CA2318434A1

CA2318434A1 - Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium

Info

Publication number: CA2318434A1
Application number: CA002318434A
Authority: CA
Inventors: L.L. Houston; Craig D. Dickinson
Original assignee: Individual
Current assignee: NuVas LLC
Priority date: 1997-12-23
Filing date: 1998-12-22
Publication date: 1999-07-01
Also published as: WO1999032143A1; AU2094199A; EP1041999A1

Abstract

Thrombosis-initiating chimeric polypeptides and conjugates, where Figure 1 portrays one of the disclosed examples of the latter, are provided, as well as compositions comprising same and nucleic acid constructs encoding same. At least one component of a chimera or a conjugate is specific for one or more external features of the vascular endothelium of vessels nourishing a tumor and at least one thrombotic component is substantially inactive when not associated with said tumor vascular endothelium, permitting specific destruction of cancer cells of solid tumors in an animal.

Description

wo ~r~zi~ Pcrms9arrms THROMBOGENIC POLYPEPIIDE CHIMERAS AND CONJUGATES HAVING ACTIVITY DEPENDENT
UPON ASSO-CIATION WITH TUMOR VASCULAR ENDOTHELIUM
FIELD OF THE INVENTION
The present invention relates to a novel strategy for the treatrnent of carcinomas and other solid tumors. In particular, the present invention relates to methods which modulate the function of endothelial cells associated with the tumor vasculature, and compounds useful therefor.
BACKGROUND OF THE IIWENTION
Even a cursory eacamination of cancer treatments demonstrates the need for better, more efficacious treatment reagents and protocols. Although significant advances in therapy have been achieved during the past 25 years, few drugs have been discovered that have a truly major impact on the course of the disease.
Conventional chemotherapy produces severe side effects that range from loss of hair to debilitating neurotoxicity. Furthermore, there has been little inroad into the discovery of new stzstegies to develop drugs that are mechanistically different and which may present better opportunities to intervene in the disease. Because cancers differ greatly in their causes and origins, drugs are not effective across a wide variety of cancers.
No single drug has been shown to be capable of treating a wide spectrum of cancers.
Because of 2 o the complexity of the causes and origins of cancer, it has been difficult.
to devise a single drug that will act on different cancers in diffemnt organ and tissue locations, particularly with solid tumors.
Solid tumors make up more than 90% of all human cancers. Yet, the delivery of 2 s drugs, antibodies and immunoconjugates to specific tumors has proven to be inefficient because pharmacological barriers exist that prevent the drugs from reaching the tumor in sufficient concentrations that they inhibit or destroy the tumor. To get enough drug into the tumor, high concentrations must be used and these produce unacceptable toxicity to wo ~r~im pcrius9sm~s - the normal cells - side effects.
Then~pies that target tumor cells using tumor antigens are also often not effective because tumors are heterogeneous, as evidenced by the lack of specific so called "tumor antigens" on all of the cells that constitute a tumor mass.
Tumor variants may be produced continuously that may lack the target to which the drug is directed.
Moreover, tumor cells can become resistant to many conventional drugs, even to the extent of developing pumps, such as glycoprotein gp170, to remove drugs from the cell.
This and other types of heterogeneity are well-known and are of great concern to 0 oncologists.
Solid tumors require a continuous supply of nutrients supplied by the continual formation of new blood vessels that are derived from older blood vessels. When the tumor mass (from metastasis, for example) reaches about 1 to 2 mm in diameter, new blood vessels must be established to support growth. This process is called tumor angiogenesis and represents a potential site for intervention and control of tumor proliferation and growth. The growth of new vessels is likely related to the response of endothelial cells to the presence of various growth factors, proteases, metalloproteinases, chemokines, cytokines, adhesion substructures, etc. that are produced by the nearby 2 o tumor cells. Because of the influence of the tumor cells, the endothelial cells within the vessels that feed the tumor mass differ from other normal endothelial cell surfaces in normal tissues and organs. These differences can be detected by the cell surface characteristics found within the blood vessels of the tumor compared to those found in normal tissues and organs. In addition, tumors are known to be procoagulant -patients 2 5 with cancer typically show evidence of hypercoagulability and may even develop thromboembolic disease.
An approach to the therapy of solid tumors is to employ high affinity immunoconjugates that target the endothelium to coagulate the vasculature of solid 3o tumors. See e.g., Huang et al. Science (1997) 275(5299):547-550. However, there are a number of disadvantages associated with such therapies. High amity targeting 3 PCT/US98J~'1498 - elements, such as antibodies, include the need for nearly absolute specificity (i.e., the target antigen may not be present on any normal tissue or toxicity will result).
Unfortunately, however, few antigens truly are tumor-specific. Moreover, few antigens are found only on one type of tissue (i.e., the endothelial surface of blood vessels).
Thus, this approach has limited applicability. For example, immunoconjugate coagulants described previously will unlikely be found adequately selective, effective and safe for use in humans.
It is clear that novel approaches are required to improve the ability of i o oncologists to successfiilly and safely eradicate or markedly regress tumors in humans.
BRIEF DESCRIPTION OF THE INVENTION
In accordance with the pn~ent invention, a novel strategy has been devised that ~ 5 clearly sets out and creates a new method of treating cancer patients.
Invention methods and reagents represent an abrupt departure from conventional approaches of therapeutically attacking tumors, which kill individual tumors on a cell-by-cell basis.
Thus, in accordance with the present invention, a method of attacking solid tumors has been developed which employs compounds which modulate the functions of tumor-2 o associated vascular endothelial cells. This strategy accomplishes global killing of the tumor by eliminating its nutritional supply. By restricting the flow of nutrients that feeds the individual cells comprising the tumor, the growth of the entire tumor mass can not be sustained, thus resulting in regression and even eradication of the tumor by necrosis.
BRIEF DESCRIPTION OF THE FIGURES
Figure I depicts the process of chemically conjugating a context-enhancing substructure and a cysteine-containing spacer substructure-TF.

WO 99/32143 PCT/US98I~~498 Figure 2 depicts the process of employing a thioether production substrocture to produce a context-dependent functional entity.
Figure 3a presents a graph illustrating the effect on plasma half life by antibody s against Tissue Factor (TF).
Figure 3b presents a graph comparing Tissue Factor half life with Selective Tumor Vasculature Thrombogen (STVT) half life.
~ o Figure 4 presents a graph illustrating the effect of differing concentrations of NV 124 (protein expressed from the E. coli expression plasmid NuV 124, see Example 7) on tumor growth.
Figure 5 depicts a graph illustrating the improved pharmacological effect of i s multiple infusions of NV 124 on tumor growth.
Figure 6 graphs the effects of NV 129 (protein expressed from the E. coli expression plasmid NuV 129, see Example 11 ) infusions on C 1300 tumor growth.
2 o Figures 7 and 8 provide graphs illustrating the effects of NV 144 (protein expressed from the E. coli expression plasmid NuV 144, see Example 12) on C

tumor growth in comparison with the effects of saline on tumor growth.
DETAILED DESCRIPTION OF THE INVENTION
2s The present invention provides novel single molecules having thrombogenic properties and context-enhancing properties. These molecules are context-dependent functional entities (also referred herein as "Selective Tumor Vasculature Thrombogen"
or "STVT") comprising substructures with thrombogenic potential and 3 o context-enhancing substructures having the ability to recognize (e.g., possessing functional complementarity) desired biologically susceptible site{s). Context-dependent WO 99/32143 PCT/US98r17498 :functional entities are characterized as imparting thrombogenic activity when positioned (e.g., fi~nctionally complemented) in the fiinction-forming-context at the biologically susceptible site(s), while having substantially no thrombogenic activity absent a fimction-forming-context at the biologically susceptible site(s). In yet another aspect of s the present embodiment, the context-dependent fiulctional entity transiently imparts activity upon formation of a transient fiuiction-forming-context at the biologically susceptible site(s).
As used herein, "substructure with thrombogenic potential" refers to one or more i o thrombosis promoting peptidyl, oligopeptidyl, protein or small organic molecule (e.g., medicinal compounds), that has the ability to selectively impart thrombogenic activity when positioned (fimctionally complemented) in a fimction-forming-context at a biologically susceptible site(s). The phrase "thrombogenic potential" refers to the ability of such substructures to selectively im~rt thrombogenic activity when localized and ~ 5 oriented in a complementary function-forming-context at a biologically susceptible sites) so as to result in thrombogenic activity. In a preferred aspect of the present embodiment, substn~chues with thmmbogenic potential include one or more domains or modules of coagulation factors. Exemplary coagulation factors include fibrinogen, prothrombin, tissue factor ('TF), factor V, factor VII through factor XIII (in addition to 2 o their activated states), von Willebrand factor, tissue plasminogen activator (tPA), streptokinase, staphylokinase, urokinase, eminase, factor C, Mac-1, EPR-1, venom-derived coagulation enzymes (e.g., Russell's viper venom), cellular enzymes (e.g., grarlzymes), and the like. Preferred coagulation factors include those involved in the coagulation promoting pathways including TF, factor V, factor VII, factor VIII, 2 5 factor IX, factor X, factor XI, activated states of such factors, combinations of co-factors (i.e., TF:factor VI1/VIIa, factors VIIIa:IXa, factors Va:Xa), and the like.
As used herein, the phrase "thrombogenic activity" refers to the selective initiation, promotion, activation and/or propagation of occlusive thrombosis (either 3 o partial or complete, transient or prolonged) at biologically susceptible site(s). A
preferred thrombogenic activity includes the function of a coagulation factor to activate WO 99l3Zi43 PGTNS9827498 or provide co-factor function for other coagulation factors in a systematic and limited proteolytic sequence (i.e., limited proteolytic cleavage to activate coagulation factors) at a biologically susceptible site(s). Examples of thrombogenic activity include conversion of factor VII to factor VIIa, factor IX to factor IXa, factor X to factor Xa, prothrombin to s thrombin, and the like.
As used herein, the phrase "occlusive thrombosis" refers to the specific and selective formation of a mass of blood elements (i.e., thrombus) that partially or completely, transiently or for a prolonged period obstructs blood flow at biologically i o susceptible site(s). Occlusive thrombosis, as contemplated by the present invention, would result in therapeutic activation of coagulation on biologically susceptible sites}
(i.e., selected endothelial cells), by formation of an occlusive thrombus to markedly reduce or even cease blood flow both upstream and downstream of the blockage as far as the points where the thrombosed vascular channel anastomoses with another, ~5 unaffected vessel (Danekamp et al. (1984) Prog Appl Microcir 4:28-38).
Thus, selective and specific occlusive thrombosis results in hypoxic cell death and ischemic necrosis of cells nourished by the affected blood vessels.
A preferred substructure with thrombogenic potential is modified or wild-type 2 o tissue factor (TF), preferably of human derivation. As used herein, "modified or wild-type tissue factor (Tl~" is a soluble TF, that in combination with factor VII, can selectively activate or initiate occlusive thrombosis only when positioned in the proper function-forming context at biologically susceptible site(s), i.e., by conversion of factor X to Xa and factor IX to IXa Preferably, the modified or wild-type TF retains the 2 5 capacity to induce factor VII/VIIa-dependent coagulation. As used herein, "modified TF" refers to truncated or native TF wherein one or more amino acids have been substituted, modified, added and/or deleted and in which carbohydrate moieties are present, absent or modified.
s o Exemplary TFs include soluble forms of TF which consist essentially of the extracellular domain of wild-type TF (as described in Edgington et al., Patent No.

- 5,110,730 (1992)), and do not contain portions of the transmembrane anchor region (i.e., TF 220-242, or amino acid residues 252 through 274 of SEQ ID NO:1 ) which anchors native TF to the cell membrane. Preferably, the TF comprises substantially the amino-terminal amino acids up to approximately residue 252 of SEQ ID NO:1.
More s preferably, the modified TF has substantially the same amino acid sequence as TF 3-211 (as set forth in residue nos. 35-243 of SEQ ID NO:1). In the presently most prefen~ed embodiment of the present invention, the modified TF is modified or further modified to increase thrombogenic activity when placed or oriented in the function-forming-context at a biologically susceptible site(s). Such modifications include substituting the amino to acid residue at one or more positions, e.g., TF 167 or position 199 of SEQ
ID NO:1, as well as residues within 15 Angstrom of TF 167 or residue 199 of SEQ ID NO:1, with a basic amino acid such as lysine, arginine, histidine, and the like.
As used herein, the term "purified" means that the molecule is substantially fi~ee i5 of contaminants normally associated with a native or natlual environment.
TF protein, or functional fi~nents thereof, useful in the practice of the present invention, can be obtained by a number of methods, e.g., precipitation, gel filtration, ion-exchange, reversed-phase, DNA affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification:
Methods in 2 o Enzymology Vol. 182, (Academic Press, 1990), which is incorporated herein by reference.
TF, and biologically active fiagments thereof, useful in the practice of the present invention can also be produced by chemical synthesis. Synthetic polypeptides 2s can be produced, for example, using Applied Biosystems, Inc. Model 430A or automatic polypeptide synthesizer and chemist<y provided by the manufacturer.
TF, and biologically active fi~agments thereof, can also be isolated directly from cells which have been transformed with the expression vectors described below in more detail.
Alternatively, a purified TF, or functional fi~agment thereof, usefi~l in the practice of the present invention, can also be obtained by well-known recombinant methods as WO 99/32143 PGTNS98/Z'1498 - described, for example, in Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley & Sons, Inc. (1993)), also incorporated herein by reference. An example of r~;ombinant means to prepare modified or wild-type TF is to express nucleic acid encoding TF, or functional fragment thereof, in a suitable host cell, such as a bacterial, yeast (e.g., Saccharomyces or Pichia), insect (e.g., Baculovirus or Drosophila) or mammalian cell, using methods well known in the art, and recovering the expressed protein, again using methods well known in the art. Thus, one embodiment of the present invention includes nucleic acid consisting essentially of the nucleic acid sequence as set forth in SEQ ID NO:1 from about position 130 at the 5' terminus to about position 918 at its 3' terminus. Preferably, the last about one hundred fifty bases are not employed. Thus, nucleic acid encoding the soluble form of TF is contemplated, in a prefen-ed aspect, i.e., a nucleic acid segment consisting essentially of the nucleotide sequence from about position 178 to about 804 as set forth in SEQ ID NO:1.
As defined herein, the phrase "context-enha~ing substrucriu~e" refers to one or more peptidyl, oligopeptidyl, protein, or small organic molecule (e.g., medicinal compounds) that enhances thrombogenic activity, and further enhances selectivity by positioning the context-dependent functional entity in a desired context at the 2 o biologically susceptible site(s). As used herein, the phrase "having the ability to recognize desired biologically susceptible site(s)" refers to the ability or capacity of the context-enhancing substructure to exhibit elements of specificity and selectivity for the biologically susceptible site(s). Preferably, the context enhancing substructure will have a low affinity for the desired biologically susceptible sites) so as to transiently activate 2 5 the thrombogenic potential of the context-dependent functional entity. The context-enhancing substructure does not contain any substantial portion of any transmembrane region, antibody, or the like, which will anchor or permanently and/or irreversibly associate the context-dependent functional entity to the biologically susceptible site(s).

WO 99/32143 PGT/US98n7498 - The function of the context-enhancing substructure is to provide transient orientation and transient localization of the thrombogenic substructiue to preferred biological susceptible sites. Exemplary context-enhancing substructures include cell surface recognition domains (i.e., from annexin), charged phospholipid-associating elements, protease inhibitors, peptidyl sequences that facilitate orientation and/or small molecules that recognize molecules or molecular assemblies enriched in tumor vasculature endothelium (e.g., all, part or modified growth factor, ligand, hormone, or lectin which have transient functional association properties so as to only transiently activate the thmmbogenic potential of the context-dependent functional entity), and the i o like. Specific examples of context-enhancing substructures include uPAR
binding antagonists (e.g., peptide%lone 20 (1994) Goodson et al. PNAS 91:7129-7133), anti-angiogenic proteins ((e.g., endostatin from collagen XVIII 20kD C-teZminus, (1997) O'Reilly et al. Cell 88(2):277-285; nucleotide sequences 1502 to 2053 from genbank I3IJMCOL18AX ACCESSION L22548), peptides derived from TSP-1 (e.g., 5 MaIIII from (1993) J Cell Biol 22:497-51 I; peptide 246, (1992) PNAS 89:3040-3044;
Laminin binding peptides (e.g., Peptide G, Guo et al. (1992) J Biol Chem 267:17743-17747; Proliferin (PLF, (1988) J Biol Chem 263(7):3521-3527, Proliferin-related-peptide (PRP, (1988) Mol Endocrinol 2(6):579-586 , membrane-binding peptide from factor VIII, (1995) Biochemistry 34(9):3022-3031, 2 o single or multiple kringle domains (e.g., kringle 1 from plasrninogen ( 1991 ) Biochemistry 30(7): 1948-1957; angiostatin, 1994 Cell 79(2):315-328, respectively, or fragments thereof (e.g., Pm Arg Lys Leu Tyr Asp), phosphatidyl-serine binding proteins (eg. annexin V J.Biol. Chem. 1995 270:21594-21599), and the like. Other context-enhancing substructures can be readily identified employing such well known 2 s methods such as phage display searches of peptide and constrained peptide combinatorial libraries (See, e.g., Ruoslahti et al., U.S. Patent No.
5,622,699 (1997) and Ruoslahti et al., U.S. Patent No. 5,206,347 (1990), Scott and Smith (1990) Science 249:386-390, Markland et al (1991) Gene 109:13-19), and the like.
s o As defined herein, the phrase "biologically susceptible site(s)" refers to one or more molecules found at the cell surface, unique to, induced or up-regulated in, vascular WO 99/32143 PCT/US98l17498 - endothelial cells in human tumors. Progressive tumor growth necessitates the development of new blood vessels (angiogenesis) to meet the nutritional needs of the expanding tumor mass. Numerous anatomical, morphological and behavioral differences between tumor-associated blood vessels and normal ones have been s documented (See, e.g., Dvorak et al. (1991) Cancer Cells 3:77-85, Jain (1988) Cancer Res 48:2641-2658, and Denekamp (1990) Cancer Metast Rev 9:267-282).
Preferably, the invention context-dependent functional entity recognizes these differences as sites in which to selectively initiate, promote, activate and/or propagate occlusive thrombosis.
1 o Examples of biologically susceptible sites include vascular structures, specific cells or tissue types associated with cancer and/or angiogenesis, wounds caused by tratuna, and other vascular pathologies, such as sites of infection by fungal, bacterial or viral agents. Additionally, examples of biologically susceptible sites include such structures, tissues and/or cells which are recognized by the context-enhancing 1 s substructure. In the case of cancer, the biologically susceptible sites) recognized by the context-dependent functional entity should preferably be molecules) on the cell surface of the tumor-associated endothelial cells and one that is not secreted into body fluids.
Preferred biologically susceptible sites) for which the context-enhancing substructure has a functional preference include tumor-associated vascular endothelial cells. Those of skill in the art can readily determine other biologically susceptible sites) which are contemplated for formation of a function-forming-context by the context-dependent functional entity employed.
As used herein, the phrase "function-forming-context" refers to the necessary 2 5 orientation and position of the context-dependent functional entity to impart thrombogenic activity at the biologically susceptible site(s). The function-forming context will depend on numerous factors including the orientation and position of the context-enhancing substructure relative to the substructure with thrombogenic potential.
Thus, the context-enhancing substructure can be positioned at the carboxy terminus of s o the substruchue with thrombogenic potential, the amino terminus of the substructure with thrombogenic potential, or between the amino terminus and the carboxy terminus wo ~r~im Pcriusssn~4ss m - of the substructure with thmmbogenic potential (i.e., inserted within a hydrophilic surface loop of the substructure with thrombogenic potential). In a preferred embodiment of the present invention, the context-dependent functional entity comprises two or more context-enhancing substructures to enhance proper or desired alignment of the substructure with thrombogenic potential at the biologically susceptible site(s).
Thus, the context-enhancing substructures) is(are) located at the carboxy terminus of the substructure with thrombogenic potential, the amino terminus of the subsr<uctwe with thrombogenic potential, between the amino terminus and the carboxy terminus of the substructure with thrombogenic potential, inserted in a hydrophilic surface loop of 1 o the substructure with thrombogenic potential, or any combinations thereof.
In yet another embodiment of the present invention, context-dependent functional entities fiuther comprise activity-modulating substructure(s). As used herein, the term "activity-modulating substructure" refers to one or more molecules which i 5 enhance the function-forming context by properly orienting and/or positioning the context-enhancing substructure relative to the substructure with thrombogenic potential (e.g., configuration of the active sites of each substructure, the relative distance between the substructures, and the like). Examples of activity modulating substructures include spacer substmch>ms, protease sites, and the like.
As used herein, the term "spacer substructure" refers to one or more spacer molecules that serve to link substruct<mes with thrombogenic potential with context-enhancing substructures. The nature of the linkage between the spacer substructure and either the subst<uctule(s) with thrombogenic potential or the 2 s context-enhancing subst<ucture(s) depends on the functionality employed in the substructures with thrombogenic potential and the context-enhancing substivctures.
Preferably, substructures with thrombogenic potential are linked to context-enhancing substz~uctures in a manner that retains the ability of the context-dependent functional entity to activate the substructure with thrombogenic potential. Typically, spacer 3 o substructures are non-immunogenic and flexible and fall in the range of about 0 to about 60 residues long, preferably about 10 amino acid residues.

WO 99/32143 PCT/U898IZ~498 - In a prefenred embodiment of the present invention, the spacer substnu~hue increases degradation of the context-dependent functional entity by releasing the substructure with thrombogenic potential from the context-enhancing substructure.
Preferably, such spacer substructures include those spacer molecules which are subject to scission when the context-dependent functional entity is extracellularly positioned in the function-forming-comext with cell surfaces at the biologically susceptible site, subjecting the spacer to degradation upon exposure to a specific enzyme.
Examples of bonds within spacer molecules include esters, peptides, amides, phosphodiesters, and even glycosidic bonds, which are hydrolysed by exposure to an esterase, protease or 1 o peptidase, amidase, phosphodiesterase and glycosidase, respectively.
Preferred context-dependent functional entities will have cysteine residues native to the spacer molecule replaced by glycine, alanine or serine. Preferred context-dependent functional entities can be engineered so that they are hydrolysed only by exposure to an enzyme known to have a precise cellular location, including enzymes associated with the vascular endothelial surface. Cleavage of the spacer substructure provides a safety factor by limiting the lifetime of the intact context-dependent functional entity at the biologically susceptible sites) andlor irreversibly inactivating the context-dependent functional entity, thus preventing dissemination of native thrombogens or access of additional entities to biologically susceptible site(s).
In a preferred embodiment of the present invention, the spacer substructure comprises homo- or hetero-bifunctional crosslinking agents or chitin oligomers.
Exemplary spacer substtuctu~~es include combinations of Gly and Ser modules, such as ((Gly)4Ser)", ((Ser)4G1Y~" and the like. Additional spacer substructm~es contemplated 2 5 are the hinge region of the heavy chain of immunoglobulin (Ig) proteins, preferably the H chain IgD sequences. Ig hinge regions have flexibility that allows the different domains in immunoglobulins to assume different geometries and orientations.
Immunoglobulin hinge regions (see, e.g., Kabat et al. Seguences of Proteins of Immunological Interest, 5 Ed., U.S. Dept. of Human Health Services) may be used as 3 o spacers in thrombogens. Examples of hinge regions that may be back converted to their respective DNA sequences obtained from conventional databases and repositories of - protein and nucleic acid sequences include Human IgD'd, Human IgG 3'Cl, Human Iggl aeCl, Human IgG eeCl, Human IGG2 a;Cl, Human Igg4 aeCl, and the like (See ICabat et al.; page 670). The cysteine residues of such hinge regions may be substituted by glycine, alanine, or serine in any combination to eliminate di~culties presented by the presence of particular cysteine thiol groups that may disrupt spacer structure or inhibit proper formation of tertiary structure of the subst<uctiwe with thrombogenic potential.
Alternatively, the present invention provides for the creation of protease sensitive sites to cleave and/or reduce activity (or inactivate) of the context-dependent functional entity after the context-dependent functional entity has performed its desired activity. Context-dependent functional entities comprising protease sensitive sites can be synthesized or manufactured employing methods well known to those of skill in the art (e.g., recombinant or chemical manufacture of integrating protease sensitive sites).
15 In yet another embodiment of the present invention, context-dependent functional entities further comprise production substlvcture(s). As used herein, the term "production substructure" refers to one or more substructural aspects which facilitate the production and/or assembly of the context-dependent functional entity.
Examples of production substructtues include restriction sites, vectors, cys residues, His-tags, and the 2 0 like.
Restriction enzyme sites can be optionally introduced to the contextdependent functional entity, as well as the individual subst<uctures that comprise the context-dependent functional entity (i.e., substtuchne(s) with thrombogenic potential, 25 context enhancing substiuctiwe(s), activity-modulating substructures) and/or cloning cassette) by additions, deletions, substitutions or modifications made at nucleic acid sequences encoding the amino-ternlinal, the carboxyl terminal and/or sequences in between to produce the function-foaming context. Unique restriction sites may be placed for the convenience of constricting a context-dependent functional entity(ies) 3o with different orientations and configurations, i.e., different combinations of the individual substructures. Unique restriction sites at the junctions of each of these WO 99/32143 PGT/US98n7498 - individual substructures can be used to clone various subtypes of the individual substructures (for example, spacer substructures of different lengths and compositions).
Examples of such modifications include placement of a specific amino acid residue at position 212 of TF or position 245 as set forth in SEQ ID NO:1, preferably a threonine, to yield a restriction site favorable to splicing with a spacer substructure and/or a context-enhancing substructure. The sequences that are presented are the subtypes of the functional substruchu~es without restriction sites. Examples of restriction sites that are prefen~ed include Xmas (CCCGGG), BamHI (GGATCC), KpnI (GGTACC), HindIII (AAGCTT), Aval (CCCGGG), EcoRI (GAATTC), AvrII (CCTAGG), or PmII
l o (CACGTG), and the like. Specific prefenred examples of context-dependent functional entity(ies) constructs are provided (e.g., SEQ ID NOs: 6, 12,15, 24 and 31).
Alternatively, the individual substructures can be conjugated chemically via production substructures to produce the context-dependent functional entity with the preferred fimction-forming-context. Preferred chemical conjugations of the individual substructures use cysteine as a production substructure to link the substructures. The cysteine thiol group provides a convenient site at which chemical links may be established, links that may be reducible (disulfide bonds) or stable (thioether). Context enhancing substructiue(s) and/or activity-modulating substructures) that contain a 2 o cysteine residue production substructure can be coupled to substxucture(s) with thrombogenic potential through the cysteine. A peptide that may act as a context enhancing substruc>ure(s) and/or activity-modulating subshvcture(s) may be modified to contain a cysteine production substructure at its amino terminus, its carboxy terminus, or at a site between the amino and carboxy terminus. Examples of each modification in 2 s a disulfide bond constrained peptide ( 1 ) are illustrated schematically in Figure 1. The preferred modification does not reduce the ability of the context enhancing substructures) and/or activity modulating subs>ructure(s) to facilitate the thrombogenic activity of the final construct.
3 o An activity-modulating substructure containing a cysteine production substructure at or near the amino terminus, or at any site within an activity-modulating substructiue, can be constructed synthetically and fused to the substructw~e with thrombogenic potential. For example, an activity-modulating substructure of the following sequence may be const<ucted between a KpnI and XmaI site:
5 GSCGGGGSGGGGSGGGGSP (SEQ ID N0:2) The cysteine in this example is place at residue number 3 when numbering from the amino terminus. It is possible, however, to place the cysteine at residue number 1 or 2 if desired. However, a thrombin-sensitive sequence such as PRG must be retained in order i o to efficiently cleave the resulting protein with thrombin. Other peptides inserted between the hexahistidine sequence and the beginning of the substructure with thrombogenic potential may be used in the construct, which would necessitate the use of a different erlzyme(s), known to those of skill in the art, to cleave and release the substructure with thrombogenic potential during its purification. The cysteine thiol of i 5 the substructure with thrombogenic substructure and the cysteine thiol of cysteine-containing activity-modulating substructure may also be linked by thioether bonds using bismaleimido groups such as that of BMH, bismaleimidohexane.
Other methods of cross linking proteins using N-hydroxysuccinimide (NHS)-ester haloacetyl cross linkers, photoreactive cross linkers, and the like that may be useful in establishing the desired bonds between facilitators and the substNCture with thrombogenic potential are known to those of skill in the art (see Figure 2).
Such chemistry is described in the Pierce Chemical Co. catalogue and in Chemistry of Protein Conjugation and Cross-linking by S.S. Wong, CRC Press, 1991, which are incorporated 2 5 herein by reference.
Context-dependent functional entity, as well as the individual substructures that comprise the context-dependent functional entity (i.e., substructure(s) with thrombogenic potential, context enhancing substructure(s), activity modulating s o substructures) and/or production substructure) useful in the practice of the present invention, can be obtained by a number of methods, e.g., solid-phase, precipitation, gel filtration, ion-exchange, reversed-phase, DNA at~nity chromatography, and the like.
Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzyarology Vol. 182, (Academic Press, 1990), which is incorporated herein by reference. Alternatively, context-dependent functional s entity(ies), as well as the individual substructures thereof, can also be obtained by well-known recombinant methods as described, for example, in Ausubel et al., Current Protocols in Molecular Biology (Greene Publishing Associates, Inc. and John Wiley &
Sons, Inc. 1993), also incorporated herein by reference. An example of recombinant means to prepare context-dependent functional entity, or the individual substnrcnrres, is 1 o to express nucleic acid encoding such entity and/or substructures) thereof, in a suitable host cell, such as a bacterial, yeast {e.g., Pichia), insect (e.g., Baculovirus) or mammalian cell, using methods well known in the art, and recovering the expressed protein, again using methods well known in the art.
15 Context-dependent functional entity, as well as the individual substructures thereof, useful in the practice of the present invention can also be produced by chemical synthesis (see, e.g., Meyers, Molecular Biology and Biotechnology: A
Comprehensive Desk Reference, VCH Publishers (1995)). Synthetic polypeptides can be produced, for example, using Applied Biosystems, Inc. Model 430A or 431A automatic polypeptide z o synthesizer and chemistry provided by the manufacturer. In addition, synthetic polypeptides can be produced by solid phase peptide synthesis employing a range of solid supports. Examples of solid supports available include those based on polyamides, polyethelene glycol (PEG) resins, and the like. Context-dependent fimctional entity, as well as the individual substruct<wes thereof can also be isolated directly finm cells 2 s which have been transformed with the expression vectors described below in more detail.
Alternatively, noncovalent links can be established to link the individual substructures together to form an assembly-dependent functional entity. For example, 3 0 leucine zippers are preferably used to hold together, by noncovalent interaction, the substnrctuze with thmmbogenic potential with the context-enhancing substructure to fore an entity with thrombogenic potential.
In a preferred embodiment of the present invention, the context-dependent functional entity, as well as the individual substructures thereof, is modified to reduce immunogenicity and/or modify biological half life. Such modifications include employing polyethelene glycol (PEG), and the like.
In yet another embodiment of the present invention, the context-dependent functional entity further comprises a cloning cassette. As used herein, the phrase "cloning cassette" refers to one or more additional substructural elements which facilitate orientation of the context-dependent fimctional entity on the biologically susceptible sites) or to add synergistic fimctions. Preferably, cloning cassettes) contemplated for inclusion into the context-dependent functional entity will be inserted into a permissive hydrophilic loop of the context-dependent finictional entity which does not adversely affect thrombogenic activity. The specific region replaced or inserted within will depend on the size and function of the cloning cassette desired, the sequence which imparts thrombogenic potential employed, and the like. Examples of amino acid residues which can be replaced include amino acid residues finm about 112 to about 123, preferably from about 115-123, of human TF as set forth in SEQ
ID NO:1.
2o Direct loop replacements can be made from about 1-30 amino acids, preferably 12-20 amino acids, so long as the entity retains thrombogenic activity. As used herein, the phrase "synergistic fiuiction" refers to functions which enhance the thrombogenic function of the substructure with thrombogenic potential or enhance the function of the context-enhancing subst<vcriue to orient the context-dependent functional entity in a 2 5 function-fon~.ming-context at the biologically susceptible site(s). Those of skill in the art can readily determine exemplary cloning cassettes which can be employed in the invention entity, including cloning cassettes identified from combinatorial libraries.
In another prefen~ed embodiment of the present invention, there are provided 3o compositions comprising context-dependent functional entities in combination with coagulation factor VIIa. Binding of factor VIIa to TF enhances the enzymatic activation of substrate factors IX and X as much as 5,000 fold (Rao et a1 (1988) PNAS
85:6687).
Factor VII can be prepared as described by Fair (1983) Blood 62:784-791.
Recombinant Factor VIIa can be purchased from Novo Biolabs (Danbury, Conn.).
s In yet another aspect of the present invention, nucleic acid constructs encoding the invention context-dependent functional entity are provided. In accordance with the methods of the present invention, an effective amount of the context-dependent functional entity can be administered in vivo as a therapeutic agent to selectively result in partial or complete occlusive thrombosis of the vasculature of solid tumors in a to subject in need thereof. The context-dependent functional entity can be administered to the subject by any means which readily permits the context-dependent functional entity to function at biologically susceptible sites) including intraperitoneal, subcutaneous, intravascular, intramuscular, intranasal or intravenous injection or infiision, implant modes of administration, and the like. In another embodiment of the present invention, 1 s the context-dependent functional entity is supplied indirectly by administering a nucleic acid segment encoding same to the subject.
As used herein, the phrase "the vasculature of solid tumors" refers to endothelial lined vascular channels of the tumor and existing vasculature adopted by invading tumor 2 o cells. Examples of tumors, also referred to herein as vascular tumors (tumors of the vasculature as well as vascular malformations), which are contemplated to be h include solid tumors such as breast, prostrate, lung, liver, colon, rectal, melanoma, kidney, stomach, pancreas, ovarian, bladder, cervical, oral, uteri, brain, and the like.
2 5 In another embodiment of the present invention, there are provided methods for obliterating vasculature malformations by administering to a subject an effective amount of the context-dependent functional entity, alone or in conjunction with aids such as coagulation factors, drugs, local hypertherniia, and the like. As used herein, the phrase "obliterating vasculature malformations" refers to the partial or complete occlusive s o thrombosis of malformations derived from or influenced by blood vessels and/or structures. Examples of vasculature malformations include hemangiomas, preferably WO 99/32143 PCTNS981s7498 w inoperable hemangiomas, aneurisms, granulomas, and the like.
Those skilled in the art will appreciate that when the compositions of the present invention are administered as therapeutic agents, it may be necessary to combine the content-dependent functional entities with a suitable pharmaceutical carrier.
The choice of pharmaceutical carrier and the preparation of the context-dependent functional entity as a therapeutic agent will depend on the intended use and mode of administration.
Suitable formulations and methods of administration of therapeutic agents can be readily be determined by those of skill in the art, including rendering the content-dependent 1 o functional entity amenable to oral delivery, intravenous delivery, intramuscular delivery, topical delivery, nasal delivery, and the like.
Depending on the mode of delivery employed, the context-dependent functional entity can be delivered in a variety of pharmaceutically acceptable forms. For example, 1 s the context-dependent functional entity can be delivered in the form of a solid, solution, emulsion, dispersion, micelle, liposome, and the like.
Pharmaceutical compositions of the present invention can be used in the form of a solid, a solution, an emulsion, a dispersion, a micelle, a liposome, and the like, 2 o wherein the resulting composition contains one or more of the compounds of the present invention, as an active ingredient, in admixture with an organic or inorganic carrier or excipient suitable for enteral or parenteral applications. The active ingredient may be compounded, for example, with the usual non-toxic, pharmaceutically acceptable carriers for tablets, pellets, capsules, suppositories, solutions, emulsions, suspensions, 2 5 and any other form suitable for use. The carriers which can be used include glucose, lactose, mannose, gum acacia, gelatin, mannitol, starch paste, magnesium trisilicate, talc, com starch, keratin, colloidal silica, potato starch, urea, medium chain length triglycerides, dextrans, and other carriers suitable for use in manufacturing preparations, in solid, semisolid, or liquid form. In addition auxiliary, stabilizing, thickening and s o coloring agents and perfumes may be used. Examples of a stabilizing dry agent includes triulose, preferably at concentrations of 0.1% or greater (See, e.g., U.S.
Patent No.

- 5,314,695). The active compound (i.e., context-dependent functional entity) is included in the pharmaceutical composition in an amount sufficient to produce the desired effect upon the processes or condition of diseases.
The pharmaceutical compositions may be in the form of a sterile injectable suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
i o Sterile, f xed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides, fatty acids (including oleic acid), naturally occurring vegetable oils like sesame oil, coconut oil, peanut oil, cottonseed oil, etc., or synthetic fatty vehicles like ethyl oleate, or the like. Buffers, preservatives, antioxidants, and the like can be incorporated as required.
One particularly preferred method for delivering the context-dependent functional entity is intravascularly to the selected vascular site via a small diameter medical catheter. When delivered by catheter, the injection rate will depend on a 2 o number of variables, including the concentration of the active ingredient, the specific substructures employed, the rate of precipitation (formation of the thrombus), total volume of the vasculature to be thrombosed, location of the biologically susceptible site(s), toxicity, side effects, and the like. When introduced into the vascular site, the context-dependent functional entity diffuses rapidly into the blood and initiates 2 s occlusive thrombosis specifically at the biologically susceptible site(s).
The dosage regimen necessary depends on a variety of factors, including type of disorder, age, weight, sex and medical condition of the patient, as well as the severity of the pathology, the route of administration, and the type of then~peutic agent used. A
3 o skilled physician or veterinarian can readily determine and prescribe the effective amount of the context-dependent functional entity or pharmaceutical required to treat - the patient. Conventionally, those of skill in the art would employ relatively low doses initially and subsequently increase the dose until a maximum response is obtained.
Typical daily doses, in general, lie within the range of from about 1 pg up to about 100 mg per kg body weight, and, preferably within the range of from 50 pg to 10 mg per kg body weight and can be administered up to four times daily. The daily N
dose lies within the range of from about 1 ~,g to about 100 mg per kg body weight, and, preferably, within the range of from 10 ~g to 10 mg per kg body weight.
to In yet another embodiment of the present invention, there are provided assembly-dependent functional complexes comprising substructures with thrombogenic potential and one or more association-enhancing substructures having the ability to transiently associate the complexes to increase local concentration at desired biologically susceptible site(s), wherein the complexes impart thrombogenic activity when positioned in the function-forming-context at the biologically susceptible site(s), and wherein such complexes have substantially no thrombogenic activity absent a fimction-forming-context at the biologically susceptible site(s). In a preferred embodiment of the present invention the complex transiently becomes functional upon 2 o association of a functional context with the biologically susceptible site(s).
Exemplary substructures with thrombogenic potential comprise a thrombogen, preferably modified or wild-type TF. Exemplary association-enhancing substructures assemble the complex in a function-forming context.
The invention will now be described in greater detail by reference to the following non-limiting examples.

- Example 1 PCR of TF cDNA
PCR is performed to amplify a DNA fiag<nent of 639 use pairs from a Marathon-Ready cDNA of human placenta origin (Clontech:(97/98 cat.#7411-1). A
100 ~L reaction contains: 2 ~L cDNA mixture, 100 pmoles each of oligos BM21 and BM33 (SEQ ID N0:3 and SEQ ID N0:4 (Oligos Etc.)), buffer, BSA, MgS04 according to manufacturer, 1 pL IOmM dNTPs and 2 units of Vent DNA polymerase (New England Biolabs 96/97 cat.#254S) which is added during the first cycle after the 1 o temperature reaches 94°C.
The thermocycling is accomplished with 35 cycles of denaturation for 1 min at 94°C, primer annealing for 1 min at 60°C, and primer extension for 1 min at 75°C. The PCR product is about 640 base pairs in length. This DNA fiagment is purified by electrophoretic separation on a 1.0% agarose gel buffered with Tris-Borate-EDTA
according to Maniatis, excision of the appropriate band, and extraction of the DNA
using the QIAEX II gel extraction kit (QIAGEN cat#20051 ) according to manufacturer's instructions for DNA Extraction from Agarose Gels (QIAEX II
Handbook 08/96). The 640 base pair DNA fiaginent concentration is estimated by 2 o agarose gel electrophoresis according to Maniatis.
The 640 base pair fragment is used as template for a second PCR amplification, this time with the oligonucleotides BM51 (SEQ ID NO:S, Oligos Etc.) and BM33.
A
100 ~,L PCR reaction contains: 10 ng 640 base pair DNA fiagment, 100 pmoles each of 2 5 oligos BM51 and BM33, buffer, BSA, MgS04 according to manufacturer, 100 N,M
dN'fPs and 2 units of Vent DNA polymerase (which is added during the first cycle after the temperature reaches 94°C). The thermocycling is accomplished with 25 cycles of denah>xation for 1 min at 94°C, primer annealing for 1 min at 60°C, and primer extension for 1 min at 75°C. The PCR product is about 670 base pairs in length. This 3 o DNA fragment is purified by passage over an Elutip-D column according to the manufacturer's instructions {Schleicher & Schuell cat# 27370). Briefly, the DNA is WO 99/32143 PGT/US98/Z949$

diluted to 1 mL volume with Low-Salt buffer (0.2M NaCI, 20mM Tris-HC1, 1 mM
EDTA pH7.4) and passed over the elutip-D column. The column is subsequently washed with 3 mL of Low-Salt buffer and eluted with 0.4 mL High-Salt buffer (1M
NaCI, 20mM Tris-HCI, I mM EDTA pH7.4). The DNA is desalted and concentrated by ethanol precipitation according to Maniatis. The result is a 640 by cDNA
fragment encoding human TF residues 3 to 211 (i.e., amino acid residues 35-243 of SEQ
ID
NO:1.
Example 2 1 o Disulfide Conjugation Different substtuchues may be joined by employing the thiol group of a cysteine production substructure. In this example, the conjugation is by the cysteine thiol groups of substructures) with thmmbogenic potential, context enhancing substructures) and/or 15 activity-modulating substructiue(s), thereby forming a disulfide bond that links the cysteine-containing spacer - TF moiety to the context enhancing substtucture(s). In absence of a cysteine, a thiol group may be artificially introduced into an amino group of context enhancing substtvcture(s), for example, using 2-iminothiolane (Treat's Reagent). To achieve the highest degree of specificity of labeling, peptides with a single 2o free amino group are preferred. The cysteine thiol of the context enhancing subslructure(s) (or the cysteine thiol of the cysteine-containing activity-modulating substructure) is formed into a mixed disulfide bond through the use of a 10-to I00-fold molar excess of 5,5'-bis(2-nitrobenzoic acid), DTNB. The amount of reaction is monitored by ultraviolet absorption of DTNB and measiued using 14,150 for the molar 25 absorption coefficient at 412 nm. After 1 hour incubation at room temperature in 0.1 M
sodium phosphate, pH 7.5, 0.1 M NaCI, 1 mM EDTA, excess DTNB is removed by gel filtration.
The mixed disulfide context enhancing substructure is mixed with the 3 o cysteine-containing activity-modulating substzucture - TF moiety or the cysteine-containing context enhancing substructures) (at different molar ratios to improve yield) and left at 4 °C to react at neutral to slightly basic pH for a period of time WO 99/31143 PCT/US98J2~498 in order to form the desired product (i.e., context-enhancing substructure-SS-spacer substructure - TF). The product is purified by a combination of gel filtration and ion exchange chromatography. The purity of the compound is judged by SDS-PAGE, HPLC using reverse phase chromatography, or by electrospray mass spectometry.
Mixed disulfides suitable for reaction with the thiol group of cysteine-containing spacers are artificially introduced into the context enhancing substructures) by modification of its amino groups) with SPDP, N-succinimidyl-3-(2-pyridylthio}-propionate, or by any of the other commonly used reagents used for this purpose, i o including without limitation SMPT, 4-succinimidly-oxycarbonyl-a,-(2-pyridyltdithio) toluene; Sulfo-LC-SMPT, sulfosuccinimdyl-6[4-succinimidly-oxycarbonyl-av-methyl-a(2-pyridyltdithio~toluamide] hexanoate; LC-SPDP, succinimidyl 6- [3- (2-pyridyldi-thio-~roprionamide) hexanoate; sulfo-LC-SPDP, sulfosuccinimdyl-6- [3(2-pyridyldi-thio)-propionamido] hexanoate; PDPH , 3-(2-pyridyldithio~propionyl hydrazide, and ~ 5 the like. These and other reagents are available from Pierce Chemical Co.
The mixed disulfide formed with any of these reagents is isolated by gel filtration chromatography and reacted with the cysteine-containing spacer at different molar concentrations under the conditions described above and analyzed for purity.
2 o A water-soluble, monitorable peptide and protein crosslinking agent, N-maleimido-6-aminocaproyl ester of 1-hydroxy-2-nitm-4-benzenesulfonic acid, (mal-sac-H1VSA), is reacted with an amino group of a peptide. In order to achieve the best specificity of labeling, the prefen:ed peptide does not contain lysine at any position other than the amino terminus (Aldwin and Nitecki (198 Anal Biochem 164:494-501).
2 5 Reaction with amino groups releases the dianion phenolate, HNSA, that can be quantitated using a spectrophotometer. The peptide is linear or its conformation may be restrained (la) by the presence of 1 or more disulfide bonds. The N-maleimido-6-aminocaproyl amide derivative is reacted with the thiol of the cysteine-containing activity-modulating substructure - TF to form a thioether bond that s o conjugates the context enhancing substructures) to TF through a spacer of variable length. Other heterobifunctional cross linkers may be used to introduce a maleimido WO 99/32143 PGTNS98I2'1498 - group into the facilitator, these include without limitation such reagents as SMCC, succinimidly 4-(N-maleimido-methyl) cyclohexane-1-carboxylate; sulfo-SMCC, sulfo-succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate; MBS, m-maleimidobenzoyl-N-hydroxysuccinimide ester; sulfo-MBS, m-maleimidobenzoyl-s N-hydroxysulfosuccinimide ester; SMPB, succinimidyl 4-(p-maieimido-phenyl) -butyrate; sulfo-SMPB, succinimidyl-4- (p-maleimidophenyl)- butyrate; GMBS, N-(-maleimidobutyryloxy) succinimide ester, sulfo-GMBS, N-(-maleimidobutyryloxy) sulfosuccinimide ester, and the like.
i o Example 3 Cloning of TF 3-211 for E.coli expression:NuV 120 670 by TF cDNA fi~nent firm PCR is prepared (described above in Example 1, in PCR of TF cDNA). The purified 670 terse pair DNA fi~nent is digested with 5 1 s units restriction endonucleases BamHI and Hi~III (New England Biolabs cat#1365 and #1045 respectively) per ~g DNA in 1X BamHI buffer (New England Biolabs) for 4 h at 37°C. Subsequently, the 659 base pair fiagtnent is purified from an agarose gel eleclmphoresis band using the QIAEX II kit as previously described for the 640 base pair fiagment. The -r659 by band is cloned into the BamHI and HindZB sites of the 2 o vector pTrcHisC (Invitrogen) by standard methods. Briefly, the pTrcHisC
DNA is digested with BamHI and HindTII and then the ~5 kb band is purified on an agarose gel.
Ligation is perform~i with about 10 ng/~L of the purified vector fiugcnent mixed with a three fold molar excess of the purified 654 by fiagment and T4 DNA ligase reaction conditions according to the manufacturer (New England Biolabs). The resulting clone 2 5 (NuV 120: SEQ ID N0:6) places the TF coding sequence in a translational reading fi~ame with the translabonal start of the vector. The clone NuV 120 is a plasmid for expression of a recombinant protein containing a (His)6 tag, a thrombin cleavage site, and TF residues 3 to 211 plus threonine at residue 212.

WO 99/32143 PCTNS9&27498 NuV 120 residues (SEQ
ID
N0:6) start colon 413-415 (His)6 tag production substructure 425-442 BamHI production substructure 513-S 18 thrombin cleavageproduction substructure 521-532 site AvaI/XmaI production substructure 533-538 TF 3-211 substructure with thrombogenic539-1165 potential PmII production substructure 1165-1170 stop colon 1169-1171 HindIII production substtvcdue 1172-1177 Example 4 s Cloning of a synthetic spacer sequence in TF: NuV 121 Mix oligonucleotides NuV005 through NuV009 (SEQ ID NOs:7-11, respectively (Oligos Etc.)) at 10 pmol/ph in 20 mM Tris-HCI, 10 mM MgCl2, 50 mM
NaCI, pH 7.5. Heat mixri~re to 80°C for 2 min then cool to 25°C
over a period of 30 i o min to allow annealing of oligonucleotides. Clone the resulting synthetic spacer into the Bam HI and Aval sites of NuV 120. The vector DNA is prepared by digesting Clone NuV 120 with BamHI and AvaI and isolation of the ~Skb fragment from an agarose gel and purification by QIAEX II (QIAGEN). The 10 pL Ggation reaction contains 100 ng of NuV 120-BamHI/AvaI fragment, 0.5 ~L of the annealed oligonucleotide mixtime, and ~ s T4 DNA ligase with buffer according to the manufacturer's recommendations (New England Biolabs). This results in NuV 121 (SEQ ID N0:12), a plasmid for expression of a recombinant protein containing a (His)6 tag, a thrombin cleavage site, a 17 amino acid spacer, and TF residues 3 to 211.

NuV 121 residues (SEQ
ID
N0:12) start colon 413-415 (His)6 tag production subsixucrure 425-442 BamHI production substivcture 513-518 thrombin cleavageproduction substructure 521-532 site spacer activity-modulating substivchu~e533-574 Aval/XmaI production substructure 575-580 TF 3-211 substructure with thmmbogenic581-1207 potential 1'm1I production subst<uch>re 1207-1212 stop colon 1211-1213 HindIII production substructwe 1214-1219 Example 5 Mutation of TF residue Thr167 to Lys: NuV 122 Oligonucleotide mutagenesis is performed on Clone NuV 121 according to the method of Deng and Nickeloff (1992) Analytical Biochem. 200:81-88] (also according to the Transformer Site-Directed Mutagenesis Kit, Clontech #K1600-1). The selection to primer (Stul, SEQ ID N0:13, (from Oligos Etc.)) changes the unique Scat site in NuV 121 to a Stul site; thus allowing selection against the parental pla~nid by restriction with ScaI. The mutagenic primer (TF+K, SEQ ID N0:14 (Oligos Etc.)) changes a Thr colon (ACA) to a Lys colon (AAA). Screening for the mutation is performed by DNA
sequencing. The modified clone is NuV 122 (SEQ ID NO:15), a plasmid for expression ~ 5 of a recombinant protein containing a (His)6 tag, a thrombin cleavage site, a 17 amino acid spacer, and TF residues 3 to 211, with residue 167 changed to Lys.

WO 99/32143 p~/p4gg NuV 122 residues (SEQ
ID
NO:15) start colon 413-415 (His)6 tag production substructure 425-442 BamHI production substructure 513-518 thrombin cleavageproduction subst<vct<ue 521-532 site spacer activity-modulating subsauchn~e533-574 Aval/XmaI production subst<~ucture 575-580 TF 3-211 substructure with thrombogenic581-1207 potential Thr167 to Lys modification 1073-1075 colon Lys mutation modification 1074 PmII production substructure 1207-1212 stop colon 1211-1213 HindIII production substzuchire 1214-1219 selection mutation 1245-1246 Example 6 Cloning of a synthetic peptide clone20 with a spacer attached to TFK: NuV 124 Oligonucleotides NuV20-1 through NuV20-8 (SEQ ID Nos:lS-23) are mixed at pmol/~L in 20 mM Tris-HCI, 10 mM MgCl2, 50 mM NaCI, pH 7.5. The mixt<n~e is heated to 80°C for 2 min, then cool to 25°C over a period of 30 min to allow annealing of oligonucleotides. The resulting synthetic spacer is cloned into the BamHI
and AvaI
t o sites of NuV 122. The vector DNA is prepared by digesting Clone NuV 122 with BamHI
and AvaI and isolation of the ~Skb fragment from an agamse gel and purification by QIAEX II (QIAGEN). The 10 microL ligation reaction contains 100 ng of NuV 122-BamHI/Aval fragment, 0.5 ~,L of the annealed oligonucleotide mixture, and T4 DNA ligase with buffer according to the manufacturer's recommendations (New England Biolabs). The resulting construct, NuV 124 (SEQ ID N0:24), is a plasmid for WO 99/32143 p~/pggg/Z~~gg expression of a recombinant protein containing a His-tag, a thrombin cleavage site, a peptide sequence refen~ed to as clone20, a spacer segment, and TF3-211 with K167.

NuV 124 - ~idues {SEQ ID
N0:24) start colon 413-415 (His)6 tag production substruchu~e 425-442 thrombin cleavageproduction substructure 521-532 site BamHI production substructure 530-535 Clone 20 context-enhancing substructure536-586 KpnI production substructure 587-592 spacer (C14S)g actlvlty-modul8tlng subshucture593-637 Aval/XmaI production substructtu~e 638-643 TF 3-211 substructure with thrombogenic644-1270 Thr167 to Lys modification 1136-1138 colon Lys mutation modification 1137 PmII production subs 1270-1275 stop colon 1274-1276 HindIII production substructure 1277-1282 selection mutation 2108-2109 ScaI
to StuI

Example 7 Characterization of NV 124 Purification of NV124.
The protein, NV 124, is expressed from the E. coli expression plasmid NuV 124. This protein is insoluble when expressed in E. coli and was recovered from inclusion bodies by solubilizing the proteins with guanidine hydrochloride (GuHCI).

WO 99/32143 PCTNS98~'1498 NV 124 was partially purified by IMAC (im~,nobilized metal anion chromatography) on a Ni-NTA column (Qiagen) in guanidine HCI. After extensive washing, low pH
(4.0) was used to release the NV 124 from the column. Folding of the protein was performed by dilution of the denatured protein into 20 mM TrisCl containing reduced s and oxidized glutathione. The final concentration of the protein after dilution in folding buffer was 50 ~g/ml. Thrombin was added under precisely controlled conditions to remove the (His)6 tag-thrombin cleavage peptide from the NV 124.
Thrombin was added at 5 microgram per mg of precursor protein. After 4 hours digestion at 37°C essentially all of the (His)6 tag-thrombin cleavage peptide was 1 o removed from the NV 124. In some studies, the (His)6 tag was not removed, yet this protein (referred to here as His-NV 124) displayed similar biological activity to the mature protein. The final step of purification was MonoQ-HR ion-exchange chromatography using FPLC and a Pharnacia 10/10 column. A gradient elution was used to elute the protein between 20 mM TrisCl, pH 8.5, and 20 mM TrisCl, pH
8.5, 1 s containing 1 M NaCI. The estimated purity at this stage is typically greater than 90%.
Yields of 20 mg NV 124 per liter of E. coli culture are typical.
Protein characterization.
2 o NV 124 and His-NV 124 were physically characterized by SDS-PAGE and mass spectrometry (MALDI). The mass of His-NV 124 determined by MALDI was 31.6 kDa and was similar to the theoretical mass of 31.7 kDa determined from the sequence.
2s TF function of NV124.
NV 124 was subjected to rigorous in vitro characterization of the function of its TF moiety. This included analysis of NV 124 dependent enhancement of factor Vila amidolytic activity, measurements of the affln'lty of NV 124 for factor VIIa, and 3 o measurements of NV 124 dependent enhancement of factor VIIa's proteolytic activity and plasma clotting activity. Table 1 summarizes some of the results obtained from the analyses of NV 124, in comparison with other STVTs characterized.
Amidolytic activity of factor VIIa complexed with NV 124 compared favorably with the complex of factor VIIa with TF1-218. This indicates that the N-terminal addition of about 20 peptide residues by way of a spacer does not interfere with subtle protein-protein interactions at the protease domain of factor VIIa that are responsible for allosteric enhancement of factor VIIa amidolytic function. The affinity of NV 124 for factor VIIa as measured by surface plasmon resonance or titration of factor VIIa amidolytic activity was also similar to that of TF1-218. Thus, the NV124 modification does not interfere with extensive interactions between TF and factor VIIa.
to It is also important that modifications to TF not disrupt proteolytic function of the TF-factor VIIa complex. This was addressed in an assay which measures the ability of the TF-factor VIIa complex to activate factor X in the fluid phase or on phospholipid vesicles. Indeed, TF directly participates in protein-protein interactions with macromolecular substrates factors X and IX. Assay of NV 124 dependent enhancement of factor VIIa hydrolysis of substrate factor X either in the presence or absence of phosphoiipid vesicles gave similar results to that obtained with TF1-218.
Additionally, NV 124 initiated plasma clotting rimes were similar to clotting times obtained with TF1-218. The results indicate that the NV124 modification does not 2 o interfere with the proteolytic cofactor function of TF. Moreover, analyses of other STVTs which were tested indicate that modifications at the N-terminus, the C-terminus and the permissive loop generally do not interfere with the cofactor functions of TF (Table 1 ).

WO 99/32143 PCT/US98/Z~498 Table l: Characterization of STYTs produced according to the invention.
Yield Kd Kd Relative of Class Protein Amidolyticfor for VIIaProteolytic ~2) VIIa s of STVT Link (mg/liter)activity (nlV1)~4)(n11~~5)activity~6) ~~) ~) TF1-218 50 1.000 4.73 3.26 1.000 NuV 123 N 47.2 0.998 5.63 4.95 0.995 NuV 125 N 0.28 ------- ------ --~_-- _ i o NuV N 21.8 0.902 5.63 3.65 1.456 NuV 129 N 6.66 0.736 4.42 4.06 0.955 NuV 135 N 23.3 ------ -------4.23 --------NuV 139 N 7.7 ------ - - -~__ __~__ NuV 141 N 31.3 1.010 11.55 7.87 0.481 1 s NuV N 50.6 1.005 15.32 7.42 0.755 NuV 144 N 13.3 0.979 4.32 3.22 0.862 NuV 128 N 6.90 0.860 3.72 10.8 -----NuV 131 C 3.66 1.031 9.70 ------- 2.082 NuV 137 C 3.89 0.941 6.03 --------0.858 2 o NuV I 62.9 1.032 6.18 --------0.977 TF-cys N 11.5 ------- ___~- _ __ ~_ 1. Linkage is the position of the facilitator relative to TF. N is amino terminus, C is carboxy terminus, and I is inserted into the permissive loop. Each STVT
represents a 25 different subclass of facilitator directed towards a different biological site. 2. Yield of refolding expressed as mg per liter of E. coli culture. 3. Normalized amidolytic activity of STVT:factor VIIa complex. 4. Apparent dissociation constant for factor VIIa determined from amidolytic activity plots at 5 nM factor VIIa concentration and varying the STVT concentration. 5. Dissociation constant determined by surface s o plasmon resonance in a BIAcore 2000. 6. Normalized factor Xa generation activity of STVT:factor VIIa complexes.
As is illustrated in Table 1, each STVT has amidolytic (measured by in vitro activity on a synthetic substrate) and factor VIIa binding activity (measured by 35 BIAcore) that is very close to that of the truncated TF. Therefore, incorporating the facilitators into the structure of TF has not damaged the inherent activity of TF.
NuV 143 has a facilitator incorporated into the permissive loop, and is a particularly interesting example of invention constructs.

WO 99/32143 PCT/US98~Z7498 Tozicity of NV124.
Soluble, truncated TF is a remarkably safe molecule to administer intravenously to mice. A dose of 1.5 mg per mouse (~75 mg/kg) has no visible effect on the mice. When a dose of 2.5 mg per mouse 0125 mg/kg) is administered, about half of the mice die. When native TF is inserted in a phospholipid vesicle and administered, death of all the mice results at a dose of about 10 ng/mouse ng/kg). Fully constituted native tissue factor is more than 250,000 times more toxic than the soluble, truncated TF used in construction of NV 124.
to When lethal doses of soluble, truncated TF or full length, reconstituted TF
are given intravenously to mice, the mice are observed to rapidly become very quiet and hunched over within 30-90 seconds, their respiration rate increases, and death occurs within 1-10 minutes. It was found that most of the radiolabelled soluble, truncated TF
is found in the lung a few minutes after IV administration. Doses of typical STVTs that kill 25-50% of 4 mice are generally around 125 to 200 micrograms per mouse (--6.25 to 10 mglkg). Therefore, NV 124 is about 12.5 to 20 times more toxic to mice than soluble, truncated TF and about at least 12,500 to 20,000 times less toxic than fully constituted TF.
Half lives of STVTs.
The half life of a STVT is short, on the order of several minutes as can be seen in the accompanying figures. Figure 3b illustrates the difference between the very short half life of radioiodinated truncated, soluble TF and the slightly longer half lives of two different STVTs injected intravenously into mice. As illustrated in Figure 3a, incubating an antibody (1OH10) that recognizes, but does not neutralize, TF
before injecting the complex into the mouse produces a much longer half life. When the mice were sacrificed soon after the last time point and their organs collected and 3o counted, most of the radiolabelled proteins were found in the lung.
Although injected, soluble TF accumulated in the lung, little thrombosis in the lung was observed. The wo ~r~m43 pc~rius9srrr49s - lack of phosphatidylserine expression in lung vesicles (see Ran et al. ( I
998) Cancer Res. 58(20):4646-53.) may account for the lack of thrombogenic activity in this tissue.
Histology The effects of various subclasses of STVT molecules on the tumor vascular system were histologically evaluated. It was desired to be able to quickly determine the effect on the vascular system by removing the tumor within 24 hours (1, 4, or 24 hours) and examining histological slides (H & E or Carstair's staining) to see the 1 o effects of the thrombogen on the vascular system. Because the tumor vessels in controls also show significant and variable tendency to thrornbose, results that differentiated control and STVT-treated tumors at early stages could not confidently be obtained. Therefore, tumor measurements over a period of 7 to 10 days were relied on.
Lack of inhibition of tumor growth by bolus doses of NV124.
Mice (strain A/~ were given C1300 neuroblastoma tumor cells subcutaneously on their flank, the tumors were allowed to grow to substantial size (up 2 o to 10 mm diameter) before treatrnent was started. After a bolus dose of selected NV 124 (in the range of 25 to 125 wg per mouse), the tumors became blue/black over a period of 1-5 minutes. In spite of the lack of inhibition of tumor growth, it is believed that change in coloration is physiologically significant and related to the NV
124, because neither saline controls nor truncated TF alone (which lacks a facilitator) produced this effect. Furthermore, not all STVTs (having different facilitators) produced this effect. Except for an occasional animal, bolus doses were generally not able to adequately infarct and inhibit the growth of tumors.

WO 99/32143 PCT/US98lZ'1498 Inhibition of C1300 growth by a single infusion of NV124 Because of these results and the short half life of the STVTs compared to the antibody based system that was previously demonstrated to work, it was decided to 5 use intravenous infusion to prolong the time that NV 124 would have to act on the tumor vascular system. In two independent experiments, tumors were grown for about 10 days before treatment was started. The volume of the tumors was calculated from {a2 * b)l2 where a is the smallest dimension of the tumor and b is the dimention of right angles to a. For comparison, a 5 x 5 mm and a 15 x 15 mm tumor yields a 1 o volume of 62.5 and 1688 mm3, respectively. The growth of the tumors was significantly slowed when the NV 124 was infused; the results of one experiment are shown in Figure 4. The tumors at the start of this experiment were about 7 x 7 mm.
A single dose 25 ~,g (closed diamond) or 125 ~g (closed square) of NV 124 was given by infusion through the tail vein over 1 hour. NV 124 contained a facilitator, clone 20 i5 described by Goodson et al (Pros. Natl. Acad. Sci. USA (1994) 91:7129-7133), which is reputed to interact with human uPAR and much less efficiently with marine uPAR.
Multiple infusion of NV124.
2 o As shown in Fig. 5, multiple infusions improved the pharmacological effect of NV 124 on tumor growth. C 1300 tumors were grown in A/J mice to about 5 x 5 mm in about 10 days. The mice had been given two infusions of NV 124 that were spaced by 4 days. NV 124 was infused through the tail vein over 1 hour at a rate of 0.2 ml per hour. One group received a single infusion of 125 pg (closed squares) and the other 2 s group received decreasing doses of 125, 75, and 50 ~g per infusion for a total of 3 infusions given on Days 0, 4, and 8. In this evaluation, the controls that were infused with saline grew from about 7 x 7 mm to about 15-17 mm in size in 8 days. The group that received 3 infusions responded better than the group that received only 1 infusion. In some animals, a hard black cap was formed over the tumors. In these 3 o particular animals, the tumors were about 1 x 1 cm in size. The cap formation, which strongly resembled a scab, was usually produced when the tumors were developing - redness just under the skin.
Histological evidence of NV124-induced effects on C1300 tumors.
Two regions of tumor loss were observed, which may correspond to scarring caused by the first administration and most recent apoptosis caused by the second administration. At least 95% of the cells. in the tumor were dead as judged from histological examination.
i o Direct observation of treated vessels.
Implanted tumor and angiogenic vessels were observed through a window established in a skin flap. This technique has been used most with hamsters in which the window is placed in the cheek pouch. The skin over the back of the animal (mice or rats) and a metal frame was placed so a fold of skin is held rigidly. A
circular piece of skin was removed from one side of the skin fold; this exposed the underside of the skin on the other side of the fold. A cover glass inserted and immobilized in the frame provides a sealed window. Vessels were easily seen by microscopic observation thmugh the window.
Before the introduction of NV 124, blood was observed flowing through arterioles and venules, individual red blood cells could be seen moving in capillaries, and white blood cells could be seen rolling along the vessel walls. The images were continuously captured on video recording tape. NV 124 produced a very rapid 2 s response; it caused rapid occlusion of arterioles, which prevented blood flow in the tumor vessels. In some instances, thromboses were visible in the vasculature.

WO 99/32143 PCT/US98l19498 - Example 8 PCR of human plasminogen cDNA fi~nents PCR is performed to amplify a DNA fi~agment of 965 base pairs from the s Clontech cDNA mixture (Marathon-Ready cDNA of Human placenta origin fiom Clontech (97/98cat.#7411-1)) which encodes kringle domains 1 and 2. A 100 pL
reaction should contain: 2 ~L cDNA mixture, 100 pmoles each of oligos Plg+63 and Plg-1005 (SEQ ID N0:25 and SEQ ID N0:26, respectively), buffer, BSA, MgS04 according to manufacturer; 1 ~L IOmM dNTPs and 2 units of Vent DNA polymerise i o which is added during the first cycle after the temperature reaches 94°C. The thermocycling is accomplished with 35 cycles of denaturation for 1 min at 94°C, primer annealing for 1 min at 55°C, and primer extension for 1 min at 75°C. The PCR product is about 965 base pairs in length. This DNA fiagtnent is purified by electrophoretic separation on a 1.0% agarose gel buffered with Tris-Borate-EDTA according to 15 Maniatis, excision of the appropriate band, and extraction of the DNA using the QIAEX
II gel extraction kit (QIAGEN cat#20051 ) according to manufacturer's instructions for DNA Extraction from Agarose Gels (QIAEX II Handbook 08/96). The 965 base pair DNA fi~agment concentration is estimated by agarose gel electrophoresis according to Maniatis.
The 965 base pair fragment is used as template for a second PCR amplification, this time with the oligonucleotides Plg+289 and Plg-523 (SEQ ID N0:27 and SEQ
ID
N0:28, respectively) to amplify only the kringle 1 domain and to add restriction sites appropriate for cloning. A 100 ~L PCR reaction contains: 10 ng 965 base pairs DNA
2 5 fisgment; 100 pmoles each of oligos Plg+289 and Plg-523, buffer, BSA, MgS04 according to manufacturer, 100 pM dNTPs and 2 units of Vent DNA polymerise (which is added during the first cycle after the temperature reaches 94°C). The thermocycling is accomplished with 25 cycles of denaturation for 1 min. at 94°C, primer annealing for 1 min at 55°C, and primer extension for 1 min at 75°C. The PCR
3 o product is about 267 by in length.

This 267 by DNA fi~Cnent is purified by passage over an elutip-D column according to the manufac~'s instructions. Briefly, the DNA is diluted to 1 mL
volume with Low-Salt buffer (0.2M NaCI, 20mM Tris-HCI, 1 mM EDTA pH7.4) and passed over the Elutip-D column. The column is subsequently washed with 3 mL
of Low-Salt buffer and eluted with 0.4 mL High-Salt buffer ( 1 M NaCI, 20mM Tris-HCI, 1 mM EDTA pH7.4). The DNA is desalted and concentrated by ethanol precipitation according to Maniatis. PCR is performed to amplify a DNA fiagment of 1677 by from the Clontech cDNA mixture which encodes kringle domains 3, 4 and 5. A 100 p,I, reaction should contain: 2 pL cDNA mixture, 100 pmoles each of oligos Plg+737 and 1 o Plg-2393 (SEQ ID N0:29 and SEQ ID N0:30, respectively), buffer, BSA, MgS04 according to the manufacturer; 1 pL IOmM dNTPs and 2 units of Vent DNA
polymerise which is added during the first cycle after the temperature reaches 94°C.
The thermocycling is accomplished with 35 cycles of denaturation for 1 min at 94°C, primer annealing for 1 min at 55°C, and primer extension for 1 min at 75°C.
The PCR product is about 1677 by in length. This DNA fragment is purified by electrophoretic separation on a 1.0% agarose gel buffered with Tris-Bon~te-EDTA
according to Maniatis, excision of the appropriate band, and extraction of the DNA
using the QIAEX II gel extraction kit {QIAGEN cat#20051) according to manufrcd~rers 2 o instivctions for DNA Extraction from Agarose Gels (QIAEX II Handbook 08/96). The 1677 by DNA fi~agment concentration is estimated by agarose gel electrophoresis according to Maniatis. The result is a 965 by cDNA fi~agment encoding plasminogen kringle domains 1 and 2, a 1677bp cDNA fragment encoding plasminogen kringle domains 3 through 5, and a 267bp cDNA fiagment encoding plasminogen kringle domain 1.
Example 9 Cloning of plasminogen kringle 1 into TFK: NuV 125 3 o The plasminogen kringle 1 fi~agment is cloned into the BamHI and KpnI
sites of NuV 124, replacing the clone 20 sequence with the plasminogen sequence. The 267 by WO 99/32143 p~~sgg,~~4gg Plasminogen cDNA fragment from PCR of human plasminogen cDNA fragments is digested with BamHI and KpnI, separated on an agarose gel, purified with QIAEX
II
resin, and ligated with NuV 124 that was prepared as follows: digestion with BamHi and KpnI, separation on an agarose gel, and purification of the -V6kb band with QIAEX II
resin. The resulting clone is designated NuV 125 (SEQ ID N0:31 ), a plasmid for expression of a recombinant protein containing a His-tag, a thrombin cleavage site, a plasminogen kringle 1, a spacer segment, and TF3-211 with K167.
NuV 125 residues (SEQ
ID
N0:31) start colon 413-415 (His)6 tag production substructure 425-442 thrombin cleavageproduction substructure 521-532 site BamHI production subshuch~re 530-535 plasminogen kringlecontext-enhancing substructure542-778 KpnI production substructure 779-784 Spacer (G4S); activity-modulating substructure785-829 AvaI/XmaI production substructure 830-835 TF 3-211 substructure with thrombogenic836-1462 potential Thr157 to Lys modification 1328-1330 colon Lys mutation modification 1329 stop colon 1466-1468 PmII production substructure 1462-1467 HindIII production substructure 1459-1474 selection mutation 2300-2301 Scat to StuI

- Example 10 Expression, refolding, and purification of TFK fusion proteins Expression in E. coli, refolding, thrombin cleavage, and purification of TFx and fusions with TFK are performed essentially as described by Stone et al. (1995) Biochem.
J. 310:605-614. TFK and variants of TFK are purified by the same basic protocol with modifications appropriate to the particular characteristics of the TFK fusion protein such as differences in elution from ion exchange chromatography and migration upon SDS-PAGE. TFK fusions are produced in yeast (see Stone et al. (1995) Biochem.
J.
io 310:605-614) or mammalian cells (see Ruf et al. (1991) J. Biol. Chem.
266:2158-2166 and Ruf et al.(1992) J. Crystal Growth 122, 253-264. These systems have the advantage that no refolding step is necessaZy. Expression in yeast may require additional mutations in TFK which alters recognition sites for the cell glycosylation machinery (Stone et al. (1995) Biochem. J. 310:605-614). A biophysical analysis is i 5 performed either by SDS PAGE or mass spectrometry (both of which are described by Stone et al. (1995) Biochem. J. 310:605-614). In vitm activity is determined either by titration of factor VIIa monitored by changes in rates of chromogenic substrate hydrolysis or by changes in rates of factor Xa activity generation (as described by Stone et al. (1995) Biochem. J. 310:605-614).
Example 11 Cloning of NuV 129 A human plasminogen fisgment encoding the fifth lcringle domain was 2 5 amplified by PCR finm a human EST clone (Genome Systems, genbank accession H61584) using Vent DNA polymerise (New England Biolabs) and the following oligonucleotides:
KS+: 5' CACACAGGATCCGAAGAAGACTGTATG 3' (SEQ ID N0:32) so KS': 5' CACACAGGTACCTGAAGGGGCCGCACA 3' (SEQ ID N0:33) - This amplified a 285 by fragment, which was digested with BamHI and KpnI and cloned into the corresponding sites of the plasmid NuV 127. Plasmid NuV 127 is a vector derived from pTrcHisC (Invitrogen) for cloning of amino terminally linked TF-fusion proteins. The resulting plasmid NuV 129 encodes a protein, NV 129, with a His-tag near the N-terminus, a thrombin cleavage site, plasminogen kringle 5 domain, a 15 residue flexible spacer, and human TF residues 3 to 211 at the C
terminus.
NuV I29 residues (SEQ
ID
N0:34) start colon 413-415 (Hiss tag production substructure 425-442 thrombin cleavageproduction substructure 521-532 site BamHI production substructure 530-535 plasminogen kringlecontext-enhancing substructure536-796 KpnI production substructure 797-802 spacer activity-modulating substructure803-847 TF 3-211 substnuture with thrombogenic854-1480 potential stop colon 1484-1486 1 o Purification and characterization.
Purification of NV 129 was performed as described for purification of NV 124 (see example 6.) Yields were in the range of 6 mg / L of E. coli culture. Mass spectrometry was performed by MALDI as previously described and resulted in a is determination of 39,305 Da in close agreement with the predicted mass of 39,303.
TF function of NV129.
Characterization of NV 129 TF activity was performed as previously described 2 o for NV 124. As shown in Table 1 although amidolytic activity of NV
129:factor VIIa WO 99132143 PGTNS98lZ?498 - was slightly lower than that of TF1-218, affinity for factor VIIa was similar to that of TF1-218. NV129:factor VIIa proteolytic activity for factor X activation was also similar to that of TF1-218:factor VIIa, as assayed both in the presence and absence of phospholipids. The results demonstrate that the TF entity of NV 129 is properly folded and functional.
C1300 neuroblastoma cells (500,000) were injected subcutaneously into the flank of A/J mice. After about 7-10 days, the tumors were grown to about 5-7 mm in diameter and were ready for treatment. A restraining device was used in which the 1 o mice were immobilized by a vest placed around their trunk and the vest was connected to the interior of a plexiglass tube. The tail of the mice was exposed from one end of the plexiglass tube. The tail was taped to a plexiglass plate and a 30-gauge needle inserted into the tail vein was connected to a Harvard precision pump.
The infusion was carried out for 60 minutes without anesthesia. Two hundred microliters i s was infused. Saline was infused into tumor bearing mice as a control.
Tumors were measured with calipers and the volume of the tumor was determined by the formula, (a~*b)l2, where a is the smallest dimension of the tumor and b is the dimension at right angles to a.
2 o A/J mice bearing C 1300 tumors of an initial size of about 6 X 6 mm were infused with 125 micrograms of NV 129 for 60 minutes. This treatment produced a statistically significant erect on the growth of C 1300 neuroblastoma tumors, as shown in Fig. 6. Saline treated control tumors grew to a volume of near 4 mls in 10 days.
Example 12 Cloning of NV 144 Oligonucleotides encoding a peptide facillitator were synthesized:
KLYD-1: S' GATCCCCGCGTAAACTGTACGACGGTAC 3' (SEQ ID N0:35) KLYD-2: S' CGTCGTACAGTTTACGCGGG 3' (SEQ ID N0:36) WO 99/32143 PGT/US98/Z~498 These oligonucleotides were annealed and cloned into the BamHI and KpnI sites of NuV 127. The resulting plasmid, NuV 144, encodes a protein, NV 144, with a His-tag near the N-terminus, a thrombin cleavage site, a six residue peptide , a 15 residue s flexible spacer, and human TF residues 3 to 211 at the C terminus.
NuV 144 residues (SEQ
ID

N0:37) start colon 413-41 S

(Hiss tag production substructure 425-442 thrombin cleavageproduction substructure 521-532 site BamHI production subshucture 530-535 peptide facilitatorcontext-enhancing substruchu~536-553 KpnI production substruchue 554-559 SpaCeI (O4S)3 activity-modulating substruchue560-604 TF 3-211 substruchu~ with thrombogenic6I 1-1240 potential stop colon 1241-1243 C1300 neuroblastoma cells (500,000) were injected subcutaneously into the flank of A/J mice. After about 7-10 days, the tumors had grown to about 5-7 mm in ~ o diameter and were ready for treatment. A restraining device was used in which the mice were immobilized by a vest placed around their trunk and the vest was connected to the interior of a plexiglass tube. The tail of the mice was exposed from one end of the plexiglass tube. The tail was taped to a plexiglass plate and a 30-gauge needle inserted into the tail vein was connected to a Harvard precision pump.
The i 5 infusion was carried out for 60 minutes without anesthesia. Two hundred microliters was infused. Saline was infi~sed into tumor bearing mice as a control. Tumors were measured with calipers and the volume of the tumor was determined by the formula, (a2*b)l2, where a is the smallest dimension of the tumor and b is the dimension at right angles to a.

Figure 7 shows the effect of multiple infusions of NV144 on C1300 tumor growth. A/J mice bearing C 1300 tumors were infused over a period of 1 hour with 125 micrograms NV 144 (also refen~ed to as KSp-TF) on Day 0 and 50 micrograms s NV 144 on Day 3. Saline controls grew rapidly with the tumors growing nearly to 3 ml in volume within 8 days. A group of 5 muscle-based tumors (triangles) exhibited reduced tumor growth rate whereas a skin based tumor (diamonds) had remarkably slowed growth rate with a secondary tumor appearing by Day 4. The higher dose of NV 144 produced better efficacy than the lower dose.
In another experiment shown in Figure 8, A/J mice bearing C 1300 tumors of an initial size of 7 X 7 mm were infi~sed over a 1 hour period on day 0 with 125 microgram NV 144 and again on Day 2 with 50 microgram NV 144. Both animals exhibited dramatic decreased tumor growth compared with saline controls 1 s (n=5). By day 7 the primary skin tumors were accompanied with large (> 5X5 mm) muscle tumors. The primary tumors developed superficial black necrotic caps by day 1. This is clear evidence for the pharmacological efficacy of NV 144.
2 o Example 13 Toxicity tests in animals Dose ranging study to identify the acute effects of test protein (TP) is performed in test animals by intravenous infusion of the drug. The maximum anticipated dosage of 2 s a therapeutic candidate molecules) in vivo is determined in adult rats, rabbits, dogs, nonhuman primates, etc. by an intravenous infiision schedule that mimics the anticipated potential therapeutic protocol in order to observe the acute effects of the drug.
s o A typical experimental design would include the following. Groups of 3 male rats are used for each dose concentration and for each control. The unanesthetized rats are gently immobilized in an approved manner with tail vein immobilized and prepared WO 99/32143 pG.j.~~~4~
- for sterile insertion of the intravenous catheter. The inserted catheter is flushed with sterile physiologic saline (SPS) or lactated Ringer's solution (LRS) at a rite of 20 L/min/100gBW. The infusion line intercepts the SPS or LRS line and under pump control permits infusion into the catheter of the test material. The control infusion of 5 SPS or LRS proceeds for 10 min. The test material shall be labeled by confidential test number and have been adjusted to five concentrations. The test samples contain differing concentrations in 3- to 10-fold concentration increments. The test material infusion pump syringe connects to the same infusion catheter by a T connector (or needle insertion) taking care that no bubbles are created that could be infused. This i o permits washing of the line past the point of test material entry. The test material, under confidential identification number and letter designation, is infused at 5 to L/min/100gBW. The duration of infusion is 0.5 to 120 min.
The rats are observed for changes in behavior, convulsions, increase of i 5 respiratory rate, or death and each shall be scored. At the end of the designated test material infusion period, the infusion is switched to SPS or LRS for 1 to 60 min. The catheter is then removed and the behavior, respiratory rate, and hemostasis at the tail vein site observed and monitored at 0.5, 1 and 2 hrs after termination of test infusion. A
sample of blood is taken at each interval for enumeration of cell types and counts. Each 2 o rat is eilthanized twenty-four hours later. The abdomen is opened and the inferior vena cava incised to take a blood sample and exsanguinate the rat followed immediately by perfusion through the heart of cold SPS or LRS containing 50 U/ml of USP
heparin.
The organs (heart, lungs, liver, kidneys, spleen, pancreas, stomach, large bowel and small bowel, adrenal, and brain are removed, cut to ~ 3 mm thick blocks and fixed in 25 10~~o neutral formalin. After processing and embedding in para~n blocks, cutting at 5 microns, sections are stained with Hematoxylin and Basin, as well as by Carstair's method for histological examination.

WO 99/32143 PCT/US98lZ'1498 . Example 14 Inhibition of human tumors grown in human skin implants in immunodeficient mice The ability of the test compounds to eradicate or reduce the size of tumors in test animals is tested as follows in a model using human skin, such as foreskin or breast reduction mammoplasty, or other transplants in severe combined immunodeficient (SC1D) mice, cats or other types of immunodeficient animals. The human foreskins are trimmed to an oval of approximately 8 mm x 13 mm and stored at 4°C in DMEM or 1 o RPMI tissue culture medium containing 10% FCS until surgery (next day).
100:1 of diluted Ketamine HCl (diluted 1:10 in sterile water) is injected intraperitoneally into mouse abdomen. A light plane of anesthesia is induced with metophane and the back of the mouse is prepared by shaving and swabbing with alcohol. After making an incision in the skin, a sample of foreskin is placed within the wound and secured by sutures. The ~ 5 wound is wrapped and dressed. Observations of the growth of the foreskin implant are made each day for 10 days. After 10 days, the bandages are removed. After 4 weeks, the implant is ready to be inoculated with an injection of tumor cells. Three million tumor cells are injected intradermally into the transplanted human skin. After approximately 2 weeks the tumors are large enough to initiate treatment of the animal 2 0 With test materials.
Example 15 Inhibition of tumors grown immunodeficient, SCID, nude or wildtype rodents The ability of the test materials to eradicate or reduce the size of tumors in test animals is tested as follows in a model using xenografts of human tumor cells implanted into SCID or nude mice or of rodent tumor cells implanted into compatible strains of wildtype rodents. Examples of mouse tumors and their host strains of rodents (i.e., mice so or rat) include colon adenocarcinoma CT-26 cells into Balb/c mice, C1300 neuroblastoma cells into AlJ mice, Hepatoma 129 cells into C3H mice, Lewis lung cells into B57BL/6 mice, and other combinations of cells and mice listed in the Division of WO 99/32143 PGT/tTS98/Z7498 Cancer Treatment, Diagnosis and Centers (DCTDC) Tumor Repository Catalogue of Transplantable Animal and Hunan Tumors that is maintained by The National Cancer Institute, Rederick Cancer Research and Development Center, PO Box B, Frederick, MD 21702. Fifty thousand to three million tumor cells are injected subcutaneously into recipient animals and the tumors allowed to grow for 3 to 30 days. Test materials are injected intravenously after the tenors have reached the desired size. Test materials over various concentrations and doses and . over different schedules may be administered. Typical doses range between 0.1 ~,g to 20 mg of test material per kg.
Typical schedules range between a course of 3 to 4 doses per day to 1 dose per week for i o a course of treatment ranging between 1 to 6 treatment cycles. The tumors are measured with calipers and the note of growth of the tumors are followed before and ailer treatment to distinguish effects of the test materials on growth. The tumor sites are removed from the mice for histological examination.
1 s Example 16 Pharn~acologic Effect in Primates Normal, healthy nonhunan primates are implanted in the superficial subdermal tissue of the volar surface of one forearm with pellets, which are approximately 100 2 o microns in diameter and containing one or more angiogenic factors, such as vascular endothelial growth factor (vascular permeability factor), basic fibroblast growth factor, etc., in three sets to produce a variation in the maturity of the vessels that are induced to grow around the pellet As a control, a similar set of pellets containing human albumin are placed in the same distribution in the other forearm. Pellets are permitted to 2 s establish local angiogenic networks for periods of 2 to 14 days. The animals then receive test materials given by intravenous injection. At intervals of 0.5 hours to 3 days following infusion of test materials, a 3 mm diameter punch biopsy is taken to include a pellet and surrounding tissue. The biopsied materials are examined histologically for any effect of the test material on the structwe of the vessels and compared with the s o control biopsy.

WO 99I321d3 PCTNS98/Z'1498 While the invention has been described in detail with reference to certain preferred embodiments thereof, it will be understood that modifications and variations are within the spirit and scope of that which is described and claimed.

WO 99/32143 PCTNS98rZ9498 SEQUENCE LISTING
<110> Houston, L.L.
Dickinson, Craig D.
<120>
<130> NuVas1100-wo <160> 37 <170> FastSEQ for Windows Version 3.0 <210> 1 <211> 2104 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (76)...(960) <400> 1 gggtgccttc agcccaacct ccccagcccc acgggcgcca cggaacccgc tcgatctcgc 60 cgccaactgg tagac atg gag acc cct gcc tgg ccc cgg gtc ccg cgc ccc 111 Met Glu Thr Pro Ala Trp Pro Arg Val Pro Arg Pro gag acc gcc gtc get cgg acg ctc ctg ctc ggc tgg gtc ttc gcc cag 159 Glu Thr Ala Val Ala Arg Thr Leu Leu Leu Gly Trp Val Phe Ala Gln gtg gcc ggc get tca ggc act aca aat act gtg gca gca tat aat tta 207 Val Ala Gly Ala Ser Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu act tgg aaa tca act aat ttc aag aca att ttg gag tgg gaa ccc aaa 255 Thr Trp Lys Ser Thr Asn Phe Lya Thr Ile Leu Glu Trp Glu Pro Lys ccc gtc aat caa gtc tac act gtt caa ata agc act aag tca gga gat 303 Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp tgg aaa agc aaa tgc ttt tac aca aca gac aca gag tgt gac ctc acc 351 Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr gac gag att gtg aag gat gtg aag cag acg tac ttg gca cgg gtc ttc 399 Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe tcc tac ccg gca ggg aat gtg gag agc acc ggt tct get ggg gag cct 447 WO 99/32143 PCTNS98r17498 Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro ctg tat gag aac tcc cca gag ttc aca cct tac ctg gag aca aac ctc 495 Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu gga cag cca aca att cag agt ttt gaa cag gtg gga aca aaa gtg aat 543 Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn gtg acc gta gaa gat gaa cgg act tta gtc aga agg aac aac act ttc 591 Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe cta agc ctc cgg gat gtt ttt ggc aag gac tta att tat aca ctt tat 639 Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr tat tgg aaa tct tca agt tca gga aag aaa aca gcc aaa aca aac act 687 Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr aat gag ttt ttg att gat gtg gat aaa gga gaa aac tac tgt ttc agt 735 Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser gtt caa gca gtg att ccc tcc cga aca gtt aac cgg aag agt aca gac 783 Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp agc ccg gta gag tgt atg ggc cag gag aaa ggg gaa ttc aga gaa ata 831 Ser Pro Val Glu Cys Met Gly Gln Glu Lys Gly Giu Phe Arg Glu Ile ttc tac atc att gga get gtg gca ttt gtg gtc atc atc ctt gtc atc 879 Phe Tyr Ile Ile Gly Ala Val Ala Phe Val Val Ile Ile Leu Val Ile atc ctg get ata tct cta cac aag tgt aga aag gca gga gtg ggg cag 927 Ile Leu Ala Ile Ser Leu His Lys Cys Arg Lys Ala Gly Val Gly Gln agc tgg aag gag aac tcc cca ctg aat gtt tca taaaggaagc actgttggag 980 Ser Trp Lys Glu Asn Ser Pro Leu Asn Val Ser ctactgcaaatgctatattgcactgtgaccgagaacttttaagaggatagaatacatgga1040 aacgcaaatgagtatttcggagcatgaagaccctggagttcaaaaaactcttgatatgac1100 ctgttattaccattagcattctggttttgacatcagcattagtcactttgaaatgtaacg1160 aatggtactacaaccaattccaagttttaatttttaacaccatggcaccttttgcacata1220 acatgctttagattatatattccgcactcaaggagtaaccaggtcgtccaagcaaaaaca1280 aatgggaaaatgtcttaaaaaatcctgggtggacttttgaaaagcttttttttttttttt1340 ttttttgagacggagtcttgctctgttgcccaggctggagtgcagtagcatgatctcggc1400 tcactgcaccctccgtctctcgggttcaagcaattgtctgcctcagcctcccgagtagct1460 wo 99r~zm3 pcrius9sizr49s gggattacaggtgcgcactaccacaccaagctaatttttgtattttttagtagagatggg1520 gtttcaccatcttggccaggctggtcttgaattcctgacctcaggtgatccacccacctt1580 ggcctcccaaagtgctagtattatgggcgtgaaccaccatgcccagccgaaaagcttttg1640 aggggctgacttcaatccatgtaggaaagtaaaatggaaggaaattgggtgcatttctag1700 gacttttctaacatatgtctataatatagtgtttaggttctttttttttttcaggaatac1760 atttggaaattcaaaacaattggcaaactttgtattaatgtgttaagtgcaggagacatt1820 ggtattctgggcaccttcctaatatgctttacaatctgcactttaactgacttaagtggc1880 attaaacatttgagagctaactatatttttataagactactatacaaactacagagttta1940 tgatttaaggtacttaaagcttctatggttgacattgtatatataattttttaasaaggt2000 tttctatatggggattttctatttacgtaggtaatattgttctatttgtatatattgaga2060 taatttatttaatatactttaaataaaggtggactgggattgtt 2104 <210> 2 <211> 19 <212> PRT
<213> Homo sapiens <400> 2 Gly Ser Cys Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro <210> 3 <211> 21 <212> DNA
<213> Homo Sapiens <400> 3 actacaaata ctgtggcagc a 21 <210> 4 <211> 33 <212> DNA
<213> Homo sapiens <400> 4 tttaagcttt cacgtgccca tacactctac cgg 33 <210> 5 <211> 51 <212> DNA
<213> Homo sapiens <400> 5 aaatggatcc tggtgcctag gggccccggg actacaaata ctgtggcagc a 51 <210> 6 <211> 5027 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413)...(1168) <400>

gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc 120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc 180 tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcgga 240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa 300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta 360 aaaattaaagaggtatatattaatgtatcgattsaataaggaggaataaacc atg ggg 418 Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag cat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys His Arg Trp atc Ctg gtg cct agg ggc cec ggg act aca aat act gtg gca gca tat 562 Ile Leu Val Pro Arg Gly Pro Gly Thr Thr Asn Thr Val Ala Ala Tyr aat tta act tgg aaa tca act aat ttc aag aca att ttg gag tgg gaa 610 Asn Leu Thr Trp Lys Ser Thr Asn Phe Lya Thr Ile Leu Glu Trp Glu ccc aaa ccc gtc aat caa gtc tac act gtt caa ata agc act aag tca 658 Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser gga gat tgg aaa agc aaa tgc ttt tac aca aca gac aca gag tgt gac 706 Gly Asp Trp Lya Ser Lye Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp ctc acc gac gag att gtg aag gat gtg aag cag acg tac ttg gca cgg 754 Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg gtc ttc tcc tac ccg gca ggg aat gtg gag agc acc ggt tct get ggg 802 Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala aly gag cct ctg tat gag aac tcc cca gag ttc aca cct tac ctg gag aca 850 Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr aac ctc gga cag cca aca att cag agt ttt gaa cag gtg gga aca aaa 898 Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys gtg aat gtg acc gta gaa gat gaa cgg act tta gtc aga agg aac aac 946 Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn act ttc cta agc ctc cgg gat gtt ttt ggc aag gac tta att tat aca 994 Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr ctt tat tat tgg aaa tct tca agt tca gga aag aaa aca gcc aaa aca 1042 Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr aac act aat gag ttt ttg att gat gtg gat aaa gga gaa aac tac tgt 1090 Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys ttc agt gtt caa gca gtg att ccc tcc cga aca gtt aac cgg aag agt 1138 Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser aca gac agc ccg gta gag tgt atg ggc acg tgaaagcttg gctgttttgg 1188 Thr Asp Ser Pro Val Glu Cys Met Gly Thr cggatgagagaagattttcagcctgatacagattaaatcagaacgcagaagcggtctgat1248 aaaacagaatttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactc1308 agaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaa1368 ctgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatct1428 gttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacg1488 ttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaactgccaggcatc1548 aaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaaactctttttg1608 tttatttttctaaatacattcaaatatgtatecgctcatgagacaataaccctgataaat1668 gcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttat1728 tcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagt1788 aaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacag1848 cggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaa1908 agttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcg1968 ccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatct2028 tacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacac2088 tgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgca2148 caacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccat2208 accaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaact2268 attaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggc2328 ggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctga2388 taaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatgg2448 taagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacg2508 aaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagacca2568 agtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatcta2628 ggtgaagatcctttttgataatctcatgaccaaaatccettaacgtgagttttcgttcca2688 ctgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcg2748 cgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccgga2808 tcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaa2868 tactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcc2928 tacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtg2988 tcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaac3048 ggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacct3108 acagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatcc3168 ggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctg3228 gtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatg3288 ctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcct3348 ggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtgga3408 taaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcg3468 cagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgca3528 tctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgc3588 atagttaagccagtatacactccgctatcgctacgtgactgggtcatggctgcgccccga3648 cacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttac3708 agacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccg3768 aaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttg3828 acaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtc3888 aattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtg3948 tctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgc4008 gggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaac4068 aactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctggccctgcacg4128 cgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtgg4188 tggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttc4248 tcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccaggatgccattg4308 ctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacac4368 ccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtggagcatctgg4428 tcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgc4488 gtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcagccgatagcgg4548 aacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatg4608 agggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgc4668 gcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacg4728 ataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcc4788 tgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagg4848 gcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgc4908 aaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttccc4968 gactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcgaattgatctg 5027 <210> 7 <211> 32 <212> DNA
<213> Homo sapiens <400> 7 gatcctggtc cctaggggag gaggcggttc ag 32 <210> 8 <211> 30 <212> DNA
<213>~Homo sapiens <400> 8 gtggtggagg taccggaggt ggaggttctc 30 <210> 9 <211> 13 <212> DNA
<213> Homo sapiens <400> 9 ccctagggac cag 13 <210> 10 <211> 13 <212> DNA
<213> Homo sapiens <400> 10 ccctagggac cag 13 <210> li <211> 21 <212> DNA
<213> Homo sapiens <400> 11 ccgggagtac ctccacctcc g 21 <210> 12 <211> 5069 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413)...(1210) <400>

gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc 60 ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc 120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc 180 tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcgga 240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa 300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta 360 aaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaacc atg ggg 418 Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His Hie His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag cat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys His Arg Trp atc ctg gtc cct agg gga gga ggc ggt tca ggt ggt gga ggt acc gga 562 Ile Leu Val Pro Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gly ggt gga ggt tct ccc ggg act aca aat act gtg gca gca tat aat tta 610 Gly Gly Gly Ser Pro Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu act tgg aaa tca act aat ttc aag aca att ttg gag tgg gaa ccc aaa 658 Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys WO 99!32143 PGTNS981S9498 ccc gtc aat caa gtc tac act gtt caa ata agc act aag tca gga gat 706 Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp tgg aaa agc aaa tgc ttt tac aca aca gac aca gag tgt gac ctc acc 754 Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr gac gag att gtg aag gat gtg aag cag acg tac ttg gca cgg gtc ttc 802 Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe tcc tac ccg gca ggg aat gtg gag agc acc ggt tct get ggg gag cct 850 Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro ctg tat gag aac tcc cca gag ttc aca cct tac ctg gag aca aac ctc 898 Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu gga cag cca aca att cag agt ttt gaa cag gtg gga aca aaa gtg aat 946 Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn gtg acc gta gaa gat gaa cgg act tta gtc aga agg aac aac act ttc 994 Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe cta agc ctc cgg gat gtt ttt ggc aag gac tta att tat aca ctt tat 1042 Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr tat tgg aaa tct tca agt tca gga aag aaa aca gcc aaa aca aac act 1090 Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr aat gag ttt ttg att gat gtg gat aaa gga gaa aac tac tgt ttc agt 1138 Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser gtt caa gca gtg att ccc tcc cga aca gtt aac cgg aag agt aca gac 1186 Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp agc ccg gta gag tgt atg ggc acg tgaaagcttg gctgttttgg cggatgagag 1240 Ser Pro Val Glu Cys Met Gly Thr aagattttcagcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaat1300 ttgcctggcggcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaa1360 cgccgtagcgccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggca1420 tcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtc1480 ggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca1540 acggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagca1600 gaaggccatcctgacggatggcctttttgcgtttctacaaactctttttgtttatttttc1660 WO 99/32143 PCT/US98lZ'1498 taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa1720 tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt1780 gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct1840 gaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc1900 cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta1960 tgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacac2020 tattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggc2080 atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaac2140 ttaettctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg2200 gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac2260 gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc2320 gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt2380 gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga2440 gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc2500 cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag2560 atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca2620 tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc2680 ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca2740 gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc2800 tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta2860 ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcctt2920 ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc2980 gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccggg3040 ttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg3100 tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag3160 ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggta~gcggc3220 agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat3280 agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg3340 gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc3400 tggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtatt3460 accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca3520 gtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggt3580 atttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagc3640 cagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaa3700 cacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg3760 tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga3820 ggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttgacaccatcga3880 atggtgcaaaacetttcgcggtatggcatgatagcgcccggaagagagtcaattcagggt3940 ggtgaatgtgaaaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatca4000 gaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagt4060 ggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcggg4120 caaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgca4180 aattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgat4240 ggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacg4300 cgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagc4360 tgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacag4420 tattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattggg4480 tcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtct4540 ggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaagg4600 cgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgt4660 tcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattac4720 cgagtccgggctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaaga4780 cagctcatgttatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggca4840 aaccagcgtggaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagct4900 gttgcccgtctcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctc4960 WO 99/32143 PCT/US98~Z7498 tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 5020 cgggcagtga gcgcaacgca attaatgtga gttagcgcga attgatctg 5069 <210> 13 <211> 28 <212> DNA
<213> Homo Sapiens <400> 13 tcaggaaaga aaaaagccaa aacaaaca 28 <210> 14 <211> 26 <212> DNA
<213> Homo sapiens <400> 14 gacttggttg aggcctcacc agtcac 26 <210> 15 <211> 5069 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413) ... (1210) <400> 15 gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc60 ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc180 tgaaatgagctgttgacaattaatcatecggctcgtataatgtgtggaattgtgagcgga240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta360 aaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaacc atg 418 ggg Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag cat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys His Arg Trp atc ctg gtc cct agg gga gga ggc ggt tca ggt ggt gga ggt acc gga 562 Ile Leu Val Pro Arg Gly Gly Gly Gly Ser Gly Gly Gly Gly Thr Gly ggt gga ggt tct ccc ggg act aca aat act gtg gca gca tat aat tta 610 Gly Gly Gly Ser Pro Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu act tgg aaa tca act aat ttc aag aca att ttg gag tgg gaa ccc aaa 658 Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys ccc gtc aat caa gtc tac act gtt caa ata agc act aag tca gga gat 706 Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp tgg aaa agc aaa tgc ttt tac aca aca gac aca gag tgt gac ctc acc 754 Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr gac gag att gtg aag gat gtg aag cag acg tac ttg gca cgg gtc ttc 802 Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe tcc tac ccg gca ggg aat gtg gag agc acc ggt tct get ggg gag cct 850 Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro ctg tat gag aac tcc cca gag ttc aca cct tac ctg gag aca aac ctc 898 Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu gga cag cca aca att cag agt ttt gaa cag gtg gga aca aaa gtg aat 946 Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn gtg acc gta gaa gat gaa cgg act tta gtc aga agg aac aac act ttc 994 Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe cta agc ctc cgg gat gtt ttt ggc aag gac tta att tat aca ctt tat 1042 Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr tat tgg aaa tct tca agt tca gga aaa aaa aaa gcc aaa aca aac act 1090 Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Lys Ala Lys Thr Asn Thr aat gag ttt ttg att gat gtg gat aaa gga gaa aac tac tgt ttc agt 1138 Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser gtt caa gca gtg att ccc tcc cga aca gtt aac cgg aag agt aca gac 1186 Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp agc ccg gta gag tgt atg ggc acg tgaaagcttg gctgttttgg cggatgagag 1240 Ser Pro Val Glu Cys Met Gly Thr aagattttca gcctgataca gattaaatca gaacgcagaa gcggtctgat aaaacagaat 1300 ttgcctggcg gcagtagcgc ggtggtccca cctgacccca tgccgaactc agaagtgaaa 1360 cgccgtagcg ccgatggtag tgtggggtct ccccatgcga gagtagggaa ctgccaggca 1420 WO 99!32143 PCT/US98r17498 tcaaataaaacgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtc1480 ggtgaacgctctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagca1540 acggcccggagggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagca1600 gaaggccatcctgacggatggcctttttgcgtttctacaaactctttttgtttatttttc1660 taaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataa1720 tattgaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattccctttttt1780 gcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgct1840 gaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatc1900 cttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgcta1960 tgtggcgcggtattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacac2020 tattctcagaatgacttggttgaggcctcaccagtcacagaaaagcatcttacggatggc2080 atgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcggccaac2140 ttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggg2200 gatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgac2260 gagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggc2320 gaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagtt2380 gcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctgga2440 gccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcc2500 cgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacag2560 atcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactca2620 tatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatc2680 ctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca2740 gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgc2800 tgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagcta2860 ccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgtcctt2920 ctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctc2980 gctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccggg3040 ttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcg3100 tgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgag3160 ctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggc3220 agggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttat3280 agtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggg3340 gggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgc3400 tggccttttgctcacatgttctttectgcgttatcccctgattctgtggataaccgtatt3460 accgcctttgagtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtca3520 gtgagcgaggaagcggaagagcgcctgatgcggtattttctccttacgcatctgtgcggt3580 atttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgcatagttaagc3640 cagtatacactccgctatcgctacgtgactgggtcatggctgcgccccgacacccgccaa3700 cacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg3760 tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcga3820 ggcagcagatcaattcgcgcgcgaaggcgaagcggcatgcatttacgttgacaccatcga3880 atggtgcaaaacctttcgcggtatggcatgatagcgcccggaagagagtcaattcagggt3940 ggtgaatgtgasaccagtaacgttatacgatgtcgcagagtatgccggtgtctcttatca4000 gaccgtttcccgcgtggtgaaccaggccagccacgtttctgcgaaaacgcgggaaaaagt4060 ggaagcggcgatggcggagctgaattacattcccaaccgcgtggcacaacaactggcggg4120 caaacagtcgttgctgattggcgttgccacctccagtctggccctgcacgcgccgtcgca4180 aattgtcgcggcgattaaatctcgcgccgatcaactgggtgccagcgtggtggtgtcgat4240 ggtagaacgaagcggcgtcgaagcctgtaaagcggcggtgcacaatcttctcgcgcaacg4300 cgtcagtgggctgatcattaactatccgctggatgaccaggatgccattgctgtggaagc4360 tgcctgcactaatgttccggcgttatttcttgatgtctctgaccagacacccatcaacag4420 tattattttctcccatgaagacggtacgcgactgggcgtggagcatctggtcgcattggg4480 tcaccagcaaatcgcgctgttagcgggcccattaagttctgtctcggcgcgtctgcgtct4540 ggctggctggcataaatatctcactcgcaatcaaattcagccgatagcggaacgggaagg4600 cgactggagtgccatgtccggttttcaacaaaccatgcaaatgctgaatgagggcatcgt4660 tcccactgcgatgctggttgccaacgatcagatggcgctgggcgcaatgcgcgccattac4720 cgagtccggg ctgcgcgttggtgcggatatctcggtagtgggatacgacgataccgaaga4780 cagctcatgt tatatcccgccgtcaaccaccatcaaacaggattttcgcctgctggggca4840 aaccagcgtg gaccgcttgctgcaactctctcagggccaggcggtgaagggcaatcagct4900 gttgcccgtc tcactggtgaaaagaaaaaccaccctggcgcccaatacgcaaaccgcctc4960 tccccgcgcg ttggccgattcattaatgcagctggcacgacaggtttcccgactggaaag5020 cgggcagtga gcgcaacgcaattaatgtgagttagcgcgaattgatctg 5069 <210> 16 <211> 37 <212> DNA
<213> Homo sapiens <400> 16 gatcttggtc cctaggggat ccgcagaacc aatgcct 37 <210> 17 <211> 36 <212> DNA
<213> Homo sapiens <400> 17 cactcgctaa acttcagtca atacctctgg tatact 36 <210> 18 <211> 36 <212> DNA
<213> Homo Sapiens <400> 18 ggtaccggag gaggcggttc aggtggtgga ggttca 36 <210> 19 <211> 16 <212> DNA
<213> Homo sapiens <400> 19 ggaggtggag gttctc 16 <210> 20 <211> 23 <212> DNA
<213> Homo sapiens <400> 20 tctgcggatc ccctagggac caa <210> 21 <211> 36 <212> DNA
<213> Homo sapiens <400> 21 aggtattgac tgaagtttag cgagtgaggc attggt 36 <210> 22 <211> 36 <212> DNA
<213> Homo sapiens <400> 22 ccacctgaac cgcctcctcc ggtaccagta taccag 3s <210> 23 <211> 30 <212> DNA
<213> Homo sapiens <400> 23 ccgggagaac ctccacctcc tgaacctcca 30 <210> 24 <211> 5132 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413)...(1273) <400> 24 gtttgacagc ttatcatcga ctgcacggtg caccaatgct tctggcgtca ggcagccatc 60 ggaagctgtg gtatggctgt gcaggtcgta aatcactgca taattcgtgt cgctcaaggc 120 gcactcccgt tctggataat gttttttgcg ccgacatcat aacggttctg gcaaatattc 180 tgaaatgagc tgttgacaat taatcatccg gctcgtataa tgtgtggaat tgtgagcgga 240 taacaatttc acacaggaaa cagcgccgct gagaaaaagc gaagcggcac tgctctttaa 300 caatttatca gacaatctgt gtgggcactc gaccggaatt atcgattaac tttattatta 360 aaaattaaag aggtatatat taatgtatcg attaaataag gaggaataaa cc atg ggg 418 Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag cat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys His Arg Trp atc ttg gtc cct agg gga tcc gca gaa cca atg cct cac tcg cta aac 562 Ile Leu Val Pro Arg Gly Ser Ala Glu Pro Met Pro His Ser Leu Asn ttc agt caa tac ctc tgg tat act ggt acc gga gga ggc ggt tca ggt 610 Phe Ser Gln Tyr Leu Trp Tyr Thr Gly Thr Gly Gly Gly Gly Ser Gly ggt gga ggt tca gga ggt gga ggt tct ccc ggg act aca aat act gtg 658 Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Gly Thr Thr Asn Thr Val gca gca tat aat tta act tgg aaa tca act aat ttc aag aca att ttg 706 Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu gag tgg gaa ccc aaa ccc gtc aat caa gtc tac act gtt caa ata agc 754 Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser act aag tca gga gat tgg aaa agc aaa tgc ttt tac aca aca gac aca 802 Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr gag tgt gac ctc acc gac gag att gtg aag gat gtg aag cag acg tac 850 Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr ttg gca cgg gtc ttc tcc tac ccg gca ggg aat gtg gag agc acc ggt 898 Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly tct get ggg gag cct ctg tat gag aac tcc cca gag ttc aca cct tac 946 Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr ctg gag aca aac ctc gga cag cca aca att cag agt ttt gaa cag gtg 994 Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val gga aca aaa gtg aat gtg acc gta gaa gat gaa cgg act tta gtc aga 1042 Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg agg aac aac act ttc cta agc ctc cgg gat gtt ttt ggc aag gac tta 1090 Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu att tat aca ctt tat tat tgg aaa tct tca agt tca gga aaa aaa aaa 1138 Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Lys gcc aaa aca aac act aat gag ttt ttg att gat gtg gat aaa gga gaa 1186 Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu aac tac tgt ttc agt gtt caa gca gtg att ccc tcc cga aca gtt aac 1234 Asn Tyr Cys Phe ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn cgg aag agt aca gac agc ccg gta gag tgt atg ggc acg tgaaagcttg 1283 Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Thr gctgttttgg cggatgagag aagattttca gcctgataca gattaaatca gaacgcagaa 1343 gcggtctgat aaaacagaat ttgcctggcg gcagtagcgc ggtggtccca cctgacccca 1403 WO 99/32143 PCTJUS98n7498 tgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcga1463 gagtagggaactgccaggcatcaaataasacgaaaggctcagtcgaaagactgggccttt1523 cgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcg1583 gatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaact1643 gccaggcatcaaattaagcagaaggccatcctgacggatggcctttttgcgtttctacaa1703 actctttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataac1763 cctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtg1823 tcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc1883 tggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacategaactgg1943 atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatga2003 gcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagc2063 aactcggtcgccgcatacactattctcagaatgacttggttgaggcctcaccagtcacag2123 aaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatga2183 gtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccg2243 cttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga2303 atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgt2363 tgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact2423 ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt2483 ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgg2543 ggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaacta2603 tggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaac2663 tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta2723 aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagt2783 tttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctt2843 tttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggttt2903 gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc2963 agataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctg3023 tagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcg3083 ataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggt3143 cgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac3203 tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg3263 acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg3323 gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat3383 ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttt3443 tacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctg3503 attctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaa3563 cgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgeggtattttc3623 tccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgct3683 ctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggc,3743 tgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggc3803 atccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc3863 gtcatcaccgaaacgcgcgaggcagcagatcaattegcgcgcgaaggcgaagcggcatgc3923 atttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccg3983 gaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagag4043 tatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttct4103 gcgaaaacgcgggaaaaagtggaagcggcgatggcggagctgaattacattcccaaccgc4163 gtggcacaacaactggcgggcaaacagtcgttgctgattggcgttgccacctccagtctg4223 gccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgccgatcaactgggt4283 gccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtg4343 cacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccag4403 gatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctct4463 gaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtg4523 gagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttct4583 gtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcag4643 ccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaa4703 WO 99!32143 PCT/US98I19498 atgctgaatg agggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctg4763 ggcgcaatgc gcgccattaccgagtccgggctgcgegttggtgcggatatctcggtagtg4823 ggatacgacg ataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacag4883 gattttcgcc tgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccag4943 gcggtgaagg gcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcg5003 cccaatacgc aaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacga5063 caggtttccc gactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcga5123 attgatctg 5132 <210> 25 <211> 21 <212> DNA
<213> Homo sapiens <400> 25 tcaccaccga ccccaacaag c 21 <210> 26 <211> 21 <212> DNA
<213> Homo sapiens <400> 26 cctcaatcca agtaacaaac c 21 <210> 27 <211> 21 <212> DNA
<213> Homo sapiens <400> 27 cctctggatg actatgtgaa t 21 <210> 28 <211> 21 <212> DNA
<213> Homo sapiens <400> 28 acttggctgt tggttgtatg g 21 <210> 29 <211> 40 <212> DNA
<213> Homo Sapiens <400> 29 cctaggggat ccggaggttg caagactggg aatggaaaga 40 <210> 30 <211> 33 <212> DNA
<213> Homo sapiens <400> 30 tcctccggta ccacactcaa gaatgtcgca gta 33 <210> 31 <211> 5324 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413)...(1465) <400>

gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc 60 ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc 120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc 180 tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcgga 240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa 300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta 360 aaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaacc atg ggg 418 Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag cat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys His Arg Trp atc ttg gtc cct agg gga tcc gga ggt tgc aag act ggg aat gga aag 562 Ile Leu Val Pro Arg Gly Ser Gly Gly Cys Lys Thr Gly Asn Gly Lys aac tac aga ggg acg atg tcc aaa aca aaa aat ggc atc acc tgt caa 610 Asn Tyr Arg Gly Thr Met Ser Lys Thr Lys Asn Gly Ile Thr Cys Gln aaa tgg agt tcc act tct ccc cac aga cct aga ttc tca cct get aca 658 Lys Trp Ser Ser Thr Ser Pro His Arg Pro Arg Phe Ser Pro Ala Thr cac ccc tca gag gga ctg gag gag aac tac tgc agg aat cca gac aac 706 His Pro Ser Glu Gly Leu Glu Glu Asn Tyr Cys Arg Asn Pro Asp Asn gat ccg cag ggg ccc tgg tgc tat act act gat cca gaa aag aga tat 754 Asp Pro Gln Gly Pro Trp Cys Tyr Thr Thr Asp Pro Glu Lys Arg Tyr gac tac tgc gac att ctt gag tgt ggt acc gga gga ggc ggt tca ggt 802 Asp Tyr Cys Asp Ile Leu Glu Cys Gly Thr Gly Gly Gly Gly Ser Gly ggt gga ggt tca gga ggt gga ggt tct ccc ggg act aca aat act gtg 850 Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Gly Thr Thr Asn Thr Val gca gca tat aat tta act tgg aaa tca act aat ttc aag aca att ttg 898 Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn Phe Lys Thr Ile Leu gag tgg gaa ccc aaa ccc gtc aat caa gtc tac act gtt caa ata agc 946 Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr Thr Val Gln Ile Ser act aag tca gga gat tgg aaa agc aaa tgc ttt tac aca aca gac aca 994 Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys Phe Tyr Thr Thr Asp Thr gag tgt gac ctc acc gac gag att gtg aag gat gtg aag cag acg tac 1042 Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp Val Lys Gln Thr Tyr ttg gca cgg gtc ttc tcc tac ccg gca ggg aat gtg gag agc acc ggt 1090 Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Asn Val Glu Ser Thr Gly tct get ggg gag cct ctg tat gag aac tcc cca gag ttc aca cct tac 1138 Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro Glu Phe Thr Pro Tyr ctg gag aca aac ctc gga cag cca aca att cag agt ttt gaa cag gtg 1186 Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln Ser Phe Glu Gln Val gga aca aaa gtg aat gtg acc gta gaa gat gaa cgg act tta gtc aga 1234 Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu Arg Thr Leu Val Arg agg aac aac act ttc cta agc ctc cgg gat gtt ttt ggc aag gac tta 1282 Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp Val Phe Gly Lys Asp Leu att tat aca ctt tat tat tgg aaa tct tca agt tca gga aaa aaa aaa 1330 Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser Ser Gly Lys Lys Lys gcc aaa aca aac act aat gag ttt ttg att gat gtg gat aaa gga gaa 1378 Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp Val Asp Lys Gly Glu aac tac tgt ttc agt gtt caa gca gtg att ccc tcc cga aca gtt aac 1426 Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro Ser Arg Thr Val Asn cgg aag agt aca gac agc ccg gta gag tgt atg ggc acg tgaaagcttg 1475 Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met Gly Thr gctgttttggcggatgagagaagattttcagcctgatacagattaaatcagaacgcagaa1535 gcggtctgataaaacagaatttgcctggcggcagtagcgcggtggtcccacctgacccca1595 tgccgaactcagaagtgaaacgccgtagcgccgatggtagtgtggggtctccccatgcga1655 gagtagggaactgccaggcatcaaataaaacgaaaggctcagtcgaaagactgggccttt1715 cgttttatctgttgtttgtcggtgaacgctctcctgagtaggacaaatccgccgggagcg1775 gatttgaacgttgcgaagcaacggcccggagggtggcgggcaggacgcccgccataaact1835 gccaggcatcaaattaagcagaaggccatcetgacggatggcctttttgcgtttctacaa1895 actctttttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataac1955 cctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtg2015 tcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc2075 tggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactgg2135 atctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatga2195 gcacttttaaagttctgctatgtggcgcggtattatcccgtgttgacgccgggcaagagc2255 aactcggtcgccgcatacactattctcagaatgacttggttgaggcctcaccagtcacag2315 aaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatga2375 gtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccg2435 cttttttgcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctga2495 atgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgt2555 tgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagact2615 ggatggaggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggt2675 ttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactgg2735 ggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaacta2795 tggatgaacgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaac2855 tgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaattta2915 aaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagt2975 tttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctt3035 tttttctgcgcgtaatctgctgcttgcasacaaaaaaaccaccgctaccagcggtggttt3095 gtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgc3155 agataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctg3215 tagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcg3275 ataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggt3335 cgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaac3395 tgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcgg3455 acaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggg3515 gaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgat3575 ttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttt3635 tacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctg3695 attctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaa3755 cgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcctgatgcggtattttc3815 tccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgct3875 ctgatgccgcatagttaagccagtatacactccgctatcgctacgtgactgggtcatggc3935 tgcgccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggc3995 atccgcttacagacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcacc4055 gtcatcaccgaaacgcgcgaggcagcagatcaattcgcgcgcgaaggcgaagcggcatgc4115 atttacgttgacaccatcgaatggtgcaaaacctttcgcggtatggcatgatagcgcccg4175 gaagagagtcaattcagggtggtgaatgtgaaaccagtaacgttatacgatgtcgcagag4235 tatgccggtgtctcttatcagaccgtttcccgcgtggtgaaccaggccagccacgtttct4295 gcgaaaacgcgggaaaaagbggaagcggcgatggcggagctgaattacattcccaaccgc4355 gtggcacaacaactggcgggcaaacagtcgttgetgattggcgttgccacctccagtctg4415 gccctgcacgcgccgtcgcaaattgtcgcggcgattaaatctcgcgcagatcaactgggt4475 gccagcgtggtggtgtcgatggtagaacgaagcggcgtcgaagcctgtaaagcggcggtg4535 cacaatcttctcgcgcaacgcgtcagtgggctgatcattaactatccgctggatgaccag4595 gatgccattgctgtggaagctgcctgcactaatgttccggcgttatttcttgatgtctct4655 gaccagacacccatcaacagtattattttctcccatgaagacggtacgcgactgggcgtg4715 gagcatctggtcgcattgggtcaccagcaaatcgcgctgttagcgggcccattaagttct4775 gtctcggcgcgtctgcgtctggctggctggcataaatatctcactcgcaatcaaattcag4835 ccgatagcggaacgggaaggcgactggagtgccatgtccggttttcaacaaaccatgcaa4895 atgctgaatgagggcatcgttcccactgcgatgctggttgccaacgatcagatggcgctg4955 ggcgcaatgcgcgccattaccgagtccgggctgcgcgttggtgcggatatctcggtagtg5015 ggatacgacgataccgaagacagctcatgttatatcccgccgtcaaccaccatcaaacag5075 gattttcgcctgctggggcaaaccagcgtggaccgcttgctgcaactctctcagggccag5135 gcggtgaagggcaatcagctgttgcccgtctcactggtgaaaagaaaaaccaccctggcg5195 cccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacga5255 caggtttcccgactggaaagcgggcagtgagcgcaacgcaattaatgtgagttagcgcga5315 attgatctg 5324 <210> 32 <211> 27 <212> DNA
<213> Homo sapiens <400> 32 cacacaggat ccgaagaaga ctgtatg 27 <210> 33 <211> 27 <212> DNA
<213> Homo sapiens <400> 33 cacacaggta cctgaagggg ccgcaca 27 <210> 34 <211> 5342 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413)...(1483) <400>

gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc60 ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc180 tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcgga240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta360 aaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaacc atg 418 ggg Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp Arg Trp atc ttg gtc cct agg gga tcc gaa gaa gac tgt atg ttt. ggg aat ggg 562 Ile Leu Val Pro Arg Gly Ser Glu Glu Asp Cys Met Phe Gly Asn Gly aaa gga tac cga ggc aag agg gcg acc act gtt act ggg acg cca tgc 610 Lys Gly Tyr Arg Gly Lys Arg Ala Thr Thr Val Thr Gly Thr Pro Cys cag gac tgg get gcc cag gag ccc cat aga cac agc att ttc act cca 658 Gln Asp Trp Ala Ala Gln Glu Pro His Arg His Ser Ile Phe Thr Pro gag aca aat cca cgg gcg ggt ctg gaa aaa aat tac tgc cgt aac cct 706 Glu Thr Asn Pro Arg Ala Gly Leu Glu Lys Asn Tyr Cys Arg Asn Pro gat ggt gat gta ggt ggt ccc tgg tgc tac acg aca aat cca aga aaa 754 Asp Gly Asp Val Gly Gly Pro Trp Cys Tyr Thr Thr Asn Pro Arg Lys ctt tac gac tac tgt gat gtc cct cag tgt gcg gcc cct tca ggt acc 802 Leu Tyr Asp Tyr Cys Asp Val Pro Gln Cys Ala Ala Pro Ser Gly Thr gga gga ggc ggt tca ggt ggt gga ggt tca gga ggt gga ggt tct cec 850 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro ggg act aca aat act gtg gca gca tat aat tta act tgg aaa tca act 898 Gly Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr aat ttc sag aca att ttg gag tgg gaa ccc aaa ccc gtc aat caa gtc 946 Asn Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val tac act gtt caa ata agc act aag tca gga gat tgg aaa agc aaa tgc 994 Tyr Thr Val Gln Ile Ser Thr Lys Ser Gly Asp Trp Lys Ser Lys Cys ttt tac aca aca gac aca gag tgt gac ctc acc gac gag att gtg aag 1042 Phe Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys gat gtg aag cag acg tac ttg gca cgg gtc ttc tcc tac ccg gca ggg 1090 Asp Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly aat gtg gag agc acc ggt tct get ggg gag cct etg tat gag aac tcc 1138 Asn Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser cca gag ttc aca cct tac ctg gag aca aac ctc gga cag cca aca att 1186 Pro Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile cag agt ttt gaa cag gtg gga aca aaa gtg aat gtg acc gta gaa gat 1234 Gln Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp gaa cgg act tta gtc aga agg aac aac act ttc cta agc ctc cgg gat 1282 Glu Arg Thr Leu Val Arg Arg Asn Asn Thr Phe Leu Ser Leu Arg Asp gtt ttt ggc aag gac tta att tat aca ctt tat tat tgg aaa tct tca 1330 Val Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser agt tca gga aaa aaa aca gcc aaa aca aac act aat gag ttt ttg att 1378 Ser Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile gat gtg gat aaa gga gaa aac tac tgt ttc agt gtt caa gca gtg att 1426 Asp Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile ccc tcc cga aca gtt aac cgg aag agt aca gac agc ccg gta gag tgt 1474 Pro Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys atg ggc acg tgaaagcttg gctgttttgg cggatgagag aagattttca 1523 Met Gly Thr gcctgatacagattaaatcagaacgcagaagcggtctgataaaacagaatttgcctggcg1583 gcagtagcgcggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcg1643 ccgatggtagtgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaa1703 cgaaaggctcagtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgct1763 ctcctgagtaggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccgga1823 gggtggcgggcaggacgcccgccataaactgccaggcatcaaattaagcagaaggccatc1883 ctgacggatggcctttttgcgtttctacaaactctttttgtttatttttctaaatacatt1943 caaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaa2003 ggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcatttt2063 gccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagt2123 tgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtt2183 ttcgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcgg2243 tattatcccgtgttgacgccgggcaagagcaactcggtcgccgcatacactattctcaga2303 atgacttggttgaggcctcaccagtcacagaaaagcatcttacggatggcatgacagtaa2363 gagaattatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctga2423 caacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaa2483 ctcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgaca2543 ccacgatgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactactta2603 ctctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccac2663 ttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagc2723 gtgggtctcgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtag2783 ttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgaga2843 taggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatacttt2903 agattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttttgata2963 atctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtag3023 aaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaa3083 WO 99/32143 PCT/US9&Z'1498 caaaaaaacc accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt 3143 ttccgaaggt aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc 3203 cgtagttagg ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa 3263 tcctgttacc agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa 3323 gacgatagtt accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc 3383 ccagcttgga gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa 3443 gcgccacgct tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa 3503 caggagagcg cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg 3563 ggtttcgcca cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc 3623 tatggaaaaa cgccagcaac gcggcctttt tacggttcct ggccttttgc ~tggccttttg 3683 ctcacatgtt ctttcctgcg ttatcccctg attctgtgga taaccgtatt accgcctttg 3743 agtgagctga taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg 3803 aagcggaaga gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 3863 gcatatggtg cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac 3923 tccgctatcg ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga 3983 cgcgccctga cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc 4043 cgggagctgc atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat 4103 caattcgcgc gcgaaggcga agcggcatgc atttacgttg acaccatcga atggtgcaaa 4163 acctttcgcg gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg 4223 aaaccagtaa cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc 4283 cgcgtggtga accaggccag ccacgtttct gcgaaaacgc gggaaaaagt ggaagcggcg 4343 atggcggagc tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg 4403 ttgctgattg gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg 4463 gcgattaaat ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga 4523 agcggcgtcg asgcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg 4583 ctgatcatta actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact 4643 aatgttccgg cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc 4703 tcccatgaag acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa 4763 atcgcgctgt tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg 4823 cataaatatc tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt 4883 gccatgtccg gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg 4943 atgctggttg ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg 5003 ctgcgcgttg gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt 5063 tatatcccgc cgtcaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg 5123 gaccgcttgc tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc 5183 tcactggtga aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg 5243 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 5303 gcgcaacgca attaatgtga gttagcgcga attgatctg 5342 <210> 35 <211> 28 <212> DNA
<213> Homo sapiens <400> 35 gatccccgcg taaactgtac gacggtac 28 <210> 36 <211> 20 <212> DNA
<213> Homo sapiens <400> 36 cgtcgtacag tttacgcggg 20 WO 99/32143 PCTNS98/Z749$
<210> 37 <211> 5099 <212> DNA
<213> Homo sapiens <220>
<221> CDS
<222> (413) . .. (1240) <400>

gtttgacagcttatcatcgactgcacggtgcaccaatgcttctggcgtcaggcagccatc 60 ggaagctgtggtatggctgtgcaggtcgtaaatcactgcataattcgtgtcgctcaaggc 120 gcactcccgttctggataatgttttttgcgccgacatcataacggttctggcaaatattc 180 tgaaatgagctgttgacaattaatcatccggctcgtataatgtgtggaattgtgagcgga 240 taacaatttcacacaggaaacagcgccgctgagaaaaagcgaagcggcactgctctttaa 300 caatttatcagacaatctgtgtgggcactcgaccggaattatcgattaactttattatta 360 aaaattaaagaggtatatattaatgtatcgattaaataaggaggaataaacc atg ggg 418 Met Gly ggt tct cat cat cat cat cat cat ggt atg get agc atg act ggt gga 466 Gly Ser His His His His His His Gly Met Ala Ser Met Thr Gly Gly cag caa atg ggt cgg gat ctg tac gac gat gac gat aag gat cga tgg 514 Gln Gln Met Gly Arg Asp Leu Tyr Asp Asp Asp Asp Lys Asp Arg Trp atc ttg gtc cct agg gga tcc ccg cgt aaa ctg tac gac ggt acc gga 562 Ile Leu Val Pro Arg Gly Ser Pro Arg Lys Leu Tyr Asp Gly Thr Gly gga ggc ggt tca ggt ggt gga ggt tca gga ggt gga ggt tct ccc ggg 610 Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Pro Gly act aca aat act gtg gca gca tat aat tta act tgg aaa tca act aat 658 Thr Thr Asn Thr Val Ala Ala Tyr Asn Leu Thr Trp Lys Ser Thr Asn ttc aag aca att ttg gag tgg gaa ccc aaa ccc gtc aat caa gtc tac 706 Phe Lys Thr Ile Leu Glu Trp Glu Pro Lys Pro Val Asn Gln Val Tyr 85 . 90 95 act gtt caa ata agc act aag tca gga gat tgg aaa agc aaa tgc ttt 754 Thr Val Gln Ile Ser Thr Lye Ser Gly Asp Trp Lys Ser Lys Cys Phe tac aca aca gac aca gag tgt gac ctc acc gac gag att gtg aag gat 802 Tyr Thr Thr Asp Thr Glu Cys Asp Leu Thr Asp Glu Ile Val Lys Asp gtg aag cag acg tac ttg gca cgg gtc ttc tcc tac ccg gca ggg aat 850 Val Lys Gln Thr Tyr Leu Ala Arg Val Phe Ser Tyr Pro Ala Gly Aen wo 99r~im rcTius9s~~49s gtg gag agc acc ggt tct get ggg gag cet ctg tat gag aac tcc cca 898 Val Glu Ser Thr Gly Ser Ala Gly Glu Pro Leu Tyr Glu Asn Ser Pro gag ttc aca cct tac ctg gag aca aac ctc gga cag cca aca att cag 946 Glu Phe Thr Pro Tyr Leu Glu Thr Asn Leu Gly Gln Pro Thr Ile Gln agt ttt gaa cag gtg gga aca aaa gtg aat gtg acc gta gaa gat gaa 994 Ser Phe Glu Gln Val Gly Thr Lys Val Asn Val Thr Val Glu Asp Glu cgg act tta gtc aga agg aac aac act ttc cta agc ctc cgg gat gtt 1042 Arg Thr Leu Val Arg Arg Asn Aan Thr Phe Leu Ser Leu Arg Asp Val ttt ggc aag gac tta att tat aca ctt tat tat tgg aaa tct tca agt 1090 Phe Gly Lys Asp Leu Ile Tyr Thr Leu Tyr Tyr Trp Lys Ser Ser Ser tca gga aaa aaa aca gcc aaa aca aac act sat gag ttt ttg att gat 1138 Ser Gly Lys Lys Thr Ala Lys Thr Asn Thr Asn Glu Phe Leu Ile Asp gtg gat aaa gga gaa aac tac tgt ttc agt gtt caa gca gtg att ccc 1186 Val Asp Lys Gly Glu Asn Tyr Cys Phe Ser Val Gln Ala Val Ile Pro tcc cga aca gtt aac cgg aag agt aca gac agc ccg gta gag tgt atg 1234 Ser Arg Thr Val Asn Arg Lys Ser Thr Asp Ser Pro Val Glu Cys Met ggc acg tgaaagcttg gctgttttgg cggatgagag aagattttca gcctgataca 1290 Gly Thr gattasatcagaacgcagaagcggtctgataaaacagaatttgcctggcggcagtagcgc1350 ggtggtcccacctgaccccatgccgaactcagaagtgaaacgccgtagcgccgatggtag1410 tgtggggtctccccatgcgagagtagggaactgccaggcatcaaataaaacgaaaggctc1470 agtcgaaagactgggcctttcgttttatctgttgtttgtcggtgaacgctctcctgagta1530 ggacaaatccgccgggagcggatttgaacgttgcgaagcaacggcccggagggtggcggg1590 caggacgcccgccataaactgccaggcatcaaattaagcagaaggccatcctgacggatg1650 gcctttttgcgtttctacaaactctttttgtttatttttctaaatacattcaaatatgta1710 tccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtat1770 gagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgt1830 ttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgcacg1890 agtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccga1950 agaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccg2010 tgttgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggt2070 tgaggcctcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatg2130 cagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcgg2190 aggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttga2250 tcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcc2310 tgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttc2370 ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 2430 ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 2490 cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 2550 gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 2610 actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 2670 aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 2730 caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 2790 aggatcttct tgagatcctt tttttctgcg cgtaatctgc tgcttgcaaa caaaaaaacc 2850 accgctacca gcggtggttt gtttgccgga tcaagagcta ccaactcttt ttccgaaggt 2910 aactggcttc agcagagcgc agataccaaa tactgtcctt ctagtgtagc cgtagttagg 2970 ccaccacttc aagaactctg tagcaccgcc tacatacctc gctctgctaa tcctgttacc 3030 agtggctgct gccagtggcg ataagtcgtg tcttaccggg ttggactcaa gacgatagtt 3090 accggataag gcgcagcggt cgggctgaac ggggggttcg tgcacacagc ccagcttgga 3150 gcgaacgacc tacaccgaac tgagatacct acagcgtgag ctatgagaaa gcgccacgct 3210 tcccgaaggg agaaaggcgg acaggtatcc ggtaagcggc agggtcggaa caggagagcg 3270 cacgagggag cttccagggg gaaacgcctg gtatctttat agtcctgtcg ggtttcgcca 3330 cctctgactt gagcgtcgat ttttgtgatg ctcgtcaggg gggcggagcc tatggaaaaa 3390 cgccagcaac gcggcctttt tacggttcct ggccttttgc tggccttttg ctcacatgtt 3450 ctttcctgcg ttatcccctg attctgtgga taaccgtatt acegcctttg agtgagctga 3510 taccgctcgc cgcagccgaa cgaccgagcg cagcgagtca gtgagcgagg aagcggaaga 3570 gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc gcatatggtg 3630 cactctcagt acaatctgct ctgatgccgc atagttaagc cagtatacac tccgctatcg 3690 ctacgtgact gggtcatggc tgcgccccga cacccgccaa cacccgctga cgcgccctga 3750 cgggcttgtc tgctcccggc atccgcttac agacaagctg tgaccgtctc cgggagctgc 3810 atgtgtcaga ggttttcacc gtcatcaccg aaacgcgcga ggcagcagat caattcgcgc 3870 gcgaaggcga agcggcatgc atttacgttg acaccatcga atggtgcaaa acctttcgcg 3930 gtatggcatg atagcgcccg gaagagagtc aattcagggt ggtgaatgtg aaaccagtaa 3990 cgttatacga tgtcgcagag tatgccggtg tctcttatca gaccgtttcc cgcgtggtga 4050 accaggccag ccacgtttet gcgaaaacgc gggaaaaagt ggaagcggcg atggcggagc 4110 tgaattacat tcccaaccgc gtggcacaac aactggcggg caaacagtcg ttgctgattg 4170 gcgttgccac ctccagtctg gccctgcacg cgccgtcgca aattgtcgcg gcgattaaat 4230 ctcgcgccga tcaactgggt gccagcgtgg tggtgtcgat ggtagaacga agcggcgtcg 4290 aagcctgtaa agcggcggtg cacaatcttc tcgcgcaacg cgtcagtggg ctgatcatta 4350 actatccgct ggatgaccag gatgccattg ctgtggaagc tgcctgcact aatgttccgg 4410 cgttatttct tgatgtctct gaccagacac ccatcaacag tattattttc tcccatgaag 4470 acggtacgcg actgggcgtg gagcatctgg tcgcattggg tcaccagcaa atcgcgctgt 4530 tagcgggccc attaagttct gtctcggcgc gtctgcgtct ggctggctgg cataaatatc 4590 tcactcgcaa tcaaattcag ccgatagcgg aacgggaagg cgactggagt gccatgtccg 4650 gttttcaaca aaccatgcaa atgctgaatg agggcatcgt tcccactgcg atgctggttg 4710 ccaacgatca gatggcgctg ggcgcaatgc gcgccattac cgagtccggg ctgcgcgttg 4770 gtgcggatat ctcggtagtg ggatacgacg ataccgaaga cagctcatgt tatatcccgc 4830 cgtcaaccac catcaaacag gattttcgcc tgctggggca aaccagcgtg gaccgcttgc 4890 tgcaactctc tcagggccag gcggtgaagg gcaatcagct gttgcccgtc tcactggtga 4950 aaagaaaaac caccctggcg cccaatacgc aaaccgcctc tccccgcgcg ttggccgatt 5010 cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga gcgcaacgca 5070 attaatgtga gttagcgcga attgatctg 5099

Claims

That which is claimed is:

1. A context-dependent functional entity comprising a substructure with thrombogenic potential and one or more context-enhancing substructure(s) having the ability to recognize desired biologically susceptible site(s), wherein said entity imparts thrombogenic activity when positioned in the function-forming-context at said biologically susceptible site(s), and wherein said entity has substantially no thrombogenic activity absent a function-forming-context at said biologically susceptible site(s).

2. A context-dependent functional entity according to claim 1, wherein said entity transiently imparts activity when positioned in a function forming-context at the biologically susceptible site.

3. A context-dependent functional entity according to claim 2, wherein said substructure with thrombogenic potential comprises a coagulation factor.

4. A context-dependent functional entity according to claim 3, wherein said clotting factor is modified or wild-type TF.

5. A context-dependent functional entity according to claim 4, wherein said TF
is derived from a human.

6. A context-dependent functional entity according to claim 5, wherein said modified TF has substantially the same amino acid(s) as set forth in sequence Nos.
35-243 of SEQ ID NO:1.

7. A context-dependent functional entity according to claim 2, wherein said modified TF is modified to increase thrombogen activity.

8. A context-dependent functional entity according to claim 6, wherein the amino acid at position 199 of SEQ ID NO:1 is a basic amino acid.

9. A context-dependent functional entity according to claim 2, wherein said context-enhancing substructure has a functional preference for vascular structures, specific cells or tissue types.

10. A context-dependent functional entity according to claim 9, wherein said context-enhancing substructure has a functional preference for tumor-associated vascular endothelial cells.

11. A context-dependent functional entity according to claim 10, wherein said context-enhancing substructure orients said entity on the cell surface of said tumor-associated vascular endothelial cells.

12. A context-dependent functional entity according to claim 11, wherein said context-enhancing substructure comprises a cell surface recognition domain.

13. A context-dependent functional entity according to claim 12, wherein said context-enhancing substructure comprises a cell surface recognition domain derived from an annexin.

14. A context-dependent functional entity according to claim 12, wherein said context-enhancing substructure comprises a protease inhibitor.

15. A context-dependent functional entity according to claim 12, wherein said context-enhancing substructure comprises a charged phospholipid-associating element.

16. A context-dependent functional entity according to claim 12, wherein said context-enhancing substructure comprises a kringle domain.

17. A context-dependent functional entity according to claim 16, wherein said kringle domain is obtained from protein selected from the group consisting of plasminogen, apolipoprotein(a), hepatocyte growth factor, urokinase, coagulation factor XIII, haptoglobin, tissue plasminogen activator (tPA) and prothrombin.

18. A context-dependent functional entity according to claim 1, wherein said context-enhancing substructure is located at the carboxy terminus of said TF, the amino terminus of said TF, between the amino terminus and the carboxy terminus of said TF or inserted in a hydrophilic surface loop of said TF.

19. A context-dependent functional entity according to claim 1, wherein said context-dependent functional entity comprises two or more context-enhancing substructures and wherein said context-enhancing substructures are located at the carboxy terminus of said TF, the amino terminus of said TF, between the amino terminus and the carboxy terminus of said TF, inserted in a hydrophilic surface loop of said TF, or any combinations thereof.

20. A context-dependent functional entity according to claim 1, wherein said entity further comprises a cloning cassette.

21. A context-dependent functional entity according to claim 20, wherein said cloning cassette further facilitates orientation of said context-dependent functional entity on said biologically susceptible site(s).

22. A context-dependent functional entity according to claim 1, wherein said cloning cassette is selected from all or part of a lectin, hormone or ligand for specific receptors on said tumor cells.

23. A context-dependent functional entity according to claim 1, wherein said entity further comprises an activity-modulating substructure.

24. A context-dependent functional entity according to claim 23, wherein said activity-modulating substructure is selected from a spacer substructure or a protease site.

25. A context-dependent functional entity according to claim 24, wherein said spacer substructure increases degradation of said entity.

26. A context-dependent functional entity according to claim 25, wherein said spacer substructure comprises homo or hetero bifunctional crosslinking agents or chitin oligomers.

27. A context-dependent functional entity according to claim 24, wherein said spacer substructure comprises a ((Gly)4Ser)n module(s) or ((Ser)4 Gly)n modules) which spaces the context-enhancing substructure and the modified TF.

28. A context-dependent functional entity according to claim 1, wherein said entity further comprises a production substructure.

29. A context-dependent functional entity according to claim 28, wherein said production substructure is selected from a His-tag, a restriction site, vector, or cys residue.

30. A context-dependent functional entity according to claim 1, wherein said context-dependent functional entity comprises an amino acid sequence substantially as set forth in SEQ ID NOs 6, 12, 15, 24, 31, 34 or 37.

31. A composition comprising a context-dependent functional entity according to claim 1 and coagulation factor VIIa.

32. A nucleic acid construct encoding a context-dependent functional entity according to claim 1.

33. A nucleic acid construct encoding a context-dependent functional entity according to claim 1, wherein said context-dependent functional entity is substantially encoded by the nucleic acid sequence set forth in SEQ ID NOs 6, 12, 15, 24, 31, 34 or 37.

34. An in vivo method to selectively thrombose the vasculature of solid tumors in a subject in need thereon said method comprising administering to said subject an effective amount of a context-dependent functional entity according to claim 1.

35. A method according to claim 34 wherein said association-dependent functional entity is supplied indirectly by administering a nucleic acid segment encoding same to said subject.

36. A method to obliterate vasculature malformations, said method comprising administering to said subject an effective amount of a context-dependent functional entity according to claim 1.

37. An assembly-dependent functional complex comprising a substucture with thrombogenic potential and one or more association-enhancing substructure(s) having the ability to assemble said complex at desired biologically susceptible site(s), wherein said complex imparts thrombogenic activity when positioned in the function-forming-context at said biologically susceptible site(s), and wherein said complex has substantially no thrombogenic activity absent a function-foaming-context at said biologically susceptible site(s).

38. An assembly-dependent functional complex according to claim 37, wherein said complex is transiently activated upon when positioned in a function-forming-context at the biologically susceptible site.

39. An assembly-dependent functional complex according to claim 38, wherein said substructure with thrombogenic potential comprises a coagulation factor.

40. An assembly-dependent functional complex according to claim 38, wherein said association-enhancing substructure assembles said complex in a function-forming context