WO2008030260A2 - Treatment of variola viral infections using a tissue factor inhibitor - Google Patents

Abstract

The present invention provides novel methods for the treatment of variola viral infections, such as smallpox and monkeypox, by administering a tissue factor inhibitor.

Description

INVENTION TITLE TREATMENT OF VARIOLA VIRAL INFECTIONS USING A TISSUE FACTOR INHIBITOR

BACKGROUND OF THE INVENTION

[Para 1 ] Variola viral infections (including smallpox virus and monkeypox virus) are serious threats to the human population and are listed as priority pathogens by the NIAID (http://www2.niaid.nih.gov/biodefense/cat.pdf)- There is growing concern that mammalian poxviruses, like monkeypox virus, may easily cross the species barrier. This is because smallpox in humans has been eradicated and vaccination against smallpox was discontinued in 1 980, rendering the majority of the human population susceptible to poxvirus infections (Lewis-Jones S. Curr Opin Infect Dis. (2004) 1 7: 81 — 89). Also, pox viruses are pathogens that may be used to carry out acts of biological terrorism, and thus cause adverse public health impact and civil disruption (Rotz LD, Khan AS, Lillibridge SR, Hughes M. Emerg Infect Dis. (2002) 8: 225-227). [Para 2] Despite these threats, the current arsenal of preventive and therapeutic strategies against variola viruses is limited. Vaccination against the viruses is efficacious, but no longer administered on a routine or general basis. Moreover, traditional vaccines suffer from rare but severe side-effects (Mayr A. Comp Immunol Microbiol Infect Dis. (2003) 26: 423-430) or are too slowly produced in the face of a terrorist act. Antiviral drugs have potential but have yet to be tested (e.g., cidofovir, Bray M, et. al. J Antimicrob Chemother. (2004) 54: 1 -5). Adequate stockpiles of vaccines and antiviral drugs do not exist. Furthermore, the development of viral resistance remains a serious threat.

[Para 3] In addition, these therapies are primarily designed as prophylactic treatments to protect the population from contracting the disease prior to an exposure event, 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 terrorist act. [Para 4] Because of the serious effect of small pox infection on a host and the potential for a cross species event from monkeypox, there exists a critical need for new treatments. It is important to find a safe and effective treatment of acute lung injury due to poxvirus infection in humans, to reduce the sequelae from the bronchopneumonia and reduce the threat of a terrorist act. SUMMARY OF THE INVENTION

[Para 5] The present invention relates to novel methods of treating a patient suffering from a variola virus infection, e.g., smallpox or monkeypox, comprising administering an inhibitor of tissue factor. Tissue factor is involved in the activation of FVII, FIX, and FX in the extrinsic coagulation pathway. This approach is designed to overcome the shortcomings inherent in previous approaches and prevent certain clinical outcomes including mortality and morbidity.

[Para 6] One aspect of the invention includes the treatment of smallpox or monkeypox infections comprising the administration of a tissue factor inhibitor which binds tissue factor and blocks the activation of the extrinsic coagulation pathway. Tissue Factor inhibitors may include antibodies, peptide mimetics, tissue factor ligand analogs, tissue factor pathway inhibitor (TFPI), and organic molecules that inhibit tissue factor. These tissue factor inhibitors also include those that do not bind directly to tissue factor per se but to the complexes of FVIIa-tissue factor, FX-tissue factor, FVIIa-FX-tissue factor, FIX-tissue factor, and FVIIa-FIX-tissue factor. [Para 7] Other aspects of the invention include the treatment of smallpox or monkeypox virus infections with a tissue factor inhibitor in combination with another antiviral agent such as, Cidofovir.

[Para 8] 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 9] 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 10] 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 1 1 ] AlI 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 1 2] 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 1 3] 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, 1 988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1 993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1 994; Sequence Analysis in Molecular Biology, von Heinje, G., Academic Press, 1 987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1 991 ; and Carillo, 30 H., and Lipman, D., SIAM J. Applied Math., 48: 1073 (1 988). 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 1 2(1 ): 387 (1 984)), BLASTP, BLASTN, and FASTA (Atschul, S. F. et al.J Molec. Biol. 21 5: 403-410 (1 990). 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 (1 990). The well-known Smith Waterman algorithm may also be used to determine identity.

[Para 14] 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 1 5] 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 (1 975), 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 (1 991 ) and Marks et al., / MoI. Biol., 222:581 -597 (1 991 ), 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. Sc/. USA, 81 :6851 -6855 (1 984)).

[Para 16] "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 1 7] 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 1 8] 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 19] 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, IgG, and IgM, and several of these may be further divided into "subclasses" (isotypes), e.g., IgGl , lgG2, lgG3, lgG4, IgA, and lgA2. The heavy-chain constant domains that correspond to the different classes

three-dimensional configurations of different classes of immunoglobulins are well known.

[Para 20] 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

-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. (1 991 )).

[Para 21 ] 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 ¹¹CDR" (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. (1 991 )) and/or those residues from a "hypervariable loop" (e.g. residues 2632 (Ll ), 50-52 (L2) and 91 -96 (L3) 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. 1 96:901 -91 7 (1 987)). "Framework Region" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined. [Para 22] "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 23] "Single-chain Fv" or "scFv" antibody fragments comprise the V_H and V_L 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 1 3, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-31 5 (1 994). Anti-ErbB2 antibody scFv fragments are described in WO93/ 161 85; U.S. Pat. No. 5,571 ,894; and U.S. Pat. No. 5,587,458. [Para 24] 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 (VO 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 1 61 ; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444- 6448 (1 993).

[Para 25] 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), 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 26] "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 humanization 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 27] Variola virus, the etiological agent of smallpox, belongs to the family Poxviridae, subfamily Chordopoxvirinae, and genus orthopoxvirus, which includes cowpox virus, vaccinia virus (VV), monkeypox virus (MPXV), and several other animal poxviruses that cross-react serologically. The poxviruses contain single, linear, double-stranded DNA molecules of 1 30-to-375-kb pairs and replicate in cell cytoplasm. They are shaped like bricks on electron micrographs and measure about 300 by 250 by 200 nm. There are two classic forms of smallpox, namely variola major and variola minor, each of which confers immunity against the other (Kuiken T, et al. Curr Opin Biotech. 14: 641 -646).

[Para 28] The incubation period for smallpox is about 1 to 2 weeks (Gooze LL et al. Semin Respir Infect. (2003) 1 8: 1 96-205). The prodromal phase, which lasts for two or three days, is characterized by severe headache, backache, and fever, all beginning abruptly. The temperature often rises to more than 40°C (104T) and then subsides over a period of two to three days. Enanthema over the tongue, mouth, and oropharynx precedes the development of a rash by a day. The rash begins as small, reddish macules, which become papules and then vesicles followed by umbilication and crusting. These lesions occur first on the face and extremities but gradually cover the body. Smallpox infection has a mortality rate of 10-30% and survivors are left with disfiguring scars and occasionally blindness. Death from smallpox is ascribed to toxemia, associated with immune complexes, and to hypotension. [Para 29] Variola major has been endemic in India for at least 2000 years, and has spread to China, Japan, Africa, and the Americas (Henderson DA, et. Al. JAMA. (1 999) 281 :21 27-21 37). Beginning in the 20th century, the less virulent form of smallpox, variola minor, spread from South Africa to Florida and the Americas, and then to Europe.

[Para 30] More than one thousand years ago people started to protect themselves by intentional inoculation with subvirulent strains of variola virus, so called "variolation". Although, this procedure offered lifelong immunity, it was a very risky practice, as disease could be transmitted to untreated individuals and the mortality rate was only 10-fold reduced compared to the natural disease. In 1 796, Edward Jenner, demonstrated that individuals could be protected against smallpox by inoculation of cowpox virus substance into the skin (Baxby D. Trans Med Soc Lond. (1 996) 1 1 3: 1 8- 22). This discovery founded the eradication of smallpox. Following a worldwide vaccination program the World Health Organization (WHO) declared smallpox to be eradicated in May 1 980. Soon thereafter, general vaccination against smallpox was discontinued. Variola virus and also monkeypox virus (MPXV, see below) are now ranked high on the list of biological agents that may be used as a bioweapon because of the high mortality rate and to date the vast majority of the population lacks protective immunity (LeDuc JW, et. al. Emerg Infect Dis. (2002) 8:743-745). In addition, there are growing concerns from the observation that other mammalian poxviruses, like cowpox virus and MPXV, may now cross the species barrier to humans more easily (Lewis-Jones S. Curr Opin Infect Dis. (2004) 1 7:81 -89). [Para 31 ] While traditional (1 ^st generation) smallpox vaccines based on replicating variola virus are efficacious and were the basis for the eradication of smallpox, they are associated with rare but severe side effects, such as postvaccination encephalitis, vaccinia gangrenosa, eczema vaccinatum, generalized vaccinia, and erythematous urticarial lesions, particularly in immune compromised individuals. Moreover, the fact that it has been estimated that at least 25% of the US population should not receive traditional smallpox vaccines in the absence of a direct threat, highlights the growing need for safe intervention strategies (Kemper AR, et. al. Eff Clin Pract. (2002) 5:84- 90). Given the relatively high rate of severe side effects associated with classical smallpox vaccination, reintroduction of general preventive smallpox vaccination is not recommended. Therefore, efficacy of new generation candidate smallpox vaccines with better safety profiles, such as the modified vaccinia virus Ankara (MVA), is being addressed at present. MVA is a highly attenuated vaccinia virus (VV), which was used as a smallpox vaccine during the end stage of the smallpox eradication program. MVA-based recombinant viruses (rMVA) have, either alone or in prime-boost regimens, been shown to be promising vaccine candidates for certain infectious diseases of human.

[Para 32] Alternatively to vaccination a promising intervention method against smallpox is the use of selective antiviral drugs. Today only the acyclic nucleotide analogue Cidofovir (HPMPC, VISTIDE®) is recommended for treatment of complications associated with smallpox vaccination and for therapy of smallpox and monkeypox virus infection. In several mouse models for poxvirus infections it has been shown that post-exposure Cidofovir treatment may delay or reduce mortality (Bray M, et. al. J Antimicrob Chemother. (2004) 54:1 -5). It has been shown that antiviral treatment with Cidofovir, initiated 24 hours after lethal intratracheal MPXV infection, resulted in significantly reduced mortality and reduced numbers of cutaneous monkeypox lesions. Although other anti-poxvirus compounds with a different mode of anti-poxvirus activity have been described, their clinical use may not be expected in the near future. Cidofovir is licensed for the treatment of cytomegalovirus retinitis in patients with HIV infection. It is not currently approved for treatment of variola virus infections in humans, but has been shown to be also active against a number of poxviruses, including vaccinia-, variola-, monkeypox-, and cowpox virus.

[Para 33] There is a growing concern that the virulence of variola virus and other poxviruses could be increased by genetic modification, enabling it to overcome vaccine-induced immunity, and would be an additional reason for developing new strategies for the treatment of poxvirus infections in humans.

[Para 34] There are a number of animal models described that can be used for testing new candidate smallpox vaccines or drugs for anti-smallpox treatment. These include infection models in different strains of mice using cowpox virus, ectromelia virus or VV.

[Para 35] A respiratory MPXV infection model has been established in cynomolgus macaques (Macaca fasciculahs). MPXV is an orthopoxvirus closely related to variola virus and can cause a smallpox-like disease in humans with a mortality rate of approximately 10% and a human-to-human transmission rate of around 50%. The disease is endemic in the rainforests of central and western Africa. Animal antibody surveys in the Democratic Republic of Congo suggested that squirrels and monkeys play a role in the life cycle of the virus. MPXV infection in cynomolgus macaques causes a disease very similar to smallpox. Different infection protocols have been described including intramuscular, intravenous and respiratory inoculation. Since MPXV, like variola virus has evolved primarily as a respiratory pathogen, it is probably most suitable for pathogenesis studies and for the evaluation of intervention strategies against smallpox and MPXV.

[Para 36] The MPXV model is based on intratracheal inoculation of MPXV as this method was reproducible and easy to standardize. FDA regulations require the use of animal models in which the animal study endpoint is clearly related to the desired human benefit: the so-called 'animal rule¹. In this model an infection dose of 10⁷ pfu was 100% lethal with mean day of death of 1 2.6, ranging from 9 - 1 9 (Stittelaar, K. J., et. al. Nature 2005 Dec 1 1 ; [Epub ahead of print]). Rise in body temperature started at day 3-4 and cutaneous lesions appeared at day 8-10. Animals showed anorexia, dyspnea, nasal discharge, conjunctivitis and the number of cutaneous lesions increased. Around day 14 the lesions could be classified as typical pustules, animals were severely lethargic and died or were euthanized when they were moribund. [Para 37] Generally, upon histopathological examination, the lungs of these animals showed acute lung injury, characterized by necrosis of the alveolar wall and flooding of the alveolar lumina with cellular and acellular components, including abundant fibrin. The association of these lesions with monkeypox virus infection was confirmed by transmission electron microscopy. Changes in other tissues in these animals included tracheitis, necrotizing glositis, lymphadenitis, splenitis with lymphoid depletion. During the infection, blood samples were collected for measurement of antibody responses, T cell responses, viral loads as well as hematological and clinical chemistry parameters. In addition, viral loads were measured in throat swabs to estimate risk for virus spread. Clinical observations were substantiated by measuring blood oxygen saturation and body temperature by telemetry. [Para 38] As mentioned above, lungs of cynomolgus macaques that died from MPXV infection (10⁷ pfu) showed fibrinonecrotic bronchopneumonia and this was also shared by animals that died despite post-exposure vaccination or post-exposure antiviral treatment. In the lungs, multifocal areas of necrosis were found that involved the terminal airways, the alveolar spaces and the interstitium. The bronchial and bronchiolar epithelia exhibited multifocal hyperplasia and vacuolation. In some areas, the mucosal epithelium was eroded and necrotic and sloughed in the lumen. Multifocal Iy, the alveolar spaces were filled by eosinophilic fibrilar material (fibrin), eosinophilic amorphous material (edema), foamy macrophages, degenerative neutrophils and necrotic cellular debris. In association with these areas small necrotic vessels were apparent. The peribronchial fibrous connective tissue was expanded by abundant number of extravasated red blood cells (hemorrhage), edema, increased number of fibroblasts and infiltrates of mixed inflammatory cells. [Para 39] Lungs of macaques that were infected with a non-lethal dose of MPXV (10⁶ pfu and lower) or that survived a lethal MPXV challenge, because they were either preventively vaccinated, post-exposure vaccinated or treated post-exposure with an antiviral compound, showed formation of connective tissue, about one week after the virus had been cleared, to different extents as a result of mild to moderate fibrosis. As previously discussed by Zaucha et al., a high incidence of bronchopneumonia was reported for fatal cases of monkeypox in humans and was also a common feature of smallpox (Zaucha CM, et. al. Lab Invest. (2001 ) 81 :1 581 -1600). [Para 40] It seems that during MPXV infection the normal anticoagulant and fibrinolytic systems were overwhelmed and did not contain the coagulation activation in the lungs. Preliminary data shows that, early during MPXV infection, endothelial cells are being activated as measured by sVCAM-1 levels in plasma. Downstream in the extrinsic coagulation cascade VCAM-I was expressed, together with NF- B, IL-I , IL- 6, IL-8, MCP-I , E-selectin and ICAM-I , as a result of PAR dependent FXa signaling. The fibrinolytic system was activated to dissolve the fibrin thrombi, resulting in consumption of plasminogen as it was converted into plasmin, and the formation of fibrin degradation products (FDP) including D-dimers as plasmin degrades fibrin clots.

[Para 41 ]Jahrling et al. (Proc Natl Acad Sci U S A (2004) 101 : 1 51 96-1 5200) have shown that D-dimer levels are markedly increased in the sera of cynomolgus macaques infected with variola virus. D-dimer levels peaked at the same time that the highest viral loads in throat swabs were measured. [Para 42] Serpins belong to a superfamily of related proteins that regulate serine proteases, which function to regulate a variety of biological activities including connective tissue turnover, coagulation and fibrinolysis. Serpin genes have been identified in cowpox virus, rabbit poxvirus and VV. It has been described that mice infected with a cowpox virus that lack a functional serpin gene show decreased pulmonary hemorrhage, reduced inflammation and an absence of alveolar edema (Thompson JP, et. al. Virology. (1993) 197:328-338). Furthermore, heparin treatment of mice that are intravenously infected with a high dose of VV circumvents coagulation and fibrin loss (Sottnek HM, et al. Lab Invest. (1 975) 33:514-521 ) [Para 43] Etiologic agents such as variola virus and monkeypox virus, which cause lung injury with abundant deposition of fibrin in alveolar spaces, can result in death at two different phases. Death may occur in the acute phase, when alveoli become flooded by fibrin mixed with oedema fluid and other inflammatory components. If patients survive this stage, death may occur in the late phase, when repair of fibrin- filled alveoli occurs due by fibrosis, which is non-functional. [Para 44] Therefore, limiting fibrin deposition in the alveolar spaces by blockade of initiation of coagulation may have therapeutic benefit in patients with lung injury from poxvirus infection, both at the acute and in the late stage. [Para 45] 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 variola 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 tracheitis, necrotizing glositis, lymphadenitis, splenitis with lymphoid depletion, multifocal hyperplasia and vacuolation of the bronchial epithelia, reduction in the mortality rates associated with these severe viral infections, and reduction in the number or severity of morbidities. [Para 46] One embodiment of the present invention is a tissue factor inhibitor 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.

[Para 47] One aspect of the invention is an anti-tissue factor antibody. Antibodies useful in the present invention may bind tissue factor, blocking or inhibiting the action of Factor VII, Factor Vila, 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-I 2255) and humanized clones of this antibody. Other anti-TF antibodies useful in the invention include those disclosed in U.S. Pat. No. 6,555,31 9; 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, Factor Vila, Factor X, or Factor IX 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 48] Peptide mimetics include fragments of tissue factor that bind Factor VII, Factor Vila, Factor IX, Factor X, or Factor Xa, thereby blocking their activation. Tissue factor ligand analogs include modified Factor VII, Factor IX, or Factor X, that bind tissue factor, preventing the binding of the corresponding wild-type ligands to tissue factor and thus inhibiting activation.

[Para 49] Other molecules useful in the present invention include molecules such as those disclosed in WO 00/1 8398 and WO 01 /30333. ANTIBODY GENERATION

[Para 50] 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. (1 988), which is hereby incorporated herein by reference in its entirety).

[Para 51 ] 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 52] One method of generating such antibodies to tissue factor may be found in U.S. Pat. No. 6,555,31 9 and 5,986,065 (which are hereby incorporated herein by reference in their entirety). In 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 53] Antibodies directed to other antigens such as Factor VII, Factor Vila, Factor IX, Factor Xa, and Factor X may be generated in a similar manner. [Para 54] 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 (1 975) 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. (1 988), by Hammerling, et al., Monoclonal Antibodies and T-CeII Hybridomas (Elsevier, N. Y., (1 981 )), 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., 1 983, Immunology Today 4:72; Cole et al., 1 983, Proc. Natl. Acad. Sci. USA 80:2026-2030), and the EBV-hybridoma technique (Cole et al., 1 985, 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 55] 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 56] 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. 4,81 6,567. [Para 57] 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 58] 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 59] 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 229:1 202 (1 985); 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; 4,81 6,567; and 4,816397, which are incorporated herein by reference in their entirety.

[Para 60] 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 (1 988), 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 (1 991 ); Studnicka et al., Protein Engineering 7(6):805-814 (1 994); Roguska. et al., PNAS 91 :969-973 (1 994)), and chain shuffling (U.S. Pat. No. 5,565,332).

[Para 61 ] 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 (1 986); Reichmann et al., Nature, 332:323-327 (1 988); Verhoeyen et al., Science, 239:1 534- 1 536 (1 988), 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 62] 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 4,71 6,1 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, (1 985); and Boerner et al., J. Immunol., 147(1 ):86-95, (1 991 )). [Para 63] 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; 6,248,51 6; and applications US200401 10941 and US2OO3O1 30496 all of which are incorporated herein by reference. [Para 64] 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 IgG, IgA, IgM and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar, Int. Rev. Immunol. 1 3:65-93 (1 995). 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; 5,625,1 26; 5,633,425; 5,569,825; 5,661 ,01 6; 5,545,806; 5,814,31 8; 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 65] 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 1 2:899-903 (1 988)).

[Para 66] 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, FASEB J. 7(5):437-444; (1 989) 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 67] 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. [Para 68] 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 (1 983). 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 1 3, 1 993, and in Traunecker et al., EMBO J., 10:3655-3659 (1 991 ).

[Para 69] 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 Enzymol, 1 21 :210 (1986).

[Para 70] 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 US200301 30496 describing the isolation of single domain fully human antibodies from phage libraries. IDENTIFICATION OF ANTI-TISSUE FACTOR ANTIBODIES

[Para 71 ] 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 (Jackson 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 72] Pharmaceutically active compounds that inhibit the action of tissue factor are described in WOOO/ 1 8398 and WOOl /30333, which are incorporated by reference in their entirety. These compounds include: Formula I:

AR-(CXY)_m-(HET)o or i-(CXⁱγⁱ)n-C(Z)p-(PO₃)3-p

[Para 73] wherein Ar is optionally substituted carbocyclic aryl or optionally substituted heteroaryl; 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 74] Formula Il (See Figure 1 ), wherein X, Y, Het, X', Y', Z, m, n and p are the same as defined above; each 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 75] Formula III (See Figure I), wherein X, Y, X', Y', Z, m, n and p are the same as defined above; 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; 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 76] Formula MIA (See Figure 1 ), 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 77] Formula IV (See Figure 1 ), 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 0 to 5; and pharmaceutically acceptable salts thereof; AND

[Para 78] Formula IVA (See Figure I), 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 79] Preparation of l -(bisphosponate)-2-amino(3-hydroxy-phenyl)ethyl, as an example, involves following the method of Degenhardt et al., y. Org. Chem., 51 : 3488-3490 (1 986) 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 ChMPOstChhChhhh (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 1 liter of dry toluene, p-toluenesulfonic 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, CH2=C(PO3(CH2CH3)2)2, can be purified if desired such as distillation. The compound CH2=C(PO3(CH2CH3h)2 can then be reacted as desired to provide compounds of the invention. In particular, to provide the title compound, CH2=C(PO3(CH2CH3)2)2, can be reacted with NH2O- 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 (1 981 )). EXAMPLE 1 - FXa-SPECIFIC SUBSTRATE ASSAY

[Para 8O] 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 TF:Vlla 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 -70°C 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 81 ] 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 Chromogenix (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 37⁰C, then 30 nM FX was added followed by an additional incubation for 10 minutes at 37°C. FXa activity was determined in 96-well titer 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 0.01 6 ml. of 5 mM S-2222 substrate. The reaction was allowed to incubate for an additional 1 5-30 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 OD₄o5nm 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 82] Mabs may be further screened by an FVMa 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 37⁰C, then admixed with 5 nM purified human FVIIa (VT= 0.1 92 ml), followed by 30 minutes incubation at 37⁰C. 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 OD405nm values from the experimental and control samples. [Para 83] 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 84] 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., Cilman et al. supra).

[Para 85] 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-HCI, 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 86] 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 87] 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 IgG 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 88] 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 89] 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 pox virus infection in macaques, as a model for the disease in humans.

[Para 9O] A lethal intratracheal infection model in cynomolgus monkeys {Macaca fascicularis) with monkeypox virus (MPXV) is used. This model is appropriate to study the possible efficacy of an anti-TF antibody to decrease the severity of lung injury caused by pox virus infection because of the similarity to human small pox infection. MPXV causes a disease similar to human smallpox. This model has been shown to measure differences in protective efficacies of classical and new generation candidate smallpox vaccines (Stittelaar KJ, et. al. J Virol. (2005) 79:7845-7851 ). This model complies with the FDA regulations requiring the use of animal models in which the animal study endpoint is clearly related to the desired human benefit: the so-called 'animal rule'. The intratracheal MPXV infection in macaques is currently the only available respiratory infection model in human primates for smallpox. [Para 91 ] The cynomolgus macaque provides unique advantages as a model due to the close similarity to humans of its pulmonary anatomy and gas exchange, resemblance of the MPXV model to human monkeypox and human smallpox and bronchopnemonia, the ability to use human reagents, and the availability of specific reagents including macaque microarrays. The intratracheal route of infection was chosen for this model system as this method is highly reproducible and easy to standardize. MPXV, like variola virus, has evolved primarily as a respiratory pathogen which replicates massively in the lungs. The MPXV strain is MSF#6 and may be obtained from various laboratories around the world.

[Para 92] The animals are placed in enhanced biosafety level 3 glove boxes and inoculated intratracheal^ with MPXV. 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 three experiments employed in this system. [Para 93] Experiment 1 is used to determine the prophylactic effect of an agent on a lethal respiratory monkeypox virus infection in cynomolgus monkeys. In this experiment, Croup 1 consists of 6 macaques administered antibody buffer and MPXV virus. Group 2 consists of 6 macaques administered an Irrelevant lgG4 and MPXV virus. Group 3 consists of 6 macaques administered anti-TF antibody and MPXV virus. [Para 94] Group 3 receives an intravenous injection of anti-TF antibody (5 mg/kg body weight) at 1 2 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 a bacteria-induced acute lung injury study in baboons) and the half life of the antibody (3-7 days) obtained from the preclinical safety studies in normal cynomolgus monkeys. Group 1 serves as a challenge control, whereas Group 2 receives a control/irrelevant human lgG4 antibody. The irrelevant human lgG4 is tested for negative reactivity and neutralization activity against MPXV by immunochemical assays and in vitro neutralization assay. At 0 dpi, Groups 1 , 2 and 3 are inoculated intratracheal^ with a lethal dose of MPXV, such as strain MSF#6 (10⁷ pfu). The macaques are euthanized at when they are moribund, which is 1 2.6 dpi for untreated animals. At 28 dpi all animals will be euthanized. This first experiment is designed to show prophylactic use of an anti-TF molecule with a "saturating" dosage schedule. [Para 95] 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 monkeypox virus infection in cynomolgus monkeys. [Para 96] Group 1 consists of 4 macaques per time point (3 dpi, 7 dpi, and 1 3 dpi) administered an irrelevant lgC4 and MPXV. Croup 2 consists of 4 macaques per time point (3 dpi, 7 dpi, and 1 3 dpi) administered anti-TF antibody and MPXV. [Para 97] The treatment of Croups 1 and 2 of this experiment corresponds to that of Groups 2 and 3 in Experiment #1 . However, the macaques are euthanized at 3, 7, and 1 3 dpi, in order to determine the effect of anti-TF antibody during the course of MPXV infection. The animals are inoculated either with a lethal dose of MPXV (10⁷ pfu) or a non-lethal dose of MPXV (10⁵ pfu) depending on the outcome of Experiment #1 . A lethal MPXV infection may causes injury that is too severe to observe meaningful changes. In that case a lower, non-lethal dose of MPXV which results in peak viral load similar to the peak viral loads observed in Cidofovir-rescued animals should be used.

[Para 98] Experiment 3 shows the effect of anti-TF antibody on post-exposure antiviral treatment with Cidofovir on the coagulation cascade, inflammatory response, viral dynamics, lung injury, and disease severity and survival during the course of MPXV infection in cynomolgus monkeys. Group 1 consists of 4 macaques administered MPXV. Group 2 consists of 6 macaques administered Cidofovir and MPXV. Croup 3 consists of 6 macaques administered an irrelevant lgG4 antibody and MPXV. Group 4 consists of 6 macaques administered anti-TF antibody plus MPXV. Group 5 consists of 6 macaques administered anti-TF antibody plus Cidofovir and MPXV.

[Para 99] A lethal dose of MPXV is used. In Groups 4 and 5, anti-TF therapy is initiated 24 hours after infection with MPXV, as is the control antibody in Group 3. Cidofovir is administered 24 hours after infection in Groups 2 and 5, but at a higher relative dose given the longer half-life of the antibody. EXPERIMENTAL ENDPOINTS, DATA ANALYSIS AND INTERPRETATION [Para 100] The efficacy of treatment with anti-TF antibody, or with the combination of anti-TF antibody and Cidofovir, is assessed by statistical comparison of drug- treated and sham-treated animals using the following endpoints as described below. [Para 101 ] 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 BL, 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 102] 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 103] Biochemical endpoints tor 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 1 3 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, TCF- , and VECF), which are implicated in the pathogenesis of acute lung injury, are measured in BALF by commercial ELISA kits.

[Para 104] Anti-TF antibody 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 105] 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: 71 3-71 9; 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 106] 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 107] Statistical power and analysis. The minimum sample size to employ 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 measurement endpoints to detect biologically meaningful differences. We generally prefer to use the lower n=6 in primates to conserve animals and to demonstrate robust, physiologically-relevant lung protection. Data was 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 will be analyzed after logarithmic (base 10) transformation using one way ANOVA. We assume that, viral loads after log transformation have a normal distribution in the separate groups as well as in the combined groups. This assumption will 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 we will first test homogeneity of the within-group variances across the groups. Then, depending on the appropriate assumption of either homogeneous or heterogeneous variances, differences in means between the groups will be tested. If and only if the overall group effect (i.e., comparing all groups simultaneously) turn out to be significant (p < 0.05), the relevant pairwise comparisons will be tested. Differences in survival between the groups will be tested using the log rank test. Again, pairwise comparisons will only be tested if the overall p-value from comparing all groups is significant (p < 0.05).

Claims

What is claimed is:

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

[Claim 2] The method of claim 1 , wherein the tissue factor inhibitor inhibits the extrinsic coagulation pathway.

[Claim 3] 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 4] The method of claim 1 , wherein the mortality associated with the viral infection is reduced.

[Claim 5] The method of claim 1 , wherein the number or severity of morbidities is reduced.

[Claim 6] The method of claim 1 , wherein lung damage associated with viral infection is reduced.

[Claim 7] The method of claiml , wherein the viral infection is caused by smallpox or monkeypox.

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

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

[Claim 1 0] The method of claim 1 , wherein the inhibitor binds a tissue factor complex comprising TF:VII, TF:Vlla, TF:IX, or TF:X and inhibits activation of Factor X to

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

[Claim 1 2] The method of claim 1 , wherein the inhibitor binds tissue factor and

inhibits the activation of Factor VII to Vila.

[Claim 1 3] The method of claim 1 , wherein the inhibitor binds Factor VII, Factor Vila, Factor IX, Factor Xa, or Factor X.

[Clai m 1 4] 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 5] The method of claim 1 5, wherein the inhibitor comprises an antibody fragment comprising a Fab, a Fab 2, or an Fv.

[Claim 1 6] The method of claim 1 , wherein the inhibitor comprises the Factor X binding site of tissue factor and binds Factor X.

[Claim 1 7] The method of claim 1 , wherein the inhibitor comprises the Factor VII binding site of tissue factor and binds Factor VII.

[Claim 1 8] The method of claim 1 , wherein the inhibitor is of the following:

Formula I

AR-(CXY)m-(HET)o or 1-(CX¹Yⁱ)_n-C(Z)P-(PO₃)B-_P wherein Ar is optionally substituted carbocyclic aryl or optionally substituted heteroaryl;

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; or a physiologically acceptable salt thereof.

[Claim 1 9] The method of claim 3, further comprising the administration of an anti-viral agent.

[Claim 20] The method of claim 1 9, wherein the anti-viral agent is Cidofovir.