MX2008005011A - Treatment for antiphospholipid-syndrome-related pregnancy complications - Google Patents

Abstract

The present application provides compositions and methods for treating antiphospholipid-syndrome-related pregnancy complications with tissue factor antagonists.

Description

TREATMENT FOR PREGNANCY COMPLICATIONS RELATED TO THE ANTIPHOSPHOLIPIDIC SYNDROME FIELD OF THE INVENTION The present invention relates to methods for treating pregnancy complications related to the antiphospholipid syndrome. BACKGROUND OF THE INVENTION The antiphospholipid syndrome (APS) is characterized by the presence of antiphospholipid antibodies (aPL), which are associated with thrombosis. PHC is also a leading cause of miscarriage and maternal and fetal morbidity. In women with PHC, pregnancies that occur in the third trimester have a high incidence of preeclampsia and subsequent restriction of fetal intrauterine growth, placental rupture and premature birth (Lima et al., Clin. Exp. Rheumatol, 14: 131-36 (nineteen ninety six)). In vivo and in vitro studies have shown that aPL antibodies trigger the activation of endothelial cells, monocytes, neutrophils and platelets, and cause inflammation, thrombosis and tissue damage. Several hypotheses have been proposed to explain the mechanism by which aPL antibodies promote thrombosis, including roles for both procoagulant and anticoagulant effects (Levine et al., N. Engl. J. Med., 346: 752-63 (2002)). ). A first theory involves the activation of endothelial cells, a second focuses on damage mediated by oxidants to the vascular endothelium, and a third proposes that aPL antibodies affect the function of the proteins involved in the regulation of coagulation (Levine et al. , supra). Although a role for the tissue factor in the pathogenesis of thrombosis related to the aPL antibody has been suggested, it remains unclear which of these mechanisms are responsible for their various pathologies (Amengual et al., Thromb. Haemost., 79: 276 -81 (1998)). However, previous studies of proteins involved in CO3. CJulación concluded that the trajectories of the placental coagulant involving the tissue factor, thrombomodulin, and annexin V, do not contribute to pregnancy complications related to APS (Lakasing et al., Am. J. Obstet. Gynecol., 181: 180- 89 (1999)). The anticoagulant heparin is the standard treatment used to prevent obstetric complications in pregnant women with APS. However, treatment with heparin is not completely effective and has some risks (Ahmed et al., Am. J. Med. Sci., 324 (5): 279-80 (2002)). Accordingly, there is a need for additional compositions and methods to treat pregnancy complications associated with APS. SUMMARY OF THE INVENTION The present invention is based in part on the surprising discovery that treatment with tissue factor antagonists blocks pregnancy complications induced by APS, including fetal loss and growth restriction. In some embodiments, the invention provides a method for treating a patient at risk for pregnancy complications related to APS, which comprises administering to said patient a tissue factor antagonist. In some embodiments, the tissue factor antagonist antagonizes an activity of the tissue factor selected from the group consisting of: binding to factor VII, binding to the factor Vlla, carrying out the proteolysis of factor VII, carrying out the proteolysis of factor IX, and carrying out the proteolysis of factor X. In some embodiments, the tissue factor antagonist is an antibody, including a monoclonal antibody. In some embodiments, the tissue factor antagonist is a peptide. In some embodiments, the tissue factor antagonist is administered in the second trimester of pregnancy. In some embodiments, the method further comprises administering to the patient an anticoagulant, including heparin. In some embodiments, the invention provides a kit comprising: (a) a tissue factor antagonist; (b) a container containing said tissue factor antagonist; and (c) a label fixed to said container, or a packaging insert included in said container, referring to the use of said antagonist in the treatment of pregnancy complications related to APS. In some embodiments, the tissue factor antagonist present in the kit is an antibody, including a monoclonal antibody. In some embodiments, the tissue factor antagonist is a peptide. In some embodiments, the kit further comprises an anticoagulant, including heparin. DETAILED DESCRIPTION OF THE PREFERRED MODALITIES The tissue factor (TF) is an integral membrane glycoprotein, which is the cofactor expressed on the cell surface for the serine protease factor Vlla (FVIIa). TF triggers blood coagulation by combining FVIIa to activate substratum factors VII IX, and X. The following coagulation reactions result in the formation of a polymerized fibrin network and aggregates of platelets, which together form a hemostatic plug. TF / FVIIa activity has been implicated in numerous diseases, including cardiovascular diseases, cancer growth and metastasis, tumor angiogenesis and in inflammatory diseases, such as sepsis, rheumatoid arthritis, and sickle cell anemia. Increased general expression of TF by endothelial cells or monocytes has been observed in patients with APS (Amenguar et al., Supra), but no diffee was observed in placentas from women with APS compared to controls (Lakasing et al. al., supra). As used herein, the term "antiphospholipid syndrome" or "APS" refers to a clinical association between antiphospholipid antibodies and a hypercoagulability syndrome (Levine et al., N. Eng. J. Med., 346: 752-63 (2002)). As used herein, the term "pregnancy complications related to the antiphospholipid syndrome" or "pregnancy complications related to PSA" refers to an increase in fetal resorption, decrease in fetal weight and / or increase in the frequency of abortions in a female mammal with antiphospholipid syndrome. In humans, the criteria for classifying a patient who has pregnancy complications related to APS include the presence of aPL antibodies and: (1) one or more unexplained morphologically normal fetuses on or after the 10th week of gestation: o (2) one or more premature births of morphologically normal neonates on or before the 34th week of gestation; or (3) three or more consecutive unexplained spontaneous abortions or miscarriages before the 10th week of gestation (Levine et al., supra).

As used herein, the terms "TF", "tissue factor", "tissue factor protein" and "mammalian tissue factor protein" refer to a polypeptide having an amino acid sequence corresponding to a mammalian tissue factor of natural origin (eg, U.S. Patent No. 6,274,142; Fisher et al., Thromb. Res., 48: 89-99 (1987); Morrisey et al., Cell, 50: 129-35 ( 1987)). TF occurs naturally in humans as well as in other animal species such as rabbits, rats, pigs, non-human primates, horses, mice and sheep (see, eg, Hartzell et al., Mol. Cell. Biol., 9: 2567 -73 (1989), Andrews et al., Gene 98: 265-69 (1991), and Takayenik et al., Biochem. Biophys. Res. Comm., 181-1145-50 (1991)). The amino acid sequences of mammalian tissue factor proteins are known or can be obtained generally by conventional techniques. As used herein, the terms "TF antagonist" and "tissue factor antagonist" refer to a substance that inhibits or neutralizes TF activity. Such antagonists can achieve this effect in various ways and do not need to act directly on TF. First, a class of TF antagonists binds to the tissue factor protein with sufficient affinity and specificity to inhibit its ability to bind factor VII or Vlla or effect proteolysis of factors VII, IX or X when they are in complex with factor VII or Vlla. Included within this group of molecules are certain antibodies and antibody fragments (such as, for example, F (ab) or F (ab ') 2 molecules). Another class of TF antagonists antagonizes TF activity by creating a complex of molecules, eg, the pathway inhibitor-1 of the naturally occurring tissue factor (TFPI-1) comprising the lipoprotein-associated coagulation inhibitor that forms a complex inactive of TF, factor VII, factor X and phospholipids (see, eg, Broze et al., Proc. Nati .. Acad. Sci., USA 84: 1886-90 (1987)). Another class of TF antagonists are fragments of TF protein, fragments of factor VII or small organic molecules, ie, peptides or peptidomimetics, which bind to TF, thus inhibiting the formation of the TF-factor VII complex or inhibiting the activation of TF. factors IX and X by TF and FVII / FVIIa (see, eg, WO 01/01749, WO 01/10892, and US Patent Publication 2001/0048924). Still another class of TF antagonists will deactivate the TF protein or tissue factor / Vlla factor complex by cleavage, e.g., a specific protease. A fifth class of antagonists blocks the binding of TF protein to factor VII, e.g., a factor VII antibody directed against a factor VII domain that is involved in TF binding.

As used herein, the term "patient" for purposes of treating, alleviating the symptoms of, or diagnosing pregnancy complications related to APS, refers to any mammal, including humans, domestic and farm animals, and zoo, sports or pet animals, such as dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, chimpanzees, baboons, monkeys, etc. Preferably, the patient is human. As used herein, the term "anticoagulant" refers to a substance that can prevent, inhibit or prolong blood coagulation in an in vitro or in vivo analysis of blood coagulation. Anticoagulants include, e.g., heparin and aspirin. Blood coagulation assays are known in the art and include, for example, prothrombin time analysis, the human model of ex vivo thrombosis described by Kirchhofer et al., Arterioscler. Thromb. Vasc. Biol., 15: 1098-1106 (1995): and Kirchhofer et al., J. Clin. Invest., 93: 2073-83 (1994), and analysis based on the measurement of factor X activation in human plasma. In some embodiments of the invention, an antibody is used for the tissue factor protein. As used herein, the term "anti-tissue factor antibody" or "ATF Ab" refers to an antibody that specifically binds to a tissue factor. In some embodiments, the ATF Ab is an antibody described in PCT publication WO 01/70984. The term "antibody" (Ab) is used herein in the broadest sense and specifically covers, for example, single monoclonal antibodies, multispecific antibodies (such as wild-type bispecific antibodies), antibody compositions with polyepitopic specificity, polyclonal antibodies, antibodies single-chain, and antibody fragments (see below) as long as they specifically bind to a natural polypeptide and / or exhibit a biological activity or an immunological activity of this invention. As used herein, an "intact" antibody is one that comprises an antigen binding site as well as a CL and at least the constant heavy chain domains CH1, CH2, and CH3. The constant domains may be constant domains of native sequence (e.g., constant domains of human native sequence) or a variant of amino acid sequence thereof. Preferably, the intact antibody has one or more effector functions. As used herein, a "species-dependent antibody" is an antibody that has a stronger binding affinity for an antigen of a first mammalian species than it does for a homologue of that antigen of a second species of mammal. mammal. Typically, the species-dependent antibody "specifically binds" to the human antigen (ie, has a binding affinity value (Kd) of no more than about 1 x 1CT7 M, preferably no more than about 1 x 1CT8 and greater preference of no more than about 1 x 10"9 M) but has a binding affinity for a homolog of the antigen of a second non-human mammal species that is at least about 50 times, or at least about 500 times, or at less than 1000 times weaker than if binding affinity for the human antigen The antibody dependent on the species can be of any of several types of antibodies as defined above, but preferably is a humanized or human antibody. present, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical except for possible mutations of natural origin that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant in the antigen. In addition to its specificity, monoclonal antibodies are advantageous in that they can be synthesized uncontaminated by other antibodies. The "monoclonal" modifier should not be interpreted as requiring the production of the antibody by any particular method. For example, monoclonal antibodies useful in the present invention can be prepared by the hybridoma methodology first described by Kohler et al., Nature 256: 495 (1975), or can be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Patent No. 4,816,567). Anti-TF monoclonal antibodies have been prepared (see e.g., Carson et al., Blood 66 (1): 152-56 (1985)). "Monoclonal antibodies" can also be isolated from phage libraries of antibodies using, for example, the techniques described in Clackson et al., Nature 352: 624-28 (1991), Marks et al., J. Mol. Biol. , 222: 581-97 (1991). Monoclonal antibodies herein include "chimeric" antibodies that in a portion of the heavy and / or light chain are identical with or homologous to the corresponding sequences in antibodies derived from a particular species or belonging to a particular class or subclass of antibody , although the rest of the chain (s) is identical with or homologous to the corresponding sequences in antibodies derived from another species or belonging to another class or subclass of antibodies, as well as fragments of such antibodies, provided that they exhibit activity of TF antagonist (see U.S. Patent No. 4, 816, 567; and Morrison et al., Proc. Nati Acad. Sci. USA 81: 6851-55 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies that comprise variable domain antigen binding sequences derived from a non-human primate (e.g., Monkey World, simian, etc.) and human sequences of constant region. . In the hybridoma method, a mouse or other appropriate host animal, such as a hamster, is immunized to emit lymphocytes that produce or are capable of producing antibodies that specifically bind to the protein used for immunization. Alternatively, the lymphocytes can be immunized in vitro. After immunization, the lymphocytes are isolated and then fused with a myeloma cell line using a suitable fusion agent, such as polyethylene glycol, to form a hybridoma cell (Goding, Monoclonal Antibodies: Principles and Practice, (Antibodies). monoclonal: principles and practice) pp. 59-103 (Academic Press, 1986)). The hybridoma cells thus prepared are seeded and cultured in a suitable culture medium, which medium preferably contains one or more substances that inhibit the growth or survival of the original unfused myeloma cells (also referred to as a fusion partner). For example, if the original myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the selective culture medium for the hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), whose substances prevent the growth of cells deficient in HGPRT. The culture medium in which the hybridoma cells are grown is analyzed by the production of monoclonal antibodies directed against the antigen. Preferably, the binding specificity of the monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). The binding affinity of the monoclonal antibody, for example, can be determined by the Scatchard analysis described in Munson et al., Anal. Biochem. , 107: 220 (1980). Once the hybridoma cells that produce antibodies of the specificity, affinity, and / or desired activity have been identified, the clones can be subcloned by limiting the dilution procedures and cultured by standard methods (Goding, Monoclonal Antibodies: Principles and Practice (Monoclonal Antibodies: Principles and practices) pp. 59-103 (Academic Press, 1986.) Culture media suitable for this purpose include, for example, D-MEM or RPMI-1640 medium.In addition, hybridoma cells can be cultured in vivo as growth tumors. ascites in an animal, eg, by ip injection of the cells in mice Monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional methods of antibody purification such as eg , affinity chromatography (eg, using protein A or protein G-Sepharose) or exchange chromatography ion, hydroxylapatite chromatography, gel electrophoresis, dialysis, etc. The DNA encoding the monoclonal antibodies is easily isolated and sequenced using conventional methods (e.g., using oligonucleotide probes that are capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). Hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed within expression vectors, which are then transfected into host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that, otherwise, they do not produce antibody protein, to obtain the synthesis of the monoclonal antibodies in the recombinant host cells. Review of the articles on recombinant expression in DNA bacteria encoding the antibody include Skerra et al., Curr. Opin. In Immunol. , 5: 256-62 (1993) and Plückthun, Immuno. Revs. , 130: 151-88 (1992). Monoclonal antibodies or antibody fragments can also be isolated from phage libraries of generated antibodies, using the techniques described in McCafferty et al., Nature 348: 552-554 (1990). Clackson et al., Nature 352: 624-28 (1991) and Marks et al., J. Mol. Biol. , 222: 581-97 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity human antibodies (nM range) by chain drag (Marks et al., Bio / Technology 10: 779-83 (1992)), as well as combinatorial infection and in vivo recombination as a strategy to construct very large phage libraries (Waterhouse et al., Nucí Acids Res., 21: 2265-66 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for the isolation of monoclonal antibodies.

The DNA encoding the antibody can be modified, for example, by substituting the heavy chain and light chain human constant domain sequences (CH and CL) for the homologous murine sequences (US Patent No. 4,816,567; and Morrison et al. , Proc. Nati, Acad. Sci. USA 81: 6851 (1984)), or by fusing the immunoglobulin coding sequence with all or part of the coding sequence for a non-immunoglobulin polypeptide. The non-immunoglobulin polypeptide sequences can be replaced by the constant domains of an antibody, or substituted by the variable domains of an antigen combining site of an antibody to create a chimeric bivalent antibody comprising an antigen combining site having specificity for an antigen and another antigen combination site that has specificity for a different antigen. The "humanized" forms of non-human antibodies (e.g., from rodents) are chimeric antibodies that contain minimal sequences derived from non-human antibodies. Methods for humanizing non-human antibodies have been described in the art. Preferably, a humanized antibody has one or more amino acid residues introduced therein from a source that is non-human. These non-human amino acid residues are often referred to as "imported" residues that are typically taken from an "imported" variable domain. Humanization can be carried out essentially following the method of inter and collaborators (Jones et al., Nature 321: 522-25 (1986); Reichmann et al., Nature 332: 323-27 (1988); Verhoeyen et al., Science 239: 1534-36 (1988)), substituting the hypervariable region sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567) wherein substantially less than an intact human variable domain has been replaced by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are replaced by residues of analogous sites in rodent antibodies. The selection of the human variable domains, both light and heavy, for use in the preparation of humanized antibodies, is very important in reducing the antigenicity and the HAMA (human anti-mouse antibody) response when the antibody is intended for human therapeutic use. According to the method called "best fit", the variable domain sequence of a rodent antibody is visualized against the full library of known human variable domain sequences. The human V domain sequence that is closest to that of the rodent is identified and the region of human structure (FR) within it is accepted for the humanized antibody (Sims et al., J. Immunol. 151: 2296 ( 1993), Chothia et al., J. Mol. Biol., 1996: 901 (1987)). Another method uses a region of particular structure derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chains. The same structure can be used for several different humanized antibodies (Carter et al., Proc. Nati. Acad. Sci. USA 89: 4285 (1992); Presta et al., J. Immunol., 151: 2623 (1993)). It is also important that the antibodies are humanized with retention of high binding affinity for the antigen and other favorable biological properties. To achieve this goal, humanized antibodies, e.g., can be prepared by a process of analysis of the original sequences and several conceptual humanized products using three-dimensional models of the original and humanized sequences. Three-dimensional immunoglobulin models are commonly available and familiar to those skilled in the art, computerized programs are available that illustrate and display probable three-dimensional conformational structures of the selected candidate immunoglobulin sequences. The inspection of these deployments allows the analysis of the probable role of the residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this manner, the FR residues can be selected and combined from the recipient and imported sequences so that the desired antibody characteristic is achieved, such as an increase in affinity for the target antigen (s). In general, the hypervariable region residues are directly and more substantially involved in influencing antigen binding. A fully humanized anti-TF antibody has been produced, D3H44 (Presta et al., Thromb. Haemost., 85, 379-79 (2001)). Consistent with the excellent potency of the murine D3 antibody from which it was derived (Kirchhofer et al., Thromb. Haemost., 84 1072-81 (2000)), D3H44 is a potent anticoagulant both in vitro (Presta et al., (2001) supra) as in vivo (Bullens et al., Thromb. Haemost., (Suppl.) Extract # P1388 (2001)). As an alternative to humanization, human antibodies can be generated. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, it has been reported that homologous deletion of the heavy chain binding region gene of the antibody (JH) in chimeric and mutant germline mice results in complete inhibition of endogenous antibody production. The transfer of the human germline immunoglobulin gene array within germline mutant mice will result in the production of human antibodies to challenge with antigen. See, e.g., Jakobovits et al., Proc. Nati Acad. Sci. USA 90: 2551 (1993); Jakobovits et al., Nature 362: 255-58 (1993); Bruggemann et al., Year in Immuno., 7:33 (1993); Patents of E.ü. Nos. 5,545,806, 5,569,825, 5,591,669, 5,545,807; and WO 97/17852. Alternatively, phage display technology (McCafferty et al., Nature 348: 552.53 (1990)) can be used to produce human antibodies and antibody fragments in vitro, from repertoires of the non-donor immunoglobulin variable domain (V) gene. immunized. According to this technique, the antibody domain V genes are cloned into a framework protein gene either larger or smaller than a filamentous bacteriophage, such as M13 or fd, and deployed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also result in the selection of the gene encoding the antibody that exhibits those properties.

Thus, the phage mimics some of the properties of the B cell. The phage display can be carried out in a variety of formats, reviewed, e.g., in Johnson and Chiswell, Curr. Opin. Struct. Biol. , 3: 564-71 (1993). Several sources of the V gene segments can be used for phage display. Clackson et al., Nature 352: 624-28 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. A repertoire of V genes from immunized human donors can be constructed and antibodies to a diverse array of antigens (including autoantigens) can be isolated essentially following the techniques described by Marks et al., J. Mol. Biol., 222: 581-97 (1991), or Griffith et al., EMBO J. 12: 725-34 (1993). See, also US Patents. Nos. 5,565,332 and 5,573,905. As discussed above, human antibodies can also be generated by human B cells activated in vitro (see, U.S. Patents 5,567,610 and 5, 229, 275). In certain circumstances, there are advantages of the use of antibody fragments, instead of whole antibodies. For example, the smaller size of the fragments allows a quick clearance and can lead to better access to certain tissues. The "antibody fragments" comprise a portion of an intact antibody, preferably the antigen binding or the variable region of the intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab') 2 and Fv fragments; diabodies; linear antibodies (see, e.g., U.S. Patent No. 5,641,870, Example 2; Zapata et al., Protein Eng., 8 (10): 1057-62 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. The term "linear antibodies" refers generally to the antibodies described in Zapata et al., (19.95) supra. Briefly, these antibodies comprise a pair of serial Fd segments (VH-CH1-VH-CH1), which, together with complementary light chain polypeptides, form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific. The papain digestion of the antibodies produces two identical antigen binding fragments called "Fab" fragments, and a residual "Fe" fragment, a designation that reflects the ability to easily crystallize. The Fab fragment consists of a complete L chain together with the variable region domain of the H chain (VH), and the first constant domain of a heavy chain (CH1). Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen-binding site. Each Fab fragment is monovalent with respect to antigen binding, i.e., has a single antigen binding site. Pepsin treatment of an antibody produces a single large F (ab ') 2 fragment corresponding approximately to two disulfide-linked Fab fragments which have a bivalent antigen-binding activity and which is still capable of cross-linking the antigen. Fab 'fragments differ from Fab fragments because they have few additional residues at the carboxy terminus of the CH1 domain including one or more cysteines from the antibody's articulation region. Fab '-SH is the designation herein for Fab' in which the cysteine residue (s) of the constant domains contain (s) a free thiol group. The F (ab ') 2 antibody fragments were originally produced as pairs of Fab' fragments having articulation cysteines between them. Other chemical couplings of antibody fragments are also known. The Fe fragment comprises the carboxy termination portions of both H chains held together by disulfides. The effector functions of the antibodies are determined by the sequences in the Fe region, whose region is also the part recognized by the Fe (FcR) receptors found. in certain types of cells. "Fv" is the minimum antibody fragment that contains a complete site of antigen recognition and antigen binding. This fragment consists of a dimer of a variable region domain of a heavy chain and a light one in close non-covalent association. From the doubling of these two domains emanate six hypervariable circuits (3 circuits of each of the H and L chain) that contribute to the amino acid residues by binding to the antigen and confer specificity of antigen binding to the antibody. Neverthelesseven a single variable domain (or half of an Fv comprising only three specific CDRs for an antigen) has the ability to recognize and bind the antigen, albeit at a lower affinity than the entire binding site. "Single-chain Fv", also abbreviated as "sFv" or "scFv", are fragments of antibody comprising the VH and VL antibody domains connected within a single chain of the polypeptide. Preferably, the sFv polypeptide further comprises a polypeptide linkage between the VH and VL domains that allows the sFv to form the desired structure for antigen binding. For a review of sFv, see Plückthun in The Pharmacology of Monoclonal Antibodies (The Pharmacology of Monoclonal Antibodies), vol. 113, Rosenburg and Moore eds. , Springer-Verlag, New York, pp. 269-315 (1994). The term "diabodies" refers to small antibody fragments prepared by constructing sFv fragments (see preceding paragraph) with short links (approximately 5-10 residues) between the VH and VL domains in such a way that inter-pair formation is achieved. chains but not intra-chains of the V domains, resulting in a bivalent fragment, ie, a fragment having two antigen binding sites. The bispecific diabodies are heterodimers of two "crossed" sFv fragments in which the VH and VL domains of the two antibodies are present in different polypeptide chains. The diabodies are more fully described, for example, in EP 404, 097; WO 93/11161; and Hollinger et al., Proc. Nati Acad. Sci., USA 90: 6444-48 (1993). Bispecific antibodies are antibodies that have binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes of the tissue factor. Other such antibodies can combine a tissue factor binding site with a binding site for another protein. Methods for producing bispecific antibodies are known in the art. The traditional production of full length bispecific antibodies is based on the coexpression of two heavy chain-immunoglobulin light chain pairs, where the two chains have different specificities (Millstein et al., Nature 305: 537-39 (1983)). ). Due to the random selection of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule, which is commonly carried out by affinity chromatography steps, is somewhat cumbersome, and the yields of the product are low. Similar procedures are described in WO 93/08829, and in Traunecker et al., E BO J. 10: 3655-59 (1991). According to a different procedure, the variable domains of antibody with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. Preferably, the fusion is with an Ig heavy chain constant domain, comprising at least part of the joint CH2 and CH3 regions. The DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and co-transfected into a suitable host cell. This provides greater flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where the unequal ratios of the three polypeptide chains used in the construction provide the optimal yield of the desired bispecific antibody. However, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector, when the expression of at least two polypeptide chains in equal ratios results in high yields, or when the they do not have a significant effect on the performance of the desired chain combination. In one embodiment of this method, bispecific antibodies are composed of an immunoglobulin heavy hybrid chain with a first binding specificity in one arm and an immunoglobulin heavy chain-light chain hybrid pair (which provides a second binding specificity). ) in the other arm. It was found that this asymmetric structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides an easy mode of separation. This procedure is described in WO 94/04690. For further details of bispecific antibody generation see, for example, Suresh et al., Meth Enzymol. 121: 210 (1986). According to another procedure described in the U.S. Patent. No. 5,731,168, the interface between a pair of antibody molecules can be fabricated to maximize the percentage of heterodimers that is recovered from the recombinant cell culture. In one embodiment, the interface comprises at least a portion of the CH3 domain. In this method, one or more small amino acid chains at the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" of identical or similar size to the chain (s) of larger sides, are created on the interface of the second antibody molecule by replacing the amino acid chains of large sides with smaller ones (e.g., alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer on other undesired end products such as homodimers. Bispecific antibodies include crosslinked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin. Such antibodies, for example, have been proposed to direct cells of the immune system to unwanted cells (U.S. Patent No. 4,676,980) and for the treatment of HIV infection (WO 91/00360, O 92/200373, and EP 0308936) . Heteroconjugate antibodies can be produced using any convenient crosslinking method. Suitable crosslinking agents are well known in the art, and are described in the U.S. Patent. No. 4,676,980, together with a number of crosslinking techniques. Techniques for generating specific antibodies from antibody fragments have also been described in the literature. For example, bispecific antibodies can be prepared using chemical bonding. Brennan et al., Science 229: 81 (1985) describe a method wherein intact antibodies are found to be proteolytically cleaved to generate F (ab ') 2 fragments. These fragments are reduced in the presence of the dithiol complexing agent, sodium arsenite, to stabilize the vicinal dithiols and prevent the formation of intermolecular disulfide. The generated Fab 'fragments are then converted into thionitrobenzoate derivatives (TNB). One of the Fab '-TNB derivatives is then reconverted to the Fab' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab '-TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as agents for the selective immobilization of enzymes. Recent progress has facilitated the direct recovery of Fab '-SH fragments from E. coli, which can be chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med., 175: 217-25 (1992) describe the production of a fully humanised F (ab ') 2 molecule of bispecific antibody. Each Fab 'fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. Various techniques for producing and isolating bispecific antibody fragments directly from the recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zipers. Kostelny et al., J. Immunol., 148 (5): 1547-53 (1992). The leucine ziper peptides of the Fos and Jun proteins were linked to the Fab 'portions of two different antibodies by fusion of the gene. the antibody homodimers were reduced in the joint region to form monomers and then re-oxidized to form the antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Nati Acad. Sci. USA 90: 6444-48 (1993) has provided an alternative mechanism for producing antibody fragments. The fragments comprise a VH connected to a VL by a link that is too short to allow the formation of pairs between the two domains on the same chain. Accordingly, the VH and VL domains of one fragment are forced to pair with the complementary VL and VH domains of another fragment, thus forming two antigen-binding sites. Another strategy has also been reported for producing bispecific antibody fragments by the use of single chain Fv (sFv) dimers. See Gruber et al., J. Immunol. , 152: 5368 (1994). The use of antibodies with more than two valences is contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol., 147: 60 (1991). Antibodies with altered glycosylation patterns are contemplated for use in the invention. Altered glycosylation patterns may include suppressing one or more carbohydrate residues found in the antibody, and / or adding one or more glycosylation sites that are not present in the antibody. The glycosylation of antibodies is typically linked to N or linked to O. Linked to N refers to the attachment of the carbohydrate residue to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, wherein X is any amino acid except proline, are recognition sequences for the enzymatic attachment of the carbohydrate residue to the asparagine side chain. Therefore, the presence of any of these tripeptide sequences in a polypeptide creates a potential glycosylation site. Glycosylation linked to 0 refers to the binding of one of the sugars N-acetylgalactosamine, galactose or xylose to an amino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used. The addition of glycosylation sites to the antibody is conveniently achieved by altering the amino acid sequence such that it contains one or more of the tripeptide sequences described above (for N-linked glycosylation sites). The alteration may also be effected by the addition of, or substitution by, one or more serine or threonine residues to the original antibody sequence (for N-linked glycosylation sites). The nucleic acid molecules that code for amino acid sequence variants of the antibodies and peptides used in this invention are prepared by a variety of methods known in the art. These methods include, but are not limited to, isolation from a natural source (in the case of amino acid sequence variants of natural origin) or preparation by oligonucleotide-mediated oligonucleotide (or site-directed) mutagenesis, PCR mutagenesis, and cassette mutagenesis, a variant prepared in advance or a non-variant version of an antibody or polypeptide used in the methods of this invention.

To increase the serum half-life of the antibody, a wild-type receptor binding epitope within the antibody (especially an antibody fragment) can be incorporated as described in the U.S. Patent. 5,739,277, for example. Other modifications of the antibody or peptides used in the invention are contemplated. For example, the antibody or polypeptide can be linked to one of a variety of non-proteinaceous polymers, e.g., polyethylene glycol, polypropylene glycol, polyoxyalkylenes or copolymers of polyethylene glycol and polypropylene glycol. The antibody can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacrylate) microcapsules, respectively), in colloidal drug delivery systems (for example). example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules), or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Oslo, A., Ed., (1980). The antibodies and polypeptides of this invention can also be formulated as immunoliposomes. A "liposome" is a small vesicle composed of several types of lipids, phospholipids and / or surfactant, which is useful for the delivery of a drug to a mammal. The components of the liposome are commonly arranged in a bilayer formation, similar to the lipid arrangement of biological membranes. Liposomes containing the antibody or polypeptide can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Nati Acad. Sci. USA 82: 3688 (1985); Hwang et al., Proc. Nati Acad. Sci., USA 77: 4030 (1980); US Patents Nos. 4,485,045 and 4, 544, 545; and WO 97/38731 published October 23, 1997. Liposomes with improved circulation time are described in the U.S. Patent. No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol and phosphatidylethanolamine derivatized with PEG (PEG-PE). The liposomes are extruded through filters of a defined pore size to produce liposomes with the desired diameter. The Fab 'fragments of the antibody of the present invention can be conjugated to the liposomes as described in Martin et al., J. Biol. Chem., 257: 286-88 (1982) by a disulfide exchange reaction. II. TREATMENT WITH TF ANTAGONISTS The tissue factor antagonist and the optional additional therapeutic used in the present invention can be provided to the recipient in combination.

Medications are considered provided "in combination" with one another if they are provided to the patient concurrently, or if the time between the administration of each medication is such that it allows an overlap of the biological activity. An amount of the tissue factor antagonist capable of treating pregnancy complications associated with APS when given to a patient is a "therapeutically effective" amount. The therapeutically effective amount of a tissue factor antagonist will be commonly administered using an amount per kilogram of the patient's weight, as determined by the physician of ordinary skill. In some embodiments, this dose can be administered by continuous intravenous infusion for a period of between 75-180 minutes at a dose of approximately in the range of 0.02-25.0 milligrams per kilogram of the patient's weight. As will be apparent to one of ordinary skill in the art, the required dose of the tissue factor antagonist for use in the treatment associated with APS and, optionally, any other therapeutic, will depend on the severity of the patient's condition, and criteria such as the patient's height, weight, age and medical history. III. PHARMACEUTICAL FORMULATIONS The therapeutic formulations of the antibodies, peptides and other molecules used in the present invention are prepared for storage by mixing those having the desired degree of purity with optional pharmaceutically acceptable vehicles, excipients or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol A. Ed., (1980)), in the form of lyophilized formulations or aqueous solutions. Acceptable carriers, excipients or stabilizers are non-toxic to the recipient at the doses and concentrations employed, and include buffers such as acetate, Tris, phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (such as octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride, benzethonium chloride, phenolic, butyl or benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m- cresol; low molecular weight polypeptides (less than about 10 residues); proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, histidine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose or dextrins, chelating agents such as EDTA, tonicizers such as trehalose and sodium chloride, sugars such as sucrose, mannitol, trehalose or sorbitol, surfactants such as polysorbate, salt-forming ions such as sodium; metal complexes (eg, Zn-protein complexes) and / or non-ionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). The formulation preferably comprises the antibody, polypeptide or other molecule in a concentration of between 5-200 mg / ml, preferably between 10-100 mg / ml. The active ingredients can also be entrapped in microcapsules prepared, for example, by coacervation techniques or by interfacial polymerization, for example, hydroxymethylcellulose or gelatin microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (eg example, liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or in macroemulsions. Such techniques are described in Remington's Pharmaceutical Sciences 16th edition, Osol, A. ed. (1980). Sustained release preparations can be prepared. Suitable examples of sustained-release preparations include semi-permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles., e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl-methacrylate), or poly (vinylalcohol), polylactides (U.S. Patent No. 3,773,919), copolymers of L-glutamic acid, and ethyl-L- glutamate, non-degradable ethylene vinyl acetate, degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT ™ (injectable microspheres composed of copolymer of lactic acid-glycolic acid and leuprolide acetate), and poly-D- (-) acid -3-hydroxybutyric The formulations for use in in vivo administration must be sterile.This is easily achieved by filtration through sterile filtration membranes IV MANUFACTURING ARTICLES In another embodiment of the invention, an article of manufacture containing materials useful for the treatment of pregnancy complications related to APS The article of manufacture comprises (1) a container, (2) a TF antagonist; and (3) a packaging label or insert comprising instructions on how to carry out the method of this invention. Another embodiment of the invention is an article of manufacture containing materials useful for carrying out a method of the invention. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed from a variety of materials such as glass or plastic. The container contains a composition that is effective to treat a condition and can have a sterile access port (for example, the container can be an intravenous solution bag or a vial having a plug pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used to treat pregnancy complications associated with APS. The label or package insert will further comprise instructions for administering a TF antagonist composition to the patient. All publications (including patents and patent applications) cited herein are incorporated herein by reference in their entirety. The commercially available reagents referred to in the Examples were used according to the manufacturer's instructions unless otherwise indicated. Unless noted otherwise, the present invention uses standard methods of recombinant DNA technology, such as those described above and in the following texts: Sambrook et al., Molecular Cloning: A laboratory Manual (Molecular cloning: a manual of laboratory) New York: Cold Spring Harbor Press, 1989; Ausubel et al., Current Protocols in Molecular Biology (Green Publishing Associates and Wiley Interscience, N.Y., 1989); Innis et al., PCR Protocole: A Guide to Methods and Applications (PCR Protocols: A Guide for Methods and Applications) (Academic Press, Inc .: N.Y. 1990); Harlow et al., Antibodies: A Laboratory Manual (Antibodies: a laboratory manual) (Cold Spring Harbor Press: Cold Spring Harbor, 1988); Gait, Oligonucleotide Synthesis (Oligonucleotide synthesis) (IRL Press: Oxford, 1984); Freshney, Animal Cell Culture, 1987 (Animal cell culture, 1987); Colligan et al., Current Protocols in Immunology, 1991. Throughout this specification and claims, it will be understood that the word "comprises" or variations such as "comprise" or "comprising", imply the inclusion of a definite integer or group of integers but not the exclusion of any other integer or group of integers. The description written above is considered sufficient to enable the person skilled in the art to practice the invention. The following Examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. In fact, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. EXAMPLES 1. Tissue factor (TF) binds to decay in a C5-dependent manner Antiphospholipid antibody (aPL) APl-IgG was obtained from patients with APS (characterized by a high titre of aPL antibodies [> 140 GPL units], thrombosis and / or pregnancy losses) and NH-IgG (normal human) of healthy individuals. The human monoclonal antibody aPL IgGl (monoclonal antibody 519) has been previously described (Ikematsu et al., Arthritis Rheum, 41: 1026-39 (1998)). IgG was purified by affinity chromatography using Protein G Sepharose® chromatography columns (Amersham Pharmacia Biotech) and treated to deplete the endotoxin with Centriprep® ultracentrifugation devices (Millipore Corporation) and determined endotoxin free using lysate analysis. of Limulus amebocyte. Mouse model for aPL-induced pregnancy loss A mouse model for a pregnancy loss induced by aPL was used as previously described (Girardi et al., Nature Med. 10: 1222-26 (2004)). On day 8 of pregnancy, females 6-8 weeks of age C5 + / + and C5 - / - (Taconic Farms) were treated with intraperitoneal injections of aPL-IgG (10 mg), human aPL mAb (1 mg) or NH -IgG (10 mg). The mice were sacrificed 60 minutes after the treatment and the deciduous were removed, frozen in the O.C.T. and they were cut into sections of 10 um. The sections were then stained using anti-tissue factor antibodies. We observed an intense coloration of deciduous TF in CD + / + mice treated with aPl-IgG. In contrast, C - / - mice did not show TF coloration of the deciduous. 2. Fetal loss induced by antiphospholipid antibody and weight restriction are dependent on C5. On days 8 and 12 of pregnancy, females 6-8 weeks of age C5 + / + and C5 - / - (Taconic Farms) were treated with Intraperitoneal injections of aPL-IgG (10 mg), aPL mAb human (1 mg) or NH-IgG (10 mg). The mice were sacrificed on day 15 of pregnancy, the fetuses were weighed and the frequencies of fetal resorption were calculated (number of resorbtions / total number of fetuses and resorbtions formed). Resorption sites are easily identified and result from the loss of a previously viable fetus. C5 + / + mice were observed an increase dependent on antiphospholipid antibody in the frequency of fetal resorption and a similar decrease in embryo weight. Mice treated with aPL-IgG had a resorption frequency of 38 + 8% while mice treated with NH-IgG had a resorption frequency of 10 + 7% (p <0.01). The average weights of the embryos were 221 + 31 mg with aPL-IgG treatment and 389 + 36 mg with NG-IgG treatment (p <0.01). In contrast, in C5 - / - no effect was observed. The C5 - / - mice treated with aPL-IgG had a resorption frequency of 11 + 3% while the mice treated with NH-IgG had a resorption frequency of 10 + 4%. The average weights of the embryos were 371 + 32 mg with a treatment of aPl-IgG and of 357 + 41 mg with treatment of NH-IgG. Two complementary effector trajectories are initiated by splitting C5: C5a, a potent anaphylatoxin and activator of endothelial cells and leukocytes, and C5b, which leads to the formation of the membrane attack complex C5b-9 (MAC). In order to distinguish which of these is involved in the dependent effects of C5, these experiments were carried out in C5aR - / - mice and in C6 - / - mice. It was observed that the results of pregnancies in C5aR - / - mice was normal while the C6 deficient mice showed no improvement in pregnancy failure induced by aPL-IgG. Additionally, it was observed that the C5aR - / - mice did not show TF production while the C6 deficient mice showed an increased TF coloration in deciduous tissues. This indicated that C5a is involved in the dependent effects of C5 and also suggested a role for TF in this process. 3. Treatment with anti-tissue factor antibody blocks fetal loss induced by antiphospholipid antibody and weight restriction To directly show that TF is an essential mediator of fetal damage induced by aPL-antibody, TF function was inhibited with a monoclonal antibody against mouse TF (1H1: Kirchhofer et al., J. Thromb. Heamost., 3: 1098-99 (2005)). Treatment with 1H1 (0.5 mg on days 6 and 10) prevented pregnancy losses induced by aPL-antibody and growth restriction. In mice treated with aPL-IgG only, a frequency of fetal resorption of 39 + 7% was observed compared to a frequency of 11 + 3% in mice treated with aPL-IgG and 1H1 (p <; 0.001). the average weights of the embryos were 234 + 37 mg with a treatment of aPL-IgG and 381 + 46 mg with a treatment of aPl-IgG plus 1H1 (p <0.005). These results confirmed that TF is a mediator of fetal damage induced by aPL-antibody and that C5a plays a critical role as a trigger for TF generation.

Claims (14)

1. CLAIMS 1. A method for treating a patient at risk of pregnancy complications related to the antiphospholipid syndrome, which comprises administering to said patient a tissue factor antagonist. The method of claim 1, wherein said tissue factor antagonist antagonizes an activity of the tissue factor selected from the group consisting of: binding to factor VII, binding to factor Vlla, carrying out factor VII proteolysis, of the proteolysis of factor IX, and carrying out the proteolysis of factor X. 3. The method of claim 1 or 2, wherein said tissue factor antagonist is an antibody. 4. The method of claim 3, wherein said antibody is a monoclonal antibody. The method of claim 1 or 2, wherein said tissue factor antagonist is a peptide. 6. The method of claim 1, wherein said tissue factor antagonist is administered in the second trimester of pregnancy. The method of any of claims 1 to 6, further comprising administering to said patient an anticoagulant. The method of claim 7, wherein said anticoagulant is heparin. 9. A kit comprising: (a) a tissue factor antagonist; (b) a container containing said tissue factor antagonist; (c) a fixed label to said container, or a packing insert included in said container, referring to the use of said antagonist in the treatment of pregnancy complications related to the antiphospholipid syndrome. 10. The kit of claim 9, wherein said tissue factor antagonist is an antibody. The kit of claim 10, wherein said antibody is monoclonal. The kit of claim 9, wherein said tissue factor antagonist is a peptide. The equipment of any of claims 9-12, further comprising an anticoagulant. 14. The kit of claim 13, wherein said anticoagulant is heparin.