTATTTGCTG TACTGATAAA AAGGAAACAG 4680 CATAGTCTCA TTCACTTTTT GAAATGGAAA TGATAAAATA AAACACATTT TGGTCATTCG 4740 GGAACAAAAT ACCCTCTCTA CTTTTATCAC ATAAAATTAA ATAAATAGAA ACCAAAATAT 4800 TTCA37ATCA ATCTTAGTTT GTGCACTTTA GGATAAAGAA TGTGTTTACC CAAATCCTTT 4860 TGGCCT33TT ACTTAGTTCA GATTTTGAAA GAAAATATAT TTGTG3CTTT TATGTGTGAA 4920 TT7AGACAA7 G3AATCCATG TGGTGCCTCG TTTTCCCTGA GATTATGTAT TAATTCAACC 4960 TGTAAATGCA AACCATCTAA TAGTCAGCGA GACCCTATAG CCCTGC7GCT TAATGGGGGC 5040 ACACAA3G3C ATGCAGCCCT CGTACCAGGC AGACTGTGTT CATATTAACA GCATCGTGGA 5100 GAAACTCATG CTGGGGGACA GGGGAGGGAG ATGTAAATGC TCAGCAGGGA GATCTGGAGA 5160 TTCCT33A3C AGGTGGAGTT GGGACCTGGC CTTGAACGAT GGGTCTGGCT CTGGCAGTCA 5220 GTAAT3CCAA A33GAAGAGC AGCATAACTG TCACTTTCCA TGG3ACA3AA GTGT3TGAAT 5283 CAAG7TGCA3 TGACGCTTCA CCTATTTATT ATTTTGGTCA TTTAGAAGAA TTTCATTGTC 5340 AGTAGAAGTC CTTTAAATCA TTTCCCCTTC AGTGACGTCT CACAAAAAAA AGATCTGTCT 5400 TTAGCTTTTT AGTCTCAGAC TTTATTAGAC AGATACTACC TGTACTCTTA TTCTGTAATC 5460 TTTGT7GG3A TGGATTCACA TCTTGCAAAG GAAGGGAGGC ATGTAGTATA ATG3GGCAAA 5520 CAGACCCA3C TCTGCCACTC GTTAGATATG TGACCTTCTG CAAGTTGCTT AGTGCCTGTG 5580 AGCTTCAGTG TCCTCATGGA TAAGAAAGAT CCAACACCTT CTTGGAAGGA TTATATCAAA 5640 TGAAGTAA TO TGAGTAAAGG GTCCAGCAGA ATACCTGGCA TATAGT33AG TCAATGAATG 57CC ATTAATAATA TTATTAATAG TGGTCATGAG AGATATATGT ATAACATGTT ATTATGTAGA 5760 CTCACTATAT AGACTCTATT CTACATAGAA TATAGAACAT TATATAACAA ACAACTATAA 5820 TAAGTAGACT ATAGTAAACA ACCTCACTTT GTCTCAGTTG CCTCATCTTG ATGGAAAACT 5880 GCTCTTTCTC TCCTGTTACC CTGACAGAGA GCGTCTACAT TCTAAAAGAA AGATATTTAA 5940 CAAAATGGTT GAGTACAGAT CCAAGAGTCA AATAGCTGTC TGGTTCAAAG TCCAGCTGTG 6000 TGATTTTGAG CTAGTCACCC AATCTCACTT TGTCTCAGTA GCCTTATTTG TAAAAACAAG 6060 GCAAATTACA GAGCCATCCC CTGGGTTGCT ATGAGGACTC AAACATGCAT CCCAAGTGCT 6120 CGGTGTTGCT AGGTATGATG GCTCACACCT GTACATTCAG CACTTTGGGA GGCCGAAGCA 6180 GAAGGATCAG CCTGGGCAAC ATAGCAGGAC CCCATCTCTA CAAAACAATG TTTAAAAAAA 6240 AGCAAAGTGC TCAGCACAGT GACTGCATCA TTAGGATTGA TTGTAGGGCT CCTGATGTTA 6300 GCACAGAACA CCACAGCCAG GAAGCAGTCT ATCTTGTTGG GTGCAAATTG TAACATTCCA 6360 TTTATGTTTC TTCCTTCTTT TCTTTCTTTA GCACTAAGTC AGGAGATTGG AAAAGCAAAT 6420 GCTTTTACAC AACAGACACA GAGTGTGACC TCACCGACGA GATTGTGAAG GATGTGAAGC € 480 AGACGTACTT GGCACGGGTC TTCTCCTACC CGGCAGGGAA TGTGGAGAGC ACCGGTTCTG 6540 CTGGGGAGCC TCTGTATGAG AACTCCCCAG AGTTCACACC TTACCTG3AG AGTAAGTGGC 6600 TTGGGCTGTA ATACCGTTCA TTCTTGTTAG AAACGTCTGA ACATTCTCGT GA7CTTGTGC 6660 CTTTAGGGGC TACAAAATTA AAAATATTTA -TCTTTTTTT CTCAGAAACT GGTATGTATC 6720 ACA3CCCTCT TCACACATTC CAGA7GTG3T AGGAGGTTCA CAGAATGTGA ACTTTTGGAG 6"90 CTGATGACAG TGTCATCAAG TAACTTTCTC CCCCAGTCTG TCCCCAGACC CTGTTACTGT 6840 CCTCAGTAAG CGGCTGAATG TGTGTTGGGA GAGGGCGGGC CAGGGAAGCG GGTAGGGATA 6 S 00 GGAAATCCAC CAAGGCCGGG GTTTTAGCT7 TTCCCTATAT ATATATCATG TATCCTGATT 6960 TTTC7GTCCC GTTATCACAC TAAAAATCCC AGTTGAGGAT TTTTCCCAAA CGGTCATAAA "320 TC.AATGAGGA AAGTCCATGG TTTCCCTC7G AGCCCATAAT TAGCCTAATT ATGCTGACCT 70 £ 0 TTTCTAATCA GTTGGCCATG ATTTGAGTTC CGT3ATGTGC CAGCACCTGC CCAGCCATCT "140 GCCTGTCACC CTCGTTCTGG TTTTGGAAAS GTG3AATACT TTCCTCCTCA GCCTTTGCCC 7200 CTGTAAGCTG GCCCTAGGAG CCAGTAAAAG AATGAAGAGA ATTCC7GTCA AGTAGGAGAT 7260 TTATTCTTTT GCCGCAACTG TGGC7C7GA3 CTAGGCAATT TAGATAAATG CATGTAGCAC "320 ATTGAGTAGA GTGAAATTAG CTTCTCTTG7 AAGGCCAGCT GGTTAGAATG AAGGTGTTGT 73 80 GTGAGTGTTA GGCCCAGCGA GAGAGAACAG TTTCTCAAGG TAGGAATG3T GAAAAGAASG 7440 GGTGGACGGA CAACCAACCA ACCATCCTCC TCTGGTATCT ACTTTGAGGG TTGAAATAGG 7500 GGGCCTGACC CCAGGTGAAT GTGGCTGCCT TCCCAGAGCC CCCATTTGCA AGACCCTCCA 7560 GACCCCCAGG TGCTTCTGCT TGTGTCTTTT GTGGCACCAG GCAAGAATGT AGCAGCGTCA 7620 GCAGCCCCTC TGGTGACTGT GGCATGGTTG ACATTCATTT CCCCCCTAAT TAATGGCATC 7680 CTCATGATTC TCTTTTATAT TAATAGTTCT TGAGTTTTTT TGTAAGCTAC TTCAAATCCT 7740 TTGTTGGTGC AAGATAGAAG ATATTTTATG TGTTTGTTTT GCATGTGCAC ACACATATTT 7800 GGCCTGTGAA TTGATGTTTG TTTTCCTGTC ATTTAACCAA AGCACATGAG ATAATTGAGC 7860 CATTGCAGAG ACCCCGTGGT TAAATCCGGC TTCTCGAGGT ACCAAGGACA TTTCCTGGGC. 7920 TTTCTCACAG CCCTACATAT TTTTGAACCT AAAATATCGT AGTTTATGCT ACCACCCTGT 7980 TCAGTATAGT AGCCACTAGC CACATGTGGC TGTTGACCAC TTGAAATATG GCTAATGCTC 8040 TAAGTATAAA GTACACACTG GAATTTAAGA AGTGTAGAAT ATCTCAAAAC TTTTTTATAT 8100 TGATTACACA TTAAAATGAT TATATTCCAG ATATATCCAG TTGACTCAAG CAATGCATGG 8160 CTGAGAGGCA CCGACTCCCT GTGCAGTTGA AAATCCGAGT ATAACTTGAC TCCCCAAAAA 8220 CTTAACTACT AATAGCCTAC CTATCGGTTG ACTGTTGACT GCAGCCTTAC CAATAAGATA 8280 AACAGTCAAT TAACACACAT TTTTCATGTT GCGTGTATTA TATACTGTAT TCTTACAATA 8340 AAGTAAGCTA GAGGAAAGAA AATGTTATTA AGAAAATTAT AAGGAAAAGA GGCTGGGCAT 8400 GGTGGCTCGT GCCTGTAATC TCAGAACTTT GGGATGCTAA GGCGG3TGGA TCACTTGAGG 8460 TCAGGAGTTC AAGACCAGCC TGGCCAACAT GGTGAAACCC CATCTCTACT AAAAATACAA 8520 AAATTAGCCA GGCGTGGTTG TGGGTGCCTG TAATCCCAGC TACTTG3GAG GCTGAGGCAG 8580 GAGAATCACT TCGACCCAGG TGGAGGAGGT TGCAGTGAAC TGAGATTGCG CCACTGCACT T640 CCGGCCTGGG TGACAGAGCG AGACTCTGTC TAAAAAAGAA AGGGAAAGAA AGAAAAAAAAA 8700 GAAAAGAAAA GAAAAGAAAG AAGGAAGGAA GAGAAAGAAT TATAA3GAAG AGAAAATATA 8760 TTTACTATTG ATAAAGTGGA AGTGGATCAT CATAAAGGTG TTCATCCTCG TCATCTTCAT 8820 GTTGAGTAGG CTGAGGAGGA GGAGGAGGAG GAAGAGCAGG GGCCACGGCA GGAGAAAAGA 8830 TGGAGGAAGT AGGAGGCGGC ACACTTGGTG 7 ACTTTTAT TTAAAAAAAT TTGCATACAA 8940 GTGGATCCAC AGAGTTCAAA CCCATGTTGT TCAGGGGTCA ACTGTCTTTG GTTAAATAAA 9000 ATATATTATT AAAATTAATT TCACCTGTTC C77TTTACTT TTTCTAATGT GACTACTAGA 9060 AAACTTAAAA T3 CATCTGA GGCTCCATTG 7377CCCC7T GGGCCAGCAC TACCACAGAA 9120 ACATGTTTTA TATGAGAGAT AATTAAGTTG TCAATTGTGA TAACAAAACA GGATTTGACT 9240 TTGTACAGAA TTCTTTGGTT CCAACCAAGC TCATTTCCTT TGTTTCAGCA AACCTCGGAC 9300 AGCCAACAAT TCAGAGTTTT GAACAGGTGG GAACAAAAGT GAATGTGACC GTAGAAGATG 9360 AACGGACTTT AGTCAGAAGG AACAACACTT TCCTAAGCCT CCGGGATGTT TTTGGCAAGG 9420 ACTTAATTTA TACACTTTAT TATTGGAAAT CTTCAAGTTC AGGAAAGGTG AGCATTTTTT 9480 AATTTGTTTT TATGACCTGT TTTAAATTGT GAATACTTGG TTTTACAACC CATTTCTTCC 9540 CCAATTCAAA AATAGCAGAA CAGAGTTGTT GAGAAGGTGA TGGAGTAGAA GGGGGAGCGC 9600 GCACTGTGGG GAGGGGTGGA CAACAGGCCT GGTCCTACCT GTGACTCTGC ACTACCCTGT. 9660 GACTCTGGCA GGGCCCCCTC GGAGACCCAG GTTCCTCAGC CAACCGGCTG GATCAGGTCA 9720 TCTCTAAAGG TCCCGCCACG CTC? CATTTC TCCCTCTATT GAGGATCCCA GGCACAAAAT 9780 TTGTTTTTGG TTCAATGCAT AATACTCCCT TCCTTTTTCT TTTACTGCAG ATATCTTCTA 9840 AAGGGGCTCA ATAGGGTTCA ATATGCCTAA ATTGGATCTT CTCAGTCTTG GAAAAGGCAT 9900 TTTTAGCAGT GATCAAGGGA AACTGATTAG CGAAGTCACT TCTAATCCTT CACGTGTCAG 9960 CTGTGTTCTT GTAGGCTTTG CTTAGAACCT AGGTTTTTAC TTCCACAGTG ACTTAATAAA 10020 GGGGAAAGAA TTGACTCAGA GCCCAGATGA ATTAAGAACT CTATCTTTTT ACAGAAAACA 10080 GCCAAAACAA ACACTAATGA GTTTTTGATT GATGTGGATA AAGGAGAAAA CTACTGTTTC 10140 AGTGTTCAAG CAGTGATTCC CTCCCGAACA GTTA? CCGGA AGAGTACAGA CAGCCCGGTA 10200 GAGTGTATGG GCCAGGAGAA AGGGGAATTC AGAGGTGAGT GGCTCTGCCA GCCATTTGCC 10260 TGGGG3TATG GGTGCTGTGG GTGACTTCTG GAGGAGTAGC TCCACCCTCA GGGCTGGGAT 10320 ATACTTCCTT GGTTAAATAT TCAGGAAAAC AAACTGCCTG GAGGTTTTTT GTTGTTATTT 10380 GTTTGTTTTG GTTTTGATTT TGCTTTGGTA CAAAAAAGAT TTTG3ACA7T TAGAAATGTT 10440 TCTGTGTTGA TTGTGCCCTT GTATTAGCAG GTGTTTTCTT GAGCACCTGT CATGTGCTAA 10500 GCCCTCTGCT GAGCACTGGA TACACAAACT GTGTTTAGGA TTTAGCAACA AGTCACAGAT 10560 TTCCCTGGGC ATTTTTTTCAT GCTTAAATTC TAATTCTGGG GGTGGCTTCT GGACCAGCTG 10620 CAACA3GACA CAGTAGACAT TCGTGAGTAC CCACTGTGGG C7GT7GCCAC AGAGGCTGTA 10680 GAGTCTAACC CATCAAGGGA AGGGATTGAG TATATCAAAT ATACCCACAT GCATGCATGT 10740 GTGTATATGG CGGACACGTG TGTGTACATG CATGTGCATA TGTTGGGAGC TCAGGCCCAT 10800 T3TGC3A3GA ACAGTCCCTA ACCG3AAGTG CTGTGGGCCT TCAGACTCTT GCA3 AA CT 10860 AGAAAAGAGT CAGGGGATAT AAACGATGGC TTACGCTGGG TGTGGTGGCT CACGCCTGTA 10980 GTCCCTGCAC TTTGGGAGGC CCAGACAGGC AAATCACTTG AGGTCAGGAG TTTGGGACCA 11040 GCCTGGCCAA CATGGTAAAA GCCCATCTCT ACTCAAAATA CAAAAAGTAG CTGGGTGTGG 11100 TTGCACGTGT CTGTAGTCCC AGCTACTCAG GAGGTTGAGG CAGGAGAATT GCTTGAACCT 11160 GGGAGGCGGA GGCTGAAGTG AGCTGAGATT GGACCACTGT ACTCCAGCCT GGGTGACAGA 11220 GCGAGATTCC ATCTCAAAAA AAAAAAAAAG AAACAACGAA AAAAGAAATG ATGGCTTAGC 11280 TACATGTGAA GATGATATTT GAACATTTTA AAACACTTTA AATAAACTGT TCTCTCCTGT 11340 TTATTGCCAC TGACAGGAGA GGTTTCTCTT TACCTCTGGT CCTGCACCCC TCTGAGCCAT. 11400 CCTACCCACA GCCTTCAGTC ATTGTCCTAA AGCCTAGCTC TAATTCCACT GCCTCTCCTT 11460 TTGTGCACAC ACACTTCTCT GCTTCCCTGG CCGTTCTCTA TCTT3GAGAG GCATTTCAAA 11520 CGCCACTTCC ACCAGAAGGC CTTGCTACTG CACCAACTAG TTACTATCTC TTCTTCACCC 11580 AAATCCTGGT AGCACTTTGG ATCTCCCACT TGCACTTAGG GTTCACCTTC CGTTATAATC 11640 ATTGCCATCA ATCTCAGCAT CGTTTTAGGC ACTTCTTTCC AGCCATTGTT CTTACCTCCA 11700 ACTACATATC TTTTCTGGAC TGTGCATTAT TCAGTTTATT AAATGCCCAT TAAATGTGTT 11760 TAGCCATTGT CAATTACTCT GAAACGTTCA GGTTTTGACA A? TTCTTTCC TAATGTAAGT 11820 GTGGTGGAAA GAGTGAAAGA AAGTCAAATT GCACAAAAAT AGGATGGTGT AATTTGGGGT 11860 TATGCCGTCA ATTTTGTCCA CTGATAAATG GGATTTGAGC TCTCCAAGTT GACTAGATGC 119 0 CCTTTATTTT TCAGAAATAT TCTACATCAT TGGAGCTGTG GTATTTGTGG TCATCATCCT 12000 TGTCATCATC CTGGCTATAT CTCTACACAA GTGTAGAAAG GCAGGAGTGG GGCAGAGCTG 12060 GAAGGAGAAC TCCCCACTGA ATGTTTCATA AAGGAAGCAC TGTTGGAGCT ACTGCAAATG 12120 CTATATTGCA CTGTGACCGA GAACTTTTAA GAGGATAGAA TACATGGAAA CGCAAATGAG 12180 TATTTCGGAG CATGAAGACC CTGGAGTTCA AAAAACTCTT GATATGACCT GTTATTACCA 12240 TTAGCATTCT GGTTTTGACA TCAGCATTAG TCACTTTGAA ATGTAACGAA TGGTACTACA 12300 ACCAATTCCA AGTTTTAATT TTTAACACCA TGGCACCTTT TGCACATAAC ATGCTTTAGA 12360 TTATATATTC CGCACTCAAG GAGTAACCAG GTCGTCCAAG CAAAAACAAA TGGGAAAATG 12420 TCTTAAAAAA TCCTGGGTGG ACTTTTGAAA AGCTTTTTTT tttttttttt TTTTTGAGAC 12480 GGAGTCTTGC TCTGTTGCCC AGGCTGGAGT GCAGTAGCAC GATCTCGGCT CACTGCACCC 12540 TCCGTCTCTC GGGTTCAAGC AATTGTCTGC CTCAGCCTCC CGAGTAGCTG GGATTACAGG 12600 TTGGCCAGGC TGGTCTTGAA TTCCTGACCT CAGGTGATCC ACCCACCTTG GCCTCCCAAA 12720 GTGCTAGTAT TATGGGCGTG AACCACCATG CCCAGCCGAA AAGCTTTTGA GGGGCTGACT 12780 TCAATCCATG TAGGAAAGTA AAATGGAAGG AAATTGGGTG CATTTCTAGG ACTTTTCTAA 12840 CATATGTCTA TAATATAGTG TTTAGGTTCT TTTTTTTTTTC AGGAATACAT TTGGAAATTC 12900 AAAACAATTG GCAAACTTTG TATTAATGTG TTAAGTGCAG GAGACATTGG TATTCTGGGC 12960 ACCTTCCTAA TATGCTTTAC AATCTGCACT TTAACTGACT TAAGTGGCAT TAAACATTTG 13020 AGAGCTAACT ATATTTTTAT AAGACTACTA TACAAACTAC AGAGTTTATG ATTTAAGGTA 13080 CTTAAAGCTT CTATGGTTGA CATTGTATAT ATAATTTTTT AAAAAGGTTT TCTATATGGG, 13140 GATTTTCTAT TTATGTAGGT AATATTGTTC TATTTGTATA TATTGAGATA ATTTATTTAA 13200 TATACTTTAA ATAAAGGTGA CTGGGAATTG TTACTGTTGT ACTTATTCTA TCTTCCATTT 13260 ATTATTTATG TACAATTTGG TGTTTGTATT AGCTCTACTA CAGTAAATGA CTGTAAAATT 13320 GTCAGTGGCT TACAACAACG TATCTTTTTC GCTTATAATA CATTTTGGTG ACTGTAGGCT 133R0 GACTGCACTT CTTCTCAATG TTTTCTCATT CTAGGATGCA AACCAATGGA GAAGCCCCTA 13440 ATTAGATCAG GGCAGAGGGA AAAACAAAAA ACTGGTAGAA ACCGGCAACC ACAGCTTCAA 13500 GCTTTAAGCC CATCTCCTAC ACTTCTGCTC TGTACGTGCC CATTGTCACT TCTGTTCACA 13560 TGC7ACTGTC CCAAGCAAGT GACCAAGCCT GACAA7ACTT TGTCTACTGG AGTCACTGCA 13620 AG CACATGA CGGGGCAGGG ATGTCGTCTT ACAGG3AAGA GAAAAGATAA TGCTCTCTAC 13680 TGCAGACTTG GAGAGATTTC TTCCCATTGG CAGTA3TTTG ACTAATTGGA GATGAGAAAA 13740 AAAG.AAACAT TCTTGGGATG ATTGTATTGA AACAAAATTA GGTAAAAGGA CAATATAGGA 13800 TAGGGAGAGA TATAAGTGGA ATGAGATCTC TAGAGTCCAT TAAAAGCAAG CTAGATTGAG 13860 13865 [2) INFORMATION FOR SEQ ID NO: 12: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 263 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) ) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 12: Ser Gly Thr Thr Asn Thr Val Wing Wing Tyr Asn Leu Thr Trp Lys Ser 1 '5 10.5 Thr Asn Phe Lys Ser Gly Asp Trp Lys Ser Lys 35 40 45 Cys Phe Tyr Thr Thr Thr Asp Thr Asp Glu He Val 50 55 60 Lys Asp Val Lye Gln Thr Tyr Leu Wing Arg Val Phe Ser Tyr Pro Wing 65 70 75 80 Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn 85 90 95 Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro .Thr 100 105 110 He Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu 115 120 125 Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg 130 135 140 Asp Val Phe Gly Lys Asp Leu He Tyr Thr Leu Tyr Tyr Trp Lys Ser 145 150 155 160 Be Ser Gly Lys Lys Thr Wing Lys Thr Asn Thr Asn Glu -Phe Leu 165 170 175 He Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Wing Val 180 185 190 He Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu 195 200 205 Cys Met Gly Gln Glu Lys Gly Glu Phe Arg Glu He Phe Tyr He He 210 215 220 Gly Ala Val Val Phe Val Val He He Leu Val He He Leu Ala He 225 230 235 240 Be Leu Kis Lys Cys Arg Lys Wing Gly Val Gly Glp Ser Trp Lys Glu 245 250 255 Asn Ser Pro Leu Asn Val Ser 260 • (2) INFORMATION FOR SEQ ID NO: 13: (i) CHARACTERISTICS OF THE SEQUENCE: (A ) LENGTH: 1440 base pairs (B) TYPE: nucleic acid (C) CHAIN TYPE: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 13: TCAACAGGCA GGGGCAGCAC TGCAGAGATT TCATCATGGT CTCCCAGGCC CTCAGGCTCC 60 TCTGCCTTCT GCTTGGGCTT CAGGGCTGCC TGGCTGCAGG CGGGGTCGCT AAGGCCTCAG 120 GAGGAGAAAC ACGGGACATG CCGTGGAAGC CGGGGCCTCA CAGAGTCTTC GTAACCCAGG 180 AGGAAGCCCA CGGCGTCCTG CACCGGCGCC GGCGCGCCAA CGCGTTCCTG GAGGAGCTGC 240 GGCCGGGCTC CCTGGAGAGG GAGTGCAAGG AGGAGCAGTG CTCCTTCGAG GAGGCCCGGG 300 AGATCTTCAA GGACGCGGAG AGGACGAAGC TGTTCTGGAT TTCTTACAGT GATGGGGACC 360 AGTGTGCCTC AAGTCCATGC CAGAATGGGG GCTCCTGCAA GGACCAGCTC CAGTCCTATA 420 TCTGCTTCTG CCTCCCTGCC TTCGAGGGCC GGAACTGTGA GACGCACAAG GATGACCAGC 480 TGATCTGTGT GAACGAGAAC GGCGGCTGTG AGCAGTACTG CAGTGACCAC ACGGGCACCA 5 0 AGCGCTCCTG TCGGTGCCAC GAGGGGTACT CTCTGCTGGC AGACGGGGTG TCCTGCACAC 600 CCACAGTTGA ATATCCATGT GGAAAAATAC CTATTCTAGA AAAAAGAAAT GCCAGCAAAC 660 CCCAAGGCCG AATTGTGGGG GGCA? GGTGT GCCCCAAAGG GGAGTGTCCA TGGCAGGTCC 720 TGTTGTTGGT GAATGGAGCT CAGTTGTGTG GGGGGACCCT GATCAACACC ATCTGGGTGG 760 TCTCCGCGGC CCACTGTTTC GACAAAATCA AGAACTGGAG GAACCTGATC GCGGTGCTGG 840 GCGAGCACGA CCTCAGCGAG CACGACGGGG ATGAGCAGAG CCGGCGGGTG GCGCAGGTCA 900 TCATCCCCAG CACGTACGTC CCGGGCACCA CCAACCACGA CATCGCGCTG CTCCGCCTGC 960 ACCAGCCCGT GGTCCTCACT GACCATGTGG TGCCCCTCTG CCTGCCCGAA CGGACGTTCT 1020 CTGAGAGGAC GCTGGCCTTC GTGCGCTTCT CATTGGTCAG CGGCTGGGGC CAGCTGCTGG 1080 ACCGTGGCGC CACGGCCCTG GAGCTCATGG TGCTCAACGT GCCCCGGCTG ATGACCCAGG 1140 ACTGCCTGCA GCAGTCACGG AAGGTGGGAG ACTCCCCAAA TATCACGGAG TACATGTTCT 1200 GTGCCGGCTA CTCGGATGGC AGCAAGGACT CCTGCAAGGG GGACAGTGGA GGCCCACATG 1260 CCACCCACTA CCGGGGCACG TGGTACCTGA CGGGCATCGT CAGCTGGGGC CAGGGCTGCG 1320 CAACCGTGGG CCACTTTGGG GTGTACACCA GGGTCTCCCA GTACATCGAG TGGCTGCAAA 1380 AGCTCATGCG CTCAGAGCCA CGCCCAGGAG TCCTCCTGCG AGCCCCATTT CCCTAGCCCA 1440 (2) INFORMATION FOR SEQ ID NO: 14: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 466 amino acids (B) TYPE: amino acid (C) TYPE OF CHAIN: (D) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 14: Met Val Ser Gln Ala Leu Arg Leu Leu Cys Leu Leu Leu Gly Leu Gln 1 5 10 15 Gly Cys Leu Wing Wing Gly Val Wing Lys Wing Wing Gly Gly Glu Thr 20 25 30 Arg Asp Met Pro Trp Lys Pro Gly Pro His Arg Val Phe Val Thr Gln 35 40 45 Glu Glu Wing His Gly Val Leu His Arg Arg Arg Arg Wing Asn Wing Phe 50 55 60 Leu Glu Glu Leu Arg Pro Gly Ser Leu Glu Arg Glu Cys Lys Glu Glu 65 70 75 80 Gln Cys Ser Phe Glu Glu Wing Arg Glu He Phe Lys Asp Wing Glu Arg 85 90 95 Thr Lys Leu Phe Trp He Ser Tyr Ser Asp Gly Asp Gln Cys Wing Ser 100 105 110 Ser Pro Cys Gln Asn Gly Gly Ser Cys Lys Asp Gln Leu Gln Ser Tyr 115 120 125 He Cys Phe Cys Leu Pro Wing Phe Glu Gly Arg Asn Cys Glu Thr Kis 130 135 140 Lys Asp Asp Gln Leu He Cys Val Asn Glu Asn Gly Gly Cys Glu Gln 145 150 155 160 Tyr Cys Ser Asp His Thr Gly Thr Lys Arg Ser Cys Arg Cys Kis Glu 165 170 175 Gly Tyr Ser Leu Leu Wing Asp Gly Val Ser Cys Thr Pro Thr Val Glu 180 185 190 Tyr Pro Cys Gly Lys He Pro He Leu Glu Lys Arg Asn Wing Ser Lys 195 200 205 Pro Gln Gly Arg He Val Gly Gly Lys Val Cys Pro Lys Gly Glu Cys 210 215 220 Pro Trp Gln Val Leu Leu Leu Val Asn Gly Wing Gln Leu Cys Gly Gly 225 230 235 240 Thr Leu He Asn Thr He Trp Val_Val Ser Wing Wing His Cys Phe Asp 245 250 255 Lys He Lys Asn Trp Arg Asn Leu He Wing Val Leu Gly Glu His Asp 260 265 270 Leu Ser Glu Kis Asp Gly Asp Glu Gln Ser Arg Arg Val Wing Gln Val 2"* = 280 2S5 He He Pro Ser Thr Tyr Val Pro Gly Thr Thr Asn His Asp He Wing 290 295 300 Leu Leu Arg Leu His Gln Pro Val Val Leu Thr Asp His Val Val Pro 305 310 315 320 Leu Cys Leu Pro Glu Arg Thr Phe Ser Glu Arg Thr Leu Ala Phe Val 325 330 335 Arg Phe Ser Leu Val Ser Gly Trp Gly Gln Leu Leu Asp Arg Gly Wing 340 345 350 Thr Wing Leu Glu Leu Met Val Leu Asn Val Pro Arg Leu Met Thr Gln 355 360 365 Asp Cys Leu Gln Gln Ser Arg Lys Val Gly Asp Ser Pro Asn He Thr 370 375 380 Glu Tyr Met Phe Cys Wing Gly Tyr Ser Asp Gly Ser Lys Asp Ser Cys 385 390 395 400 Lys Gly Asp Ser Gly Gly Pro His Wing Thr His Tyr Arg Gly Thr Trp 405 410 415 Tyr Leu Thr Gly He Val Ser Trp Gly Gln Gly Cys Wing Thr Val Gly 420 425 430 His Phe Gly Val Tyr Thr Arg Val Ser Gln Tyr He Glu Trp Leu Gln 435 440 445, and s Leu Met Arg Ser Glu Pro Arg Pro Gly Val Leu Leu Arg Ala Pro 450 455 460 Phe Pro 465 (2) INFORMATION FOR SEQ ID NO: 15: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 27 base pairs (B) TYPE: nucleic acid (OR CHAIN TYPE: simple (C) TOPOLOGY: linear ( xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 15: GTCATGCCAT GGCCTCAGGC ACTACAA 27 (2) INFORMATION FOR SEQ ID NO: 16: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: acid n.ucl.eic z Ci ^ (C) TYPE OF CHAIN: simple . - (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 16: TGACAAGCTT ATTCTCTGAA TTCCCCTTTC. T .: ^ 31 (2) INFORMATION FOR SEQ ID NO: 17: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 47 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 17: CTCATGCCAT GGCCCTGGTG CCTCGTGCTT CTGGCACTAC AAATACT 47 (2) INFORMATION FOR SEQ ID NO: 18: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 18: GTCATGCCAT GGCCCTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT 50 (2) INFORMATION FOR SEQ ID NO: 19: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 53 base pairs ( B) TYPE: nucleic acid (C) CHAIN TYPE: simple_ (C) TOPOLOGY: linear (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 19: CGCGGATCCA CCGCCACCAG ATCCACCGCC TCCTTCTCTG AATTCCCCTT TCT53 (2) INFORMATION FOR SEQ ID NO: 20:. (i) CHARACTERISTICS OF THE SEQUENCE: -. (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear -. (xi) SEQUENCE DESCRIPTION: SEQ ID N0: -2a: CGCGGATCCG GCGGTGGAGG CTCTTCAGGC ACTACAAATA CTGT 44 (2) INFORMATION FOR SEQ ID NO: 21: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 21: TGACAAGCTT ATTCTCTGAA TTCCCCTTTC T 31 (2) INFORMATION FOR SEQ ID NO: 22: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 50 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 22: GTCATGCCAT GGCCCTGGTG CCTCGTGGTT CTTGCGGCAC TACAAATACT 50 (2) INFORMATION FOR SEQ ID NO: 23: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 31 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 23: TGACAAGCTT ATTCTCTGAA TTCCCCTTTC T 31 (2) INFORMATION FOR SEQ ID NO: 24: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 24: GTCATGCCAT GGCCCTGGTG CCTCGTGGTT GCACTACAAA TACT 44 (2) INFORMATION FOR SEQ ID NO: 25: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 34 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 25: TGACAAGCTT AGCATTCTCT GAATTCCCCT TTCT 34 (2) INFORMATION FOR SEQ ID NO: 26: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 19 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 26: CAAGTTCAGC CAAGAAAAC (2) INFORMATION FOR SEQ ID NO: 27: (i) CHARACTERISTICS OF THE SEQUENCE: (A) LENGTH: 36 base pairs (B) TYPE: nucleic acid (C) TYPE OF CHAIN: simple- (C) TOPOLOGY: linear (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 27: ACACTTTATT ATCGGAAATC TTCAGCTTCA GGAAAG 36

Claims (9)

1. NOVEPAD DB LA IFLVBUCIQN Having described the foregoing invention, the following CLAIMS are considered as a novelty and, therefore, claimed as property 1. A composition comprising a biologically effective amount of a deficient coagulation tissue factor compound that it is at least 100 times less active in coagulation than the original full-length tissue factor, which has been modified to increase its biological half-life; characterized in that the modification does not consist in binding the Tissue Factor compound to an antibody or an antigen that binds the region thereof that binds to the diseased cell, a component of the vasculature associated with the disease or a component of the stroma associated with the disease.
2. 2. The composition of culture with that claimed in claim 1, for its use to promote coagulation preferentially in the tumor vasculature of an animal.
3. 3. The use of a composition according to claim 1 or 2 in the manufacture of a medicament for use in the treatment of vascularized tumors by promoting coagulation in the vasculature of an animal's tumor.
4. 4. The use of a composition comprising a biologically effective amount of coagulation tissue factor-in-deficient compound in the manufacture of a medicament for use in the treatment of vascularized tumors by promoting coagulation in the vasculature of an animal's tumor; characterized in that the deficient coagulation tissue factor compound is at least about 100 times less active in coagulation than the original, full-length tissue factor, and is not bound to an antibody binding region or antigen thereof which is one to a diseased cell, a vasculature component associated with the disease or a stromal component associated with the disease.
5. 5. The use of conformity: as claimed in claim 4, characterized in that the deficient coagulation tissue factor compound has been modified to increase its biological half-life. 6 ^. The use according to claim 1, in any of claims 3, 4 or 5 characterized in that the deficient coagulation tissue factor compound has been modified to increase its biological half-life by binding to a protein carrier molecule or not. protein. The use according to claim 6, characterized in that the deficient coagulation tissue factor compound binds to an albumin or globulin carrier molecule. 8. Use according to claim 6, characterized in that the deficient coagulation tissue factor compound binds to an antibody, or to a portion thereof. 9. The use according to claim 8, characterized in that the deficient coagulation tissue factor compound binds to an immunoglobulin G antibody, an Fc portion of an antibody or is inserted into an immunoglobulin G molecule in place of the CH3 domain. The use as claimed in any of claims 3 to 9, characterized in that the deficient coagulation tissue factor compound is between about 100 times and about 1,000,000 times less active in coagulation than the tissue factor. original, full length. 11. The use as claimed in any of claims 3 to 10, characterized in that the deficient coagulation tissue factor compound is at least about 1,000 times less active coagulation than the original tissue factor, full length 12. The use as claimed in any of claims 3 to 11, characterized in that the deficient coagulation tissue factor compound is at least 10,000 times less active coagulation than the original tissue factor., full length. 13. The use as claimed in any of claims 3 to 12, characterized in that the deficient coagulation tissue factor compound is at least 100,000 times less active coagulation than the original tissue factor, in length complete 14. Use according to claim 3, characterized in that the deficient coagulation tissue factor compound is at least 500,000 times less active coagulation than the original tissue factor, in length. complete 15. The use as claimed in any of claims 3 to 14, characterized in that the deficient coagulation tissue factor compound is at least 1,000,000 times less active coagulation than the original tissue factor, in length. complete 1
6. 6. The use as claimed in any of claims 3 to 15, characterized in that the deficient coagulation tissue factor compound is a mutant tissue factor compound deficient in its ability to activate Factor VII. . 1
7. 7. The use as claimed in claim 16, characterized in that the deficient coagulation tissue factor compound is a mutant tissue factor compound that includes at least one first mutation in the amino acid region between approximately the position 157 and approximately position 167 of Identification Sequence No. 1. 1
8. 8. The use as claimed in claim 17, characterized in that the deficient coagulation tissue factor compound is a mutant tissue factor compound in which Trp at position 158 changes to Arg; Being in position 162 changes to Ala, - Gly in position 164 changes to Ala; or in which Trp in the position 158 changes to Arg and Ser in position 162 changes to Ala. 1
9. 9. The use as claimed in any of claims 3 to 18, characterized in that the deficient coagulation tissue factor compound is a deficient tissue factor compound for binding to a phospholipid surface. 20. The use as claimed in any of claims 3 to 19, characterized in that the deficient coagulation tissue factor compound is a truncated tissue factor compound. 21. The use as claimed in any of claims 3 to 20, characterized in that the deficient coagulation tissue factor compound is a truncated tissue factor compound of approximately 219 amino acids in length. 22. The use as claimed in any of claims 3 to 21, characterized in that the deficient coagulation tissue factor compound is a homodimeric, heterodimeric or polymeric tissue factor compound. 23. The use as claimed in any of claims 3 to 22, characterized in that the deficient coagulation tissue factor compound is: (a) a mutant tissue factor compound consisting essentially of. amino acid sequence Identification Sequence No. 8 or Identification Sequence No. 9; or (b) a truncated Tissue Factor compound consisting essentially of the amino acid sequence Identification Sequence No. 1 24. The use as claimed in any of claims 3 to 23, characterized in that the deficient coagulation tissue factor compound is a human tissue factor compound. 25. The use as claimed in any of claims 3 to 24, characterized in that the deficient coagulation tissue factor compound is a tissue factor compound prepared by recombinant expression. 26. The use according to claim as claimed in any of claims 3 to 25, characterized in that the composition comprises a second deficient coagulation tissue factor compound. 27. The use according to claim as claimed in any of claims 3 to 26, in combination with a biologically effective amount of at least one Factor Vlla or a Factor VII activator. 28. Use according to claim 27, in combination with Factor Vlla. 29. The use according to claim 28, characterized in that the Vlla Factor consists essentially of the amino acid sequence from amino acid 61 to amino acid 212 of the polypeptide sequences of Factor VII of the Identification Sequence. DO NOT. 14. The use as claimed in any of claims 27 to 29, characterized in that the deficient coagulation tissue factor compound is combined with Vlla Factor in a preformed tissue factor-Vlla factor complex. . 31. The use as claimed in any of claims 3 to 30, in combination with a therapeutically effective amount of an anticancer substance. 32. Use according to claim 31, characterized in that the anticancer substance is a chemotherapeutic substance. 33. Use in accordance with the claim in claim 32, characterized in that the anticancer substance is a chemotherapeutic substance listed in Table II. 34. Use according to claim 33, characterized in that the anticancer substance is etoposide. 35. The use according to claim 31, characterized in that the anticancer substance is an antibody construct comprising an antibody or antigen binding region that specifically binds to a component of a tumor cell, tumor vasculature. or tumor stroma, the antibody is operatively linked to a cytotoxic agent or to a coagulation factor. 36. The use as claimed in claim 35, characterized in that the anticancer substance is an antibody construct that specifically binds to a tumor vasculature or tumor stroma component. 37. The use as claimed in claim 35, characterized in that the anticancer substance is an antibody construct comprising a cytotoxic substance. 38. The use as claimed in claim 35, characterized in that the anticancer substance is an antibody construct comprising a coagulation factor. 39. The use as claimed in claim 38, characterized in that the anticancer substance is an antibody construct comprising Tissue Factor or a Tissue Factor derivative. 40. The use according to claim claimed in any of claims 3 to 39, characterized in that the drug is for use in the promotion of coagulation in the tumor vasculature associated with a malignant tumor of medium or large size vascularized of an animal. 41. The use according to claim claimed in any of claims 3 to 40, characterized in that the medicament is formulated for systemic administration to an animal. 42. Use as claimed in any of claims 3 to 41, characterized in that the medicament is formulated for intravenous injection into the animal. 43. The use according to any of claims 3 to 42, characterized in that the medicament is intended for administration to a human subject. 44. A therapeutic kit comprising, in a convenient container element: (a) a biologically effective combination of a composition in accordance with claim 1 or claim 2, and at least one among Factor Vlla, an activator of Factor VII or an anticancer substance; or (b) a biologically effective combination of an anticancer substance and a deficient coagulation tissue factor compound that is at least 100 times less active in coagulation than the original, full-length tissue factor, and is not bound to a region of antibody or antigen binding thereof that binds to a diseased cell, a component of the vasculature associated with the disease or a stromal component associated with the disease. 45. The kit according to claim 44, comprising a biologically effective combination of a composition according to claim 1 or 2, and at least one between Factor Vlla, an activator of Factor VII or an anticancer substance. 46. The kit according to claim 44, which comprises a biologically effective combination of an anticancer substance and a deficient coagulation tissue factor compound that is at least about 100 times less active in coagulation than a coagulation factor. Original, full-length tissue, and does not bind to an antibody or antigen binding region thereof that binds to a diseased cell, a vasculature component associated with the disease or a stromal component associated with the disease. 47. The kit according to claim 44, which comprises a biologically effective combination of (a) a deficient coagulation tissue factor compound that is at least 100 times less active in coagulation than the original tissue factor. , full length, and is not bound to an antibody or antigen binding portion thereof that binds to a diseased cell, a vasculature component associated with the disease or a stromal component associated with the disease; (b) at least one between the Factor Vlla or a Factor VII activator; and (c) an anticancer substance.