WO2007076091A2 - Treatment of viral infections using a tissue factor inhibitor - Google Patents

Abstract

The present invention provides novel methods for the treatment of viral infections, such as influenza virus both human and avian, by administering a tissue factor inhibitor.

Description

INVENTION ^"N I L. TREATMENT OF VIRAL INFECTIONS USING A TISSUE FACTOR INHIBITOR

BACKGROUND OF THE INVENTION

[Para 1] Viral infections, including human and avian influenza viruses and coronaviruses, such as the SARS virus, are serious threats to the human population. Influenza virus, including the currently circulating avian influenza virus of the H5N1 subtype and the recently reconstituted Spanish influenza virus from 1918, are listed as priority pathogens by the NIAID. Influenza is a globally important and potentially deadly disease. About 20% of children and 5% of adults worldwide develop symptomatic influenza A or B each year. In the U.S., the average seasonal incidence of influenza-related deaths was 9.1 per 100,000 people during the years 1 972-1992 (Snacken R. Vaccine. (1999) 1 7: S61 -S63).

[Para 2] However, mortality incompletely reflects the total health burden and economic costs of influenza because many severe cases of influenza, although they do not lead to death, still require hospitalization. In the US, influenza virus infections were associated with annual averages of about 95,000 cases of primary pneumonia and influenza hospitalizations, and about 226,000 cases of primary respiratory and circulatory hospitalizations from the 1979-1 980 through the 2000-2001 seasons. These represented 8.6% of all primary pneumonia and influenza hospitalizations, and 2.6% of all primary respiratory and circulatory hospitalizations (Thompson WA, et al. J Am Med Assoc. (2004) 292: 1333-1340). Furthermore, influenza is listed as a Category C priority pathogen for biological terrorism by the National Institute of Allergy and Infectious Diseases.

[Para 3] Severe acute respiratory syndrome (SARS) is caused by a coronovirus and has the potential to set off a global pandemic. In 2003, the WHO reported a cumulative total of 8202 probable cases with 725 deaths from 28 countries (www.who.int/csr/en) More than half the individuals affected (51 -72%) report general influenza-like symptoms, including chills, malaise, loss of appetite, etc. Moreover, the fatality rate is 1 5% for patients under the age of 60 and can be as high as 50% in patients over 60. Nearly 40% of patients develop respiratory failure requiring assisted ventilation. In addition, SARS is difficult to distinguish from other viral infections when the patient first present symptoms. There is currently no approved vaccination for SARS.

[Para 4] Vaccination is the primary method for preventing influenza. Current vaccines against influenza virus infection are produced from virus grown in fertile hens' eggs and are inactivated by either formalin or beta-propiolactone. They consist of whole virus, detergent-treated split product, or purified hemagglutinin and neuraminidase surface antigen formulations of the three virus strains currently recommended by WHO.

[Para 5] Besides vaccination, there are two types of antiviral drugs available to manage influenza. Amantidine and rimantidine inhibit the M2 ion channel of influenza A virus. This channel regulates the internal pH of the virus and is crucial during early viral replication. There is no high-quality evidence from randomized controlled trials that amantidine is effective. Amantidine has three important limitations: its range of activity excluded influenza B virus; it has adverse side-effects, including insomnia, hallucinations, and headache; and drug resistance emerges rapidly during treatment. Rimantidine is not available in most parts of the world.

[Para 6] The other type of antiviral drugs is the neuraminidase inhibitors, such as zanamivir and oseltamivir. Neuraminidase catalyzes the cleavage of hemagglutinin to the cell surface receptor, thereby assisting in the release of progeny virions from infected cells. Zanamivir results in 1.3 days earlier alleviation of symptoms in influenza virus-positive patients compared to patients treated with placebo. The limitations of zanamivir are that it requires the use of an inhaler for delivery and that it may cause bronchospasm. Osaltamivir is an orally active drug and has a similar effectiveness as zanamivir. Besides reducing the duration of symptoms, treatment with osaltamivir reduces the frequencies of otitis media, antibiotic use, pneumonia, and hospital admissions. As with amantidine, drug resistance may occur, but appears to be less common.

[Para 7] There is a continuing threat of a new influenza pandemic, either from mutation of an avian influenza virus directly transmitted from birds to humans, or from recombination of gene segments from an avian influenza virus and a human influenza virus (Kuiken T, et al. Curr Opin Biotech. (2003) 14: 641 -646). As was first seen in 1997 in Hong Kong and more widely in South-East Asia since 2002, the domestic chicken can act as an intermediate host for the transmission of avian influenza A (H5N1 ) virus from aquatic birds to humans. In the 1997 outbreak of H5N1 virus infection in Hong Kong, seven of the first 12 hospitalized patients had clinical and radiological evidence of pneumonia, while six developed ARDS. Full autopsies were performed on two of the six patients who died from H5N1 virus infection. Histological sections of the lungs of both patients showed organizing diffuse alveolar damage, interstitial fibrosis, and cystically dilated air spaces. In the 2003 outbreak of H7N7 virus infection in The Netherlands, one person died from H7N7 virus infection. Histologically, the patient's lungs showed severe diffuse alveolar damage, characterized by serosanguineous fluid in alveolar lumina, mixed with fibrin and neutrophils. As of 5 August 2005, 1 12 cases of H5N1 virus infection, mainly due to direct transmission from birds, have been recorded in humans, of whom 57 (51 %) have died. The clinical presentation was characterized by fever, respiratory symptoms, and lymphopenia. Main respiratory symptoms were cough, shortness of breath, and a rapid respiratory rate. Abnormalities on chest radiographs included extensive bilateral infiltration, lobar collapse, and focal consolidation. A full autopsy has been performed on one patient. Histologically, the lungs showed a proliferative phase of diffuse alveolar damage, interstitial pneumonia, focal haemorrhage, and bronchiolitis. By immunohistochemistry, influenza virus infection was localized to type 2 pneumocytes in the alveoli (Uiprasertkul M, et al. Emerg Infect Dis. (2005) 1 1 : 1036-1041 ). [Para 8] A major concern with avian influenza is that the virus becomes easily transmissible among humans, either through mutation or through reassortment — in humans or other host species— with other strains of influenza virus. This would pave the way for a new influenza pandemic. The emergence of these avian influenza viruses in the human population as well as the threat of a new pandemic are important reasons why influenza has been specifically named in Category C (emerging infectious disease threats) of the list of priority pathogens for biological terrorism by the National Institute of Allergy and Infectious Diseases.

[Para 9] Current antiviral strategies are insufficient to prevent a future viral pandemic. The capacity and preparation time to produce vaccines against influenza virus, including the more recent avian H5N1 virus strains are insufficient to protect the human population. By using the traditional approaches, fewer than 500 million people could currently be vaccinated in the event of a pandemic influenza outbreak. The current main method of vaccine production, based on virus replication in embryonated chicken eggs, is too time-consuming and too dependent on a steady supply of eggs to be reliable in the face of a pandemic emergency. Even during interpandemic periods, 6 months is required to organize sufficient fertile chicken eggs for annual vaccine manufacture.

[Para 10] Similarly, the stockpiles of antiviral drugs are limited. For example, the current WHO global stockpiles of oseltamivir are limited to 120,000 treatment courses. Besides, antiviral agents limit viral replication, but do not eliminate virus excretion. Although strategies for targeted mass prophylactic use of antiviral agents have been evaluated to halt a pandemic in its early stages, adequate stockpiles of antiviral drugs and global networks for their rapid distribution have yet to be established (Longini IM et al.. Science (2005) 309: 1083-1087). Despite these threats, the current arsenal of preventive and therapeutic strategies against human and avian influenza is limited.

[Para 1 1 ] In addition, these therapies are primarily designed as prophylactic treatments to protect the population from contracting the disease, or in the case of antivirals such as amantidine and rimantidine, act in the early stages of the disease. There is no treatment for those individuals that have already contracted the disease and have not been administered treatment in the early phases of the disease. This would be especially true in the case of a pandemic in many parts of the world. [Para 12] Because of the serious effect of influenza infection on a host and the widespread infection of influenza, as well as the threat for a new pandemic outbreak of influenza, avian influenza or SARS, there exists a critical need for new treatments.

SUMMARY OF THE INVENTION [Para 13] The present invention relates to novel methods of treating a patient suffering from a viral infection comprising administering an inhibitor of tissue factor.

This approach is designed to overcome the shortcomings inherent in previous approaches and prevent certain clinical outcomes including mortality and morbidity.

[Para 14] One aspect of the invention includes the treatment of human or avian influenza infections or corona virus infections, such as SARS.

[Para 15] Tissue factor inhibitors may include antibodies, peptide mimetics, tissue factor ligand analogs, TFPI, and organic molecules that inhibit tissue factor.

[Para 16] Other aspects of the invention include the treatment of viral infections with a tissue factor inhibitor in combination with another antiviral agent such as amantidine, rimantidine, or neuraminidase inhibitors, such as zanamivir and oseltamivir.

[Para 17] Numerous other advantages and aspects of the invention will become apparent to the skilled artisan upon consideration of the following detailed description of the invention.

DETAILED DESCRIPTION OF THE INVENTION

[Para 18] This invention is not limited to the particular methodology, protocols, cell lines, vectors, or reagents described herein because they may vary. Further, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the present invention. As used herein and in the appended claims, the singular forms "a", "an", and "the" include plural reference unless the context clearly dictates otherwise, e.g., reference to "a host cell" includes a plurality of such host cells. [Para 19] Unless defined otherwise, all technical and scientific terms and any acronyms used herein have the same meanings as commonly understood by one of ordinary skill in the art in the field of the invention. Although any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention, the exemplary methods, devices, and materials are described herein.

[Para 20] All patents and publications mentioned herein are incorporated herein by reference to the extent allowed by law for the purpose of describing and disclosing the proteins, enzymes, vectors, host cells, and methodologies reported therein that might be used with the present invention. However, nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention. DEFINITIONS

[Para 21] The term "amino acid sequence variant" refers to polypeptides having amino acid sequences that differ to some extent from a native sequence polypeptide. Ordinarily, amino acid sequence variants will possess at least about 70% homology, or at least about 80%, or at least about 90% homology to the native polypeptide. The amino acid sequence variants possess substitutions, deletions, and/or insertions at certain positions within the amino acid sequence of the native amino acid sequence. [Para 22] The term "identity" or "homology" is defined as the percentage of amino acid residues in the candidate sequence that are identical with the residue of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions nor insertions shall be construed as reducing identity or homology. Methods and computer programs for the alignment are well known in the art. Sequence identity can be readily calculated by known methods, including but not limited to those described in (Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991 ; and Carillo, 30 H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). Methods to determine identity are designed to give the largest match between the sequences tested. Computer program methods to determine identity between two sequences include, but are not limited to, the GCG program package (Devereux, J., et al., Nucleic Acids Research 12(1 ): 387 (1984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al., J Molec. Biol. 215: 403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLASTManual, Altschul, S., et al, NCBI NLM NIH Bethesda, Md. 20894, Altschul, S., et al., J. MoI. Biol. 21 5: 403-410 (1990). The well-known Smith Waterman algorithm may also be used to determine identity.

[Para 23] The term "antibody" herein is used in the broadest sense and specifically covers intact monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g. bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. [Para 24] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations which include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the present invention may be made by the hybridoma method first described by Kohler et al, Nature, 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in Clackson et al., Nature, 352:624-628 (1991 ) and Marks et al., J. MoI. Biol., 222:581 -597 (1991 ), for example. The monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Pat. No. 4,816,567; and Morrison et al, Proc. Natl. Acad. ScI. USA, 81 :6851 -6855 (1984)). [Para 25] "Antibody fragments" comprise a portion of an intact antibody comprising the antigen-binding or variable region thereof. Examples of antibody fragments include Fab, Fab 2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragment(s).

[Para 26] An "intact" antibody is one which comprises an antigen-binding variable region as well as a light chain constant domain (CL) and heavy chain constant domains, CHI , CH2 and CH3. The constant domains may be native sequence constant domains (e.g. human native sequence constant domains) or amino acid sequence variant thereof. The intact antibody may have one or more effector functions. [Para 27] Antibody "effector functions" refer to those biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody. Examples of antibody effector functions include Cl q binding; complement dependent cytotoxicity; Fc receptor binding; antibody- dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down regulation of cell surface receptors (e.g. B cell receptor; BCR), etc.

[Para 28] Depending on the amino acid sequence of the constant domain of their heavy chains, intact antibodies can be assigned to different "classes". There are five- major classes of intact antibodies: IgA, IgD, IgE, IgC, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGI , lgC2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes and three-dimensional configurations of different classes of immunoglobulins are well known. [Para 29] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions both in the light chain and the heavy chain variable domains. These hypervariable regions are also called complementarity determining regions or CDRs. The more highly conserved portions of variable domains are called the framework regions (FRs). The variable domains of native -sheet configuration, connected by three hypervariable regions, which form loops

-sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and, with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991 )).

[Para 30] The term "hypervariable region" when used herein refers to the amino acid residues of an antibody which are responsible for antigen-binding. The hypervariable region generally comprises amino acid residues from a "complementarity determining region" or "CDR" (e.g. residues 24-34 (Ll ), 50-56 (L2) and 89-97 (L3) in the light chain variable domain and 31 -35 (Hl), 50-65 (H2) and 95-102 (H3) in the heavy chain variable domain; Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and/or those residues from a "hypervariable loop" (e.g. residues 2632 (LI ), 50-52 (L2) and 91 -96 (13) in the light chain variable domain and 26-32 (Hl ), 53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothia and Lesky. MoI. Biol. 196:901 -91 7 (1987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[Para 31 ] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy chain and one light chain variable domain in tight, non-covalent association. It is in this configuration that the three hypervariable regions of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although at a lower affinity than the entire binding site. [Para 32] "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. Preferably, the Fv polypeptide further comprises a polypeptide linker between the V_H and VL domains which enables the scFv to form the desired structure for antigen binding. For a review of scFv see Plϋckthun in The Pharmacology of Monoclonal Antibodies, vol. 1 13, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994). Anti-ErbB2 antibody scFv fragments are described in WO93/16185; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458. [Para 33] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, which comprise a variable heavy domain (VH) connected to a variable light domain (V_L) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are described more fully in, for example, EP 404,097; WO 93/1 1 161 ; and Hollinger et al., Proc. Natl. Acad. ScL USA, 90:6444- 6448 (1 993).

[Para 34] A "single-domain antibody" is synonymous with "dAb" and refers to an immunoglobulin variable region polypeptide wherein antigen binding is effected by a single variable region domain. A "single-domain antibody" as used herein, includes i) an antibody comprising heavy chain variable domain (VH), or antigen binding fragment thereof, which forms an antigen binding site independently of any other variable domain, ii) an antibody comprising a light chain variable domain (VL), or antigen binding fragment thereof, which forms an antigen binding site independently of any other variable domain, iii) an antibody comprising a VH domain polypeptide linked to another VH or a VL domain polypeptide (e.g., VH-VH or VHx-VL), wherein each V domain forms an antigen binding site independently of any other variable domain, and iv) an antibody comprising VL domain polypeptide linked to another VL domain polypeptide (VL-VL)₅ wherein each V domain forms an antigen binding site independently of any other variable domain. As used herein, the VL domain refers to both the kappa and lambda forms of the light chains.

[Para 35] "Humanized" forms of non-human (e.g., rodent) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies are human immunoglobulins wherein the hypervariable regions are replaced by residues from a hypervariable region of a non-human species, such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, and capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the human antibody or in the non-human antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Examples of humanizatioπ technology may be found in, e.g., Queen et al. U.S. Pat. No. 5,585,089, 5,693,761 ; 5,693,762; and 6,1 80,370, which are incorporated herein by reference. VIRAL INFECTION

[Para 36] The epidemiological behavior of influenza is related to the two types of antigenic variation of its envelope glycoproteins— antigenic drift and antigenic shift. During antigenic drift, new strains of virus evolve by accumulation of point mutations in the surface glycoproteins. The new strains are antigenic variants but are related to those circulating during preceding epidemics. This feature enables the virus to evade immune recognition, leading to repeated outbreaks during inter-pandemic years. Antigenic shift occurs with the emergence of a "new", potentially pandemic, influenza A virus that possesses a novel hemagglutinin alone or with a novel neuraminidase. The new virus is antigenically distinct from earlier human viruses and could not have arisen from them by mutation. Pandemics have occurred three times in the 20^th century: the 1918 'Spanish flu' A (HI Nl ) pandemic was particularly severe, causing 40-50 million deaths world-wide, while the more recent pandemics, A (H2N2) 'Asian flu' in 1957 and A (H3N2) in 1968, were associated with moderately increased mortality.

[Para 37] The onset of inflammatory disease associated with viral infection is often initially seen by the development of the systemic inflammatory response syndrome (SIRS) which is characterized by an increase in proinflammatory cytokine levels. The inflammatory response can progress to acute lung injury (ALI) which is manifested by involvement of the pulmonary system displaying decreased pulmonary compliance, increased levels of hypoxia and deposition of fibrin in the lungs (Idell S., Crit Care Med. (2003) 31 : S213-20). Progression from ALI to acute respiratory distress syndrome (ARDS) occurs when the condition becomes more severe as lung function continues to deteriorate and the level of hypoxia continues to increase. The function of other organs such as the kidney may also decline during ARDS (Gunther A, et al., Seminar Respir Crit Care Med. (2001 ) 22: 247-58). Severe sepsis is characterized by progressive deterioration leading to multiple organ failure, microvascular thrombosis, disseminated intravascular coagulation and ultimately death in the majority of patients.

[Para 38] Infections induce the production of proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6), interleukin-8 (IL-8), and interleukin-1 (IL-I ) from the innate immune system and endothelial cells (Damas P, et al.,. Crit Care Med. (1997) 25: 405-412). These inflammatory cytokines then cause the endothelial cells and monocytes to express tissue factor (TF), the initiator protein in the extrinsic pathway of coagulation, on their surfaces (Esmon CT, et al., Haematologica. (1999) 84: 254-259). Exposure of TF ultimately leads to the production of thrombin. Thrombin itself and components of the extrinsic coagulation pathway, such as TF, factor X (FX) and activated factor X (FXa), activate the protease activated receptors (PARs) displayed on platelets and endothelial cells leading to further production of proinflammatory cytokines (Riewald M, et al., Crit Care. (2003) 7: 123-129). Thrombin generation also leads to fibrinogen cleavage and platelet activation leading to the formation of fibrin clots in the microvasculature. The proflammatory cytokines IL-I and TNF-alpha also lead to the production of plasminogen activator inhibitor-! , a potent inhibitor of fibrinolysis. Exposure of tissue factor and the liberation of cytokines in the lungs thus begin an overwhelming inflammatory response leading to ALI/ARDS.

[Para 39] Pathophysiological features of ALI and ARDS also include damage to the vascular endothelium and alveolar epithelium resulting in local activation of the extrinsic coagulation cascade and inhibition of fibrinolysis in the lung (Welty-Wolf KE, et al., Thromb Haemostat. (2002) 88: 17-25). During endotoxemia, exposure of monocytes to LPS resulted in increased tissue factor and plasminogen activator inhibitor-! expression leading to a procoagulant environment. As injury to the lung evolves, these perturbations in hemostasis lead to fibrin deposition in the microvascular, interstitial, and alveolar spaces of the lung leading to capillary obliteration and hyaline membrane formation. Components of the extrinsic coagulation pathway such as tissue factor, thrombin, and fibrin signal alterations in inflammatory cell traffic and increase vascular permeability. In addition, procoagulant molecules and fibrin also promote other key events including complement activation, production of pro-inflammatory cytokines, inhibition of fibrinolysis and remodeling of injured lung tissue.

[Para 40] The understanding of the pathophysiology of systemic inflammatory conditions, complicated by coagulation and fibrinolytic involvement, has led to the development of the present invention as a new approach to the inhibition of the systemic inflammatory response and the prevention of ALI /ARDS in patients suffering from severe viral infections. Abnormalities in the coagulation cascade are an important component of acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) associated with severe viral infections (Ware LB, et al. N Engl J Med. (2000) 342: 1334-1349). Clinical and animal data indicate that ALI/ARDS are important causes of fatality in subjects infected with influenza (Claas EC, et al. Lancet. (1998) 351 : 472-477; Fouchier RA, et al. Proc Natl Acad Sci USA. (2004) 101 : 1356- 1361 ; Fenner F, et al. Geneva, Switzerland: World Health Organization (1988). [Para 41] Tissue factor ("TF") is a 47 kDa transmembrane glycoprotein that is the major cellular trigger of blood coagulation under physiologic conditions. The factor Vila-tissue factor ("fVII/TF") catalytic complex is able to generate factor Xa via direct activation of factor X, and indirectly through the activation of factor IX, thus initiating thrombin generation. It has been reported that tissue factor also plays an important role in disease processes resulting from the activation of the coagulation pathway. [Para 42] In addition, a number of viruses have been reported to activate the coagulation system following infection; such activation may also be triggered by the up regulation of TF expression (Bowman, et al., European J. Clin. Invest. (2002) 32: 759-766; Baugh et al., J. Biol. Chem. (1998) 273: 4378-4386; Taylor et al., Blood (1998) 91 : 1609-161 5). [Para 43] The present invention is designed to address the problem of treatment in individuals who have already contracted the disease and no longer can be treated with prophylactic methods. Therefore, the present invention relates to an alternative intervention strategy for the treatment of serious viral infections. The present invention relates to the use of tissue factor inhibitors as a therapy to fill this unmet medical need. Another aspect of the invention is to provide other benefits such as shortened stays in the ICU, reduction in the time of hospitalization, shortened time on assisted ventilation, reduced incidence of complications, such a severe diffuse alveolar damage, interstitial pneumonia, focal haemorrhage, and bronchiolitis, reduction in the mortality rates associated with these severe viral infections, and reduction in the number or severity of morbidities.

[Para 44] One embodiment of the present invention is an anti-tissue factor antibody that binds to human TF or the TF-Factor Vila (FVIIa) complex preventing binding and/or activation of Factor X (FX) and Factor IX (FIX), thereby inhibiting thrombin generation. By blocking the initiating events of extrinsic coagulation activation, their effects on pro-inflammatory events in the lungs and disordered fibrin deposition may be minimized and the evolution of severe structural and functional injury may be averted during the course of viral infection. Antibodies useful in the present invention may bind tissue factor, blocking or inhibiting the action of either Factor VII, Factor IX or Factor X. The antibody may be monoclonal and may be chimeric, humanized, or human. The antibody may also be a single-domain antibody. Examples of such antibodies of the invention that inhibit TF function by effectively blocking FX binding or access to TF molecules, include H36.D2.B7 (secreted by hybridoma ATCC HB-12255) and humanized clones of this antibody. Other anti-TF antibodies useful in the invention include those disclosed in U.S. Pat.

No. 6,555,319; 5,986,065; 5,223,427; 6,677,436; 6,703,494; or PCT application

WO2004/039842, which are incorporated by reference. Antibodies may also be directed to Factor VII or Factor X thereby inhibiting tissue factor by blocking the ligand necessary for activation. Examples of such antibodies have been disclosed in

5,506,1 34 and 6,835,81 7.

[Para 45] Peptide mimetics include fragments of tissue factor that bind Factor VII,

Factor IX or Factor X, thereby blocking their activation. Tissue factor ligand analogs include modified Factor VII, FactorlX or Factor X, that bind tissue factor but do not allow activation.

[Para 46] Other molecules useful in the present invention include molecules such as those disclosed in WOOO/18398 and WOOl /30333.

ANTIBODY GENERATION

[Para 47] The antibodies of the present invention may be generated by any suitable method known in the art. The antibodies of the present invention may comprise polyclonal antibodies. Methods of preparing polyclonal antibodies are known to the skilled artisan (Harlow, et al., Antibodies: a Laboratory Manual, (Cold spring Harbor

Laboratory Press, 2nd ed. (1988), which is hereby incorporated herein by reference in its entirety).

[Para 48] For example, antibodies may be generated by administering an immunogen comprising the antigen of interest to various host animals including, but not limited to, rabbits, mice, rats, etc., to induce the production of sera containing polyclonal antibodies specific for the antigen. [Para 49] One method of generating such antibodies to tissue factor may be found in U.S. Pat. No. 6,555,319 and 5,986,065 (which are hereby incorporated herein by reference in their entirety).ln brief, monoclonal antibodies directed to human tissue factor can be raised by immunizing rodents (e.g. mice, rats, hamsters and guinea pigs) with a purified sample of native TF, typically native human TF, or a purified recombinant human tissue factor (rhTF). Truncated recombinant human tissue factor or "rhTF" (composed of 243 amino acids and lacking the cytoplasmic domain) may be used to generate anti-TF antibodies. The antibodies also can be generated from an immunogenic peptide that comprises one or more epitopes of native TF that are not exhibited by non-native TF. References herein to "native TF" include such TF samples, including such rhTF.

[Para 50] Antibodies directed to other antigens such as Factor VII or Factor X may be generated in a similar manner.

[Para 51] The antibodies useful in the present invention comprise monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma technology, such as those described by Kohler and Milstein, Nature, 256:495 (1975) and U.S. Pat. No. 4,376,1 10, by Harlow, et al., Antibodies: A Laboratory Manual, (Cold spring Harbor Laboratory Press, 2.sup.nd ed. (1988), by Hammerling, et al., Monoclonal Antibodies and T-CeII Hybridomas (Elsevier, N.Y., (1981 )), or other methods known to the artisan. Other examples of methods that may be employed for producing monoclonal antibodies include, but are not limited to, the human B-cell hybridoma technique (Kosbor et al., 1983, Immunology Today 4:72; Cole et al., 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Such antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD and any subclass thereof. The hybridoma producing the antibodies of this invention may be cultivated in vitro or in vivo.

[Para 521 Using typical hybridoma techniques, a host such as a mouse, a humanized mouse, a mouse with a human immune system, hamster, rabbit, camel or any other appropriate host animal, is typically immunized with an immunogen to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the antigen of interest. Alternatively, lymphocytes may be immunized in vitro with the antigen. Hybridoma technology is well known in the art.

[Para 53] A variety of methods exist in the art for the production of monoclonal antibodies and thus, the invention is not limited to their sole production in hydridomas. For example, the monoclonal antibodies may be made by recombinant

DNA methods, such as those described in U.S. Pat. No. 4,816,567.

[Para 54] The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art. For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain. The heavy chain is truncated generally at any point in the Fc region so as to prevent heavy chain cross-linking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent cross-linking.

[Para 55] Antibody fragments which recognize specific epitopes may be generated by known techniques. For example, Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments). F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.

[Para 56] For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125:191 -202; U.S. Pat. Nos. 5,807,71 5; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.

[Para 57] Humanized antibodies are antibody molecules generated in a non-human species that bind the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework (FR) regions from a human immunoglobulin molecule. Often, framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding. These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entireties). Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 /09967; U.S. Pat. Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1 994); Roguska. et al., PNAS 91 :969-973 (1994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[Para 58] Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be essentially performed following the methods of Winter and co-workers (Jones et al., Nature, 321 :522-525 (1986); Reichmann et al., Nature, 332:323-327 (1 988); Verhoeyen et al., Science, 239:1 534- 1 536 (1988), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possible some FR residues are substituted from analogous sites in rodent antibodies.

[Para 59] Completely human antibodies are particularly desirable for therapeutic treatment of human patients. Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,1 1 1 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91 /10741 ; each of which is incorporated herein by reference in its entirety. The techniques of Cole et al., and Boerder et al., are also available for the preparation of human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Riss, (1985); and Boerner et al., J. Immunol., 147(l ):86-95, (1 991)). [Para 60] Human antibodies can also be single-domain antibodies having a VH or VL domain that functions independently of any other variable domain. These antibodies are typically selected from antibody libraries expressed in phage. These antibodies and methods for isolating such antibodies are described in U.S. Pat. No. 6,595,142; 6,248,516; and applications US200401 10941 and US2OO3O130496 all of which are incorporated herein by reference.

[Para 61] Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. The mouse heavy and light chain immunoglobulin genes may be rendered nonfunctional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring which express human antibodies. The transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention. Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically useful IgC, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,41 3,923; 5,625,126; 5,633,425; 5,569,825; 5,661 ,016; 5,545,806; 5,814,318; 5,885,793; 5,916,771 ; and 5,939,598, which are incorporated by reference herein in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.), Genpharm (San Jose, Calif.), and Medarex, Inc. (Princeton, NJ.) can be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above. [Para 62] Also human MAbs could be made by immunizing mice transplanted with human peripheral blood leukocytes, splenocytes or bone marrows (e.g., Trioma techniques of XTL). Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection." In this approach a selected non-human monoclonal antibody, e.g., a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope. (Jespers et al., Bio/technology 12:899-903 (1988)).

[Para 63] Further, antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEBJ. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991 )). For example, antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand. Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand. For example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.

[Para 64] The antibodies of the present invention may be bispecific antibodies. Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present invention, one of the binding specificities may be directed towards tissue factor, the other may be for any other antigen, and preferably for a cell-surface protein, receptor, receptor subunit, tissue-specific antigen, virally derived protein, virally encoded envelope protein, bacterially derived protein, or bacterial surface protein, etc. Bispecific antibodies may also comprise two or more single-domain antibodies. W

[Para 65] Methods for making bispecific antibodies are well known. Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulin heavy-chain/ light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature, 305:537-539 (1983). Because of the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecule is usually accomplished by affinity chromatography steps. Similar procedures are disclosed in WO 93/08829, published May 13, 1993, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991 ).

[Para 66] Antibody variable domains with the desired binding specificities (antibody-antigen combining sites) can be fused to immunoglobulin constant domain sequences. The fusion preferably is with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. It may have the first heavy-chain constant region (CHl) containing the site necessary for light-chain binding present in at least one of the fusions. DNAs encoding the immunoglobulin heavy-chain fusions and, if desired, the immunoglobulin light chain, are inserted into separate expression vectors, and are co-transformed into a suitable host organism. For further details of generating bispecific antibodies see, for example Suresh et al., Meth. In Enzym., 121 :210 (1986).

[Para 67] Heteroconjugate antibodies are also contemplated by the present invention. Heteroconjugate antibodies are composed of two covalently joined antibodies. Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980). It is contemplated that the antibodies may be prepared in vitro using known methods in synthetic protein chemistry, including those involving cross-linking agents. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioester bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, for example, in U.S. Pat.

No. 4,676,980.In addition, one can generate single-domain antibodies to tissue factor. Examples of this technology have been described in WO9425591 for antibodies derived from Camelidae heavy chain Ig, as well in US2OO3O130496 describing the isolation of single domain fully human antibodies from phage libraries.

IDENTIFICATION OF ANTI-TISSUE FACTOR ANTIBODIES

[Para 68] Plates for the ELISA assay were coated with 100 microliters of recombinant tissue factor (0.25 μg/ml) in a carbonate based buffer. All steps were performed at room temperature. Plates were blocked with BSA, washed, and then the test samples and controls were added. Antigen/antibody binding was detected by incubating the plate with goat anti-mouse HRP conjugate Gackson ImmunoResearch

Laboratories) and then using an ABTS peroxidase substrate system (Kirkegaad and

Perry Laboratories). Absorbance was read on an automatic plate reader at a wavelength of 405 nm.

PHARMACEUTICALLY ACTIVE INHIBITORS OF TISSUE FACTOR

[Para 69] Pharmaceutically active compounds that inhibit the action of tissue factor are described in WOOO/18398 and WOOl /30333, which are incorporated by reference in their entirety. These compounds include: Formula I:

[Para 70] AR-(CXY)m-(HET)-(CXⁱγ')n-C(Z)p-(PO₃)₃-p [Para 71] wherein Ar is optionally substituted carbocyclic aryl or optionally substituted heteroaryl;

[Para 72] Het is optionally substituted N, O or S; each X, each Y, each X', each Y' and each Z are each independently hydrogen; halogen; hydroxyl; sulfhydryl; amino; optionally substituted alkyl preferably; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; or optionally substituted alkylamino; m and n each is independently an integer of from 0 to 4; p is

1 or 2; and pharmaceutically acceptable salts thereof.

[Para 73] Formula Il (See Figure 1)

[Para 74] wherein X₁ Y, Het, X', Y', Z, m, n and p are the same as defined above;

[Para 75] each R' is independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl; and q is an integer of from 0 to 5; and pharmaceutically acceptable salts thereof.

[Para 76] Formula III (See Figure I)

[Para 77] wherein X, Y, X', Y', Z, m, n and p are the same as defined above;

[Para 78] W is hydrogen, optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl;

[Para 79] R¹ is independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl; q is an integer of from 0 to 5; and pharmaceutically acceptable salts thereof. [Para 80] Formula IHA (See Figure 1)

[Para 81] wherein R¹ is independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl; and q is an integer of from 0 to 5; and pharmaceutically acceptable salts thereof. [Para 82] Formula IV (See Figure 1)

[Para 83] wherein X, Y, X', Y', Z, m, n and p are the same as defined above; R¹ is independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl; q is an integer of from O to 5; and pharmaceutically acceptable salts thereof; AND

[Para 84] Formula IVA (See Figure I):

[Para 85] wherein X', Y', and n are the same as defined above; R¹ is independently halogen; amino; hydroxy; nitro; carboxy; sulfhydryl; optionally substituted alkyl; optionally substituted alkenyl; optionally substituted alkynyl; optionally substituted alkoxy; optionally substituted alkylthio; optionally substituted alkylsulfinyl; optionally substituted alkylsulfonyl; optionally substituted alkylamino; optionally substituted alkanoyl; optionally substituted carbocyclic aryl; or optionally substituted aralkyl; and q is an integer of from 0 to 5; and pharmaceutically acceptable salts thereof. [Para 86] Preparation of 1 -(bisphosponate)-2-amino(3-hydroxy-phenyl)ethyl, as an example, involves following the method of Degenhardt et al.,y. Org. Chem., 51 : 3488-3490 (1986) to produce the compound. Briefly, paraformaldehyde (104.2 g, 3.47 mol) and di-ethylamine (50.8 g, 0.69 mol) are combined in 2 liters of methanol and the mixture warmed until clear. The heat is removed and ChMPC^ChhCHshb (200 g, 0.69 mol) is added. The mixture is refluxed for 24 hours, and then an additional 2 liters of methanol is added, and the solution concentrated under reduced pressure at 35⁰C. 1 liter of toluene is added to the concentrate, and the resulting solution concentrated, and the toluene addition and concentration repeated. The resulting intermediate is then dissolved in T liter of dry toluene, p-toluenesulfoπic acid monohydrate (0.50 g) is added and the mixture is refluxed. Resulting methanol is removed, e.g. via a Dean-Stark trap or molecular sieves. After 14 hours the solution can be concentrated, diluted in chloroform, washed with water (2 x 1 50 ml), dried over MgSO₄ and concentrated. The resulting compound,

W 2

can be purified if desired such as distillation. The compound CH2=C(PO3(CH2CH₃)2)2 can then be reacted as desired to provide compounds of the invention. In particular, to provide the title compound,

can be reacted with NH2(3- hydroxyphenyl) in a Michael reaction. The phosphono di-ester can be converted to the di-acid by treatment with bromotrimethylsilane (see, e.g. Morita et al., Bull. Chem. Soc.Jpn., 54:267 (1981 )). EXAMPLE 1 - FXa-SPECIFIC SUBSTRATE ASSAY

[Para 87] In general, the experiments described herein may be used to determine if a given antibody inhibits the activity of tissue factor. Experiments were conducted using rhTF lipidated with phosphatidycholine (0.07 mg/ml) and phosphatidylserine (0.03 mg/ml) at a 70/30 w/w ratio in 50 mM Tris-HCI, pH 7.5, 0.1% bovine serum albumin (BSA) for 30 minutes at 37⁰C. A stock solution of preformed TFrVIIa complex was made by incubating 5 nM of the lipidated rhTF and 5 nM of FVIIa for 30 minutes at 37°C. The TF:Vlla complex was aliquoted and stored at -70⁰C until needed. Purified human factors VII, Vila, and FX were obtained from Enyzme Research Laboratories, Inc. The following buffer was used for all FXa and FVIIa assays: 25 mM Hepes-NaOH, 5 mM CaCI₂, 1 50 mM NaCI, 0.1 % BSA, pH 7.5.

[Para 88] Mabs were tested for their capacity to block TF:Vlla-mediated activation of FX to FXa. The FX activation was determined in two discontinuous steps. In the first step (FX activation), FX conversion to FXa was assayed in the presence of Ca⁺². In the second step (FXa activity assay), FX activation was quenched by EDTA and the formation of FXa was determined using a FXa-specific chromogenic substrate (S- 2222). The S-2222 and S-2288 (see below) chromogens were obtained from Chrσmogenix (distributed by Pharmacia Hepar Inc.). FX activation was conducted in 1.5 ml microfuge tubes by incubating the reaction with 0.08 nM TF:Vlla, either pre- incubated with an anti-rhTF antibody or a buffer control. The reaction was subsequently incubated for 30 minutes at 37°C, then 30 nM FX was added followed by an additional incubation for 10 minutes at 37°C. FXa activity was determined in 96-well titre plates. Twenty microlitres of sample was withdrawn from step one and admixed with an equal volume of EDTA (500 mM) in each well, followed by addition of 0.144 ml of buffer and 0.016 ml of 5 mM S-2222 substrate. The reaction was allowed to incubate for an additional 15-30 minutes at 37⁰C. Reactions were then quenched with 0.05 ml of 50% acetic acid, after which, absorbance at 405 nm was recorded for each reaction. The inhibition of TF:Vlla activity was calculated from OD4θsnm values in the experimental (plus antibody) and control (no antibody) samples. In some experiments, an anti-hTF antibody, TF/Vlla, and FX were each added simultaneously to detect binding competition. H36.D2 MAb inhibited TF:/Vlla activity toward FX to a significantly greater extent (95%) than other anti-rHTF Mabs tested. EXAMPLE 2 - FVIIa-SPECIFIC SUBSTRATE ASSAY

[Para 89] Mabs may be further screened by an FVIIa specific assay. In this assay, 5 nM lipidated rhTF was first incubated with buffer (control) or 50 nM antibody (experimental) in a 96-well titre plate for 30 minutes at 37°C, then admixed with 5 nM purified human FVIIa (V_τ= 0.192 ml), followed by 30 minutes incubation at 37°C. Eight microliters of a 20 mM stock solution of the FVIIa specific substrate S-2288 was then added to each well (final concentration, 0.8 mM). Subsequently, the reaction was incubated for one hour at 37⁰C. Absorbance at 405 nm was then measured after quenching with 0.06 ml of 50% acetic acid. Percent inhibition of TF/Vlla activity was calculated from OD405πm values from the experimental and control samples. [Para 90] H36 antibody did not significantly block TF/Vlla activity toward the S- 2288 substrate when the antibody was either pre-incubated with TF (prior to Vila addition) or added to TF pre-incubated with Vila (prior to adding the antibody). This indicates that H36 does not interfere with the interaction (binding) between TF and FVIIa, and that H36 also does not inhibit TF:Vlla activity toward a peptide substrate. EXAMPLE 3 - PROTHROMBIN TIME (PT) ASSAY

[Para 91 ] Calcified blood plasma will clot within a few seconds after addition of thromplastin (TF); a phenomenon called the "prothrombin time" (PT). A prolonged PT is typically a useful indicator of anti-coagulation activity (see e.g., Gilman et al. supra).

[Para 92] The H36.D2 antibody was investigated for capacity to affect PT according to standard methods using commercially available human plasma (Ci-Trol Control, Level I obtained from Baxter Diagnostics Inc.). Clot reactions were initiated by addition of lipidated rhTF in the presence of Ca⁺+. Clot time was monitored by an automated coagulation timer (MLA Electra 800). PT assays were initiated by injecting 0.2 ml of lipidated rhTF (in a buffer of 50 mM Tris-HCl, pH 7.5, containing 0.1 % BSA, 14.6 mM CaCb, 0.07 mg/ml of phosphatidylcholine, and 0.03 mg/ml of phosphatidylserine) into plastic twin-well cuvettes. The cuvettes each contained 0.1 ml of the plasma preincubated with either 0.01 ml of buffer (control sample) or antibody (experimental sample) for 1 -2 minutes. The inhibition of TF-mediated coagulation by the H36.D2 antibody was calculated using a TF standard curve in which the log [TF] was plotted against log clot time.

[Para 93] H36.D2 antibody substantially inhibits TF-initiated coagulation in human plasma. The H36.D2 antibody increased PT times significantly, showing that the antibody is an effective inhibitor of TF-initiated coagulation (up to approximately 99% inhibition).

EXAMPLE 4 - SPECIFIC BINDING OF THE H36.D2 ANTIBODY TO NATIVE rhTF [Para 94] Evaluation of the binding of an antibody to Native tissue factor may be assayed according to the following protocol. H36.D2 binding to native and non- native rhTF was performed by a simplified dot blot assay. Specifically, rhTF was diluted to 30 μg/ml in each of the following three buffers: 10 mM Tris-HCI, pH 8.0; 10 mM Tris-HCI, pH 8.0 and 8 M urea; and 10 mM Tris-HCI, pH 8.0, 8 M urea and 5 mM dithiothreitol. Incubation in the Tris buffer maintains rhTF in native form, whereas treatment with 8M urea and 5nM dithiothreitol produces non-native (denatured) rhTF. Each sample was incubated for 24 hours at room temperature. After the incubation, a Millipore lmmobilon (7x7cm section) membrane was pre- wetted with methanol, followed by 25 mM Tris, pH 10.4, including 20% methanol. After the membranes were air-dried, approximately 0.5 μl, 1 μl, and 2 μl of each sample (30 μg/ml) was applied to the membrane and air-dried. After blocking the membrane by PBS containing 5% (w/v) skim milk and 5% (v/v) NP-40, the membrane was probed with H36.D2 antibody, followed by incubation with a goat anti-mouse IgC peroxidase conjugate (obtained from Jackson ImmunoResearch Laboratories, Inc.). After incubation with ECL Western Blotting reagents in accordance with the manufacturer's instructions (Amersham), the membrane was wrapped with plastic film (Saran Wrap) and exposed to X-ray film for various times.

[Para 95] H36.D2 Mab binds a conformational epitope on native TF in the presence of Tris buffer or Tris buffer with 8M urea. (See U.S. Pat. No. 6,555,319) The autoradiogram was exposed for 40 seconds. EXAMPLE 5 - VIRAL INFECTION MODEL

[Para 96] The following study is used to assess the effect of an exemplary tissue factor inhibitor, an anti-TF antibody, on the coagulation cascade, inflammatory response, viral dynamics, and lung damage due to avian influenza H5N1 virus infection in macaques, as a model for the disease in humans.

[Para 97] This model is appropriate to study the possible efficacy of an anti-TF antibody to decrease the severity of lung injury from influenza virus infection in humans. For these studies, typically 6 macaques per group are used to get a satisfactory statistical power.

[Para 98] The animal experiments are carried out under conditions that meet the standards for animal experimentation care of the animals in the center. Macaques are sedated for all procedures requiring handling. This is performed by intramuscular injection of ketamine HCI at a dose of 10-15 mg per kg body weight. This dose provides deep sedation for about 20 to 40 minutes and reduces the hazard of accidental bite wound or exposure to hazardous agents for personnel handling the macaques. Euthanasia is performed by administering a dose of sodium pentobarbital, intravenously, under ketamine sedation.

[Para 99J A cynomolgus macaque model is used to investigate whether the lung injury from avian influenza H5N1 virus infection is reduced as a result of blockade of the extrinsic coagulation pathway. Although other animal models (mouse, ferret, and cat) exist, the macaque model is especially suitable because the immunological, anatomical, and physiological resemblance of this species to humans is greater than that of other experimental animals. Also, the clinical signs, pathological changes, and tissue distribution of H5N1 virus infection in macaques mimic that in humans. In contrast, H5N1 virus infection in other animal models often causes prominent pathological changes associated with virus replication in other organ systems. [Para 100] For these studies, cynomolgus macaques are placed in enhanced biosafety level 3 glove boxes and inoculated intratracheal^ with influenza virus A/HongKong/483/97 (H5N1 ). Anti-TF antibody is given intravenously in a loading dose before virus inoculation at 5 mg/kg body weight, with repeated doses at regular intervals after infection to maintain the anti-coagulation effect. Antibody levels are monitored, as well as plasma fibrinogen, complete blood counts, PT and PTT in all macaques. There are 3 experiments in the study:

[Para 101] Experiment 1 shows the effect of an anti-TF antibody on the coagulation cascade, inflammatory response, viral dynamics, and lung damage in the acute phase of H5N1 virus infection in cynomolgus monkeys. Group 1 consists of 3 macaques administered antibody buffer in the absence of virus in a mock infection. Group 2 consists of 6 macaques administered antibody buffer and H5N1 virus. Group 3 consists of 6 macaques administered an Irrelevant lgG4 and H5N1 virus. Croup 4 consists of 6 macaques administered anti-TF antibody and H5N1 virus. [Para 102] Group 4 receives an intravenous injection of anti-TF antibody (5 mg/kg body weight) at 12 hours before virus inoculation, and lower doses (0.5 mg/kg body weight) at 1 and 2 dpi (days after inoculation of virus). This dose of anti-TF antibody is chosen based on the effective dose (5 mg/kg over 34 hrs in the baboon ALI study) and the half life of the antibody (3-7 days) obtained from the preclinical safety studies in normal cynomolgus monkeys. Group 2 receives intravenous injections of the antibody vehicle at the same time points, whereas Group 3 receives a control/irrelevant human lgG4 antibody. The irrelevant human lgG4 is tested for negative reactivity and neutralization activity against avian influenza virus by immunochemical assays and in vitro neutralization assay. At 0 dpi, Croups 2, 3 and 4 are inoculated intratracheal Iy with influenza virus A/HongKong/483/97 (H5N1 ) (2.5 x 10⁴ TCIDso). The macaques are euthanized at 3 dpi, which is one day after the expected peak of virus replication in the lungs. At this time point, acute lung injury, with abundant fibrin in the alveolar spaces, is expected to be pronounced in the Groups 2 and 3. Group 1 is a negative control group that is used for comparison with the other groups in the experiments.

[Para 103] Experiment 2 shows the effect of an anti-TF antibody on the coagulation cascade, inflammatory response, viral dynamics, and lung damage during the course of H5N1 virus infection in cynomolgus monkeys. Group 1 consists of 3 macaques per time point (3 dpi , 7 dpi, and 14 dpi) administered an irrelevant lgG4 and H5N1 virus. Group 2 consists of 3 macaques per time point(3 dpi , 7 dpi, and 14 dpi) administered anti-TF antibody and H5N1 virus.

[Para 104] The treatment of Groups 1 and 2 of this experiment corresponds to that of Groups 3 and 4 in Experiment #1 . However, the group size is larger and macaques are euthanized at 3, 7, and 14 dpi, in order to determine the effect of anti-TF antibody during the course of H5N1 virus infection. Based on previous studies with H5N1 virus, virus replication in the lungs is expected to end by about 7 dpi. In Group 1 , the lung injury is expected to change from acute at 3 dpi, with abundant fibrin in the alveolar spaces, to subacute at 7 dpi, with pronounced type 2 pneumocyte hyperplasia, and finally to chronic at 14 dpi, with a combination of re-epithelialization of alveoli and alveolar fibrosis. [Para 105] Experiment 3 shows the effect of anti-TF antibody on the coagulation cascade, inflammatory response, viral dynamics, and lung damage in the acute phase of H5N1 virus infection, in combination with a current antiviral agent oseltamivir in cynomolgus monkeys. Group 1 consists of 3 macaques administered Irrelevant lgG4 and H5N1 virus. Group 2 consists of 6 macaques administered anti-TF antibody and H5N1 virus. Group 3 consists of 6 macaques administered Oseltamivir and H5N1 virus. Group 4 consists of 6 macaques administered anti-TF antibody plus Oseltamivir and H5N1 virus.

[Para 106] The treatment of Groups 1 and 2 of this experiment corresponds to that of Groups 3 and 4 in Experiment #1. Group 3 receives oseltamivir (1 mg/kg body weight, per os) on -1 , 0, 1 , and 2 dpi, unless the results of an independent experiment on the pharmacokinetics of oseltamivir in cynomolgus monkeys indicates otherwise. Group 4 receives both anti-TF antibody and oseltamivir at the dose regime indicated for groups 2 and 3. The macaques are euthanized at 3 dpi, at which time pronounced acute lung injury is expected. EXPERIMENTAL ENDPOINTS, DATA ANALYSIS AND INTERPRETATION [Para 107] The efficacy of treatment with anti-TF antibody, or with the combination of anti-TF antibody and oseltamivir, is assessed by statistical comparison of drug- treated and sham-treated animals using the following endpoints as described below. [Para 108] Histopathology endpoints for lung injury are based on histological evaluation of postmortem lung tissue. Per macaque, one lung is inflated with 10% neutral-buffered formalin and samples are selected in a standard manner from cranial, medial, and caudal parts of the lung. Influenza virus antigen expression in the lung is determined by immunohistochemistry (Kuiken T, et al. Veterinary Pathology. (2003) 40:304-310; Rimmelzwaan et al.. J Virol (2001) 75; 6687-6691 ), and scored per animal as the number of positive fields per 100 fields (Haagmans BL₁ et al. Nat Med. (2004) 1 0:290-293). Inflammatory lesions are scored in a semiquantitative manner, based on the number and size of inflammatory foci and the severity of inflammation. The presence of polymerized fibrin and collagen within these foci are assessed by use of phosphotungstic acid-hematoxylin stain and Masson's trichrome stain, respectively.

[Para 109] Virology endpoints for virus replication and excretion are based on virological examination of swabs collected during the experiment and lung tissue collected at necropsy. Nasal swabs and pharyngeal swabs are collected under ketamine anesthesia at 0, 1 , 2, 3, 5, 7, 10, and 14 dpi. Lung specimens for virological examination are collected at necropsy. Both lung specimens and swabs are tested for the presence and quantity of influenza virus RNA by use of a quantitative real time PCR assay .

[Para 1 10] Biochemical endpoints for inflammation and the coagulation cascade are measured in broncho-alveolar lavage fluid (BALF) collected at necropsy, and in serum collected under ketamine anesthesia at 0, 1 , 2, 3, 5, 7, 10, and 14 dpi. BALF is not collected during the course of infection, because it is known to influence the course of viral infection in the lung. Cytokines (TNF-rl , IL-I , IL-6, IL-8, TGF- , and VEGF), which are implicated in the pathogenesis of acute lung injury, are measured in BALF by commercial ELISA kits.

[Para 1 1 1] Anti-TFantibody levels and anti-coagulant activities are measured by established assays. Sensitive ELISAs are used to measure TF and anti-TF antibody. Procoagulant activity in plasma and BALF are determined by prothrombin time (PT), and by ELISAs for fibrinogen, FDP, and thrombin-antithrombin (TAT) complexes. Anti- TF antibody levels are compared statistically to pro-coagulant and fibrinolytic activity in plasma and BALF at the end of the experiments.

[Para 1 12] Determination of gene and protein expression by gene-chip microarrays and proteomics are performed on whole blood and lung tissues obtained from the monkeys (Experiment #2). Gene and protein expression are analyzed, focusing on biomarkers related to inflammation and coagulation/fibrinolytic systems using, e.g., Affymetrix's gene chips (for cynomolgus monkeys). Gene expression using microarrays is now used across diverse biological applications and is playing an increasingly important role in the study of virus-host interactions (Kato-Maeda, M et al., Cell Microbiol. (2001 ) 3: 713-719; Manger ID, et al., Curr Opin Immunol. (2000) 1 2: 21 5-21 8). Such studies are yielding many new insights into how viruses interact with the cell and mechanisms of disease pathogenesis.

[Para 1 13] By proteomics, a search for proteins and peptides that are differentially expressed in the lung tissue of different experimental groups is done. Because of the enormous complexity of the proteome and the dynamic range of proteins, samples may be pre -fractionated by, e.g., nano liquid chromatography techniques. The resulting fractions are compared by, e.g., Fourier transform mass spectrometry. The resulting peptides that are differentially expressed can be identified by MS/MS approaches.

[Para 1 14] Statistical power and analysis. The minimum sample size that is employed is six animals per group. For two normally distributed samples with equal variances (max s 0.25), the estimated sample size needed to detect a mean difference of 40% is 5.6 for a<0.05 and b 0.8. For a mean difference of 33%, sample size is 7.8. Thus group sizes of 6-8 provide adequate power for the measurement endpoints to detect biologically meaningful differences. It is generally preferred to use the lower n=6 in primates to conserve animals and to demonstrate robust, physiologically- relevant lung protection. Data is analyzed using SPSS version 1 1. The outcome variables viral load at certain time points and AUC of viral load over a defined time period are analyzed after logarithmic (base 10) transformation using one-way ANOVA. It is assumed that viral loads after log transformation have a normal distribution in the separate groups as well as in the combined groups. This assumption can be verified by inspection of the distribution (mainly concerning symmetry) and by using the Kolmogorov-Smirnov test, yielding p-values around 0.70. For AUC analysis, homogeneity of the within-group variances is tested across the groups. Then, depending on the appropriate assumption of either homogeneous or heterogeneous variances, differences in means between the groups can be tested. If the overall group effect (i.e., comparing all groups simultaneously) turns out to be significant (p < 0.05), the relevant pairwise comparisons are tested.

Claims

What is claimed is:

[Claim 1 ] A method of treating a patient suffering from a viral infection comprising administering a tissue factor Inhibitor.

[Claim 2] The method of claim 1 , wherein the tissue factor inhibitor comprises an antibody, a protein or peptide mimetic, a tissue factor ligand analog or an organic molecule.

[Claim 3] The method of claim 1 , wherein the mortality associated with the viral infection is reduced.

[Claim 4] The method of claim 1 , wherein the number or severity of morbidities is reduced.

[Claim 5] The method of claim 1 , wherein lung damage associated with viral infection is reduced.

[Clai m 6] The method of claiml , wherein the viral infection is caused by influenza virus, avian influenza virus, or a coronovirus.

[Claim 7] The method of claim 1 , wherein the inhibitor binds tissue factor and inhibits binding of Factor X to tissue factor.

[Claim 8] The method of claim 1 , wherein the inhibitor binds tissue factor and inhibits the activation of Factor X to Xa.

[Claim 9] The method of claim 1 , wherein the inhibitor binds tissue factor and inhibits binding of Factor VII to tissue factor.

[Claim 1 0] The method of claim 1 , wherein the inhibitor binds tissue factor and inhibits the activation of Factor VII to Vila. [Claim 1 1 ] The method of claim 1 , wherein the inhibitor binds Factor VII or Factor

X.

[Claim 1 2] The method of claim 1 , wherein the inhibitor comprises a single domain antibody, a monoclonal antibody, a human antibody, a humanized antibody, a single chain antibody or a binding fragment thereof.

[Claim 1 3] The method of claim 12, wherein the inhibitor comprises an antibody fragment comprising a Fab, a Fab 2, or an Fv.

[Claim 1 4] The method of claim 1 , wherein the inhibitor comprises the Factor X binding site of tissue factor and binds Factor X.

[Claim 1 5] The method of claim 1 , wherein the inhibitor comprises the Factor VII binding site of tissue factor and binds Factor VII.

[Claim 1 6] The method of claim 1 , wherein the inhibitor is 1 -(bisphosponate)-2- amino(3-hydroxy-phenyl)ethyl.

[Claim 1 7] The method of claim 2, further comprising the administration of an anti-viral agent.

[Claim 1 8] The method of claim 3, wherein the anti-viral agent is amantidine, rimantidine, or a neuraminidase inhibitor, such as zanamivir and oseltamivir.