WO1999032143A1 - Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium - Google Patents
Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium Download PDFInfo
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- WO1999032143A1 WO1999032143A1 PCT/US1998/027498 US9827498W WO9932143A1 WO 1999032143 A1 WO1999032143 A1 WO 1999032143A1 US 9827498 W US9827498 W US 9827498W WO 9932143 A1 WO9932143 A1 WO 9932143A1
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6424—Serine endopeptidases (3.4.21)
- C12N9/6435—Plasmin (3.4.21.7), i.e. fibrinolysin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
- A61K47/64—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent
- A61K47/6425—Drug-peptide, drug-protein or drug-polyamino acid conjugates, i.e. the modifying agent being a peptide, protein or polyamino acid which is covalently bonded or complexed to a therapeutically active agent the peptide or protein in the drug conjugate being a receptor, e.g. CD4, a cell surface antigen, i.e. not a peptide ligand targeting the antigen, or a cell surface determinant, i.e. a part of the surface of a cell
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K49/00—Preparations for testing in vivo
- A61K49/0004—Screening or testing of compounds for diagnosis of disorders, assessment of conditions, e.g. renal clearance, gastric emptying, testing for diabetes, allergy, rheuma, pancreas functions
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/745—Blood coagulation or fibrinolysis factors
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/21—Serine endopeptidases (3.4.21)
- C12Y304/21007—Plasmin (3.4.21.7), i.e. fibrinolysin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
Definitions
- the present invention relates to a novel strategy for the treatment of carcinomas and other solid tumors.
- the present invention relates to methods which modulate the function of endothelial cells associated with the tumor vasculature, and compounds useful therefor.
- Solid tumors make up more than 90% of all human cancers. Yet, the delivery of 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 drag into the tumor, high concentrations must be used and these produce unacceptable toxicity to the normal cells - side effects.
- 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.
- tumor cells can become resistant to many conventional drugs, even to the extent of developing pumps, such as glycoprotein gpl70, to remove drugs from the cell.
- pumps such as glycoprotein gpl70
- Solid tumors require a continuous supply of nutrients supplied by the continual formation of new blood vessels that are derived from older blood vessels.
- 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 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.
- tumors are known to be procoagulant - patients with cancer typically show evidence of hypercoagulability and may even develop thromboembolic disease.
- a novel strategy has been devised that 15 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.
- a method of attacking solid tumors has been developed which employs compounds which modulate the functions of tumor- 20 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.
- Figure 1 depicts the process of chemically conjugating a context-enhancing substructure and a cysteine-containing spacer substructure-TF.
- Figure 2 depicts the process of employing a thioether production substructure to produce a context-dependent functional entity.
- Figure 3 a presents a graph illustrating the effect on plasma half-life by antibody against Tissue Factor (TF).
- Figure 3b presents a graph comparing Tissue Factor half-life with Selective Tumor Vasculature Thrombogen (STVT) half-life.
- Figure 4 presents a graph illustrating the effect of differing concentrations of
- NV124 protein expressed from the E. coli expression plasmid NuV124, see Example 7) on tumor growth.
- Figure 5 depicts a graph illustrating the improved pharmacological effect of multiple infusions of NV 124 on tumor growth.
- Figure 6 graphs the effects of NV129 (protein expressed from the E. coli expression plasmid NuV129, see Example 11) infusions on C1300 tumor growth.
- Figures 7 and 8 provide graphs illustrating the effects of NV144 (protein expressed from the E. coli expression plasmid NuV144, see Example 12) on C1300 tumor growth in comparison with the effects of saline on tumor growth.
- NV144 protein expressed from the E. coli expression plasmid NuV144, see Example 12
- 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 context-enhancing substructures having the ability to recognize (e.g., possessing functional complementarity) desired biologically susceptible site(s).
- Context-dependent functional entities are characterized as imparting thrombogenic activity when positioned (e.g., functionally complemented) in the function-forming-context at the biologically susceptible site(s), while having substantially no thrombogenic activity absent a function-forming-context at the biologically susceptible site(s).
- the context-dependent functional entity transiently imparts activity upon formation of a transient function-forming-context at the biologically susceptible site(s).
- substructure with thrombogenic potential refers to one or more thrombosis promoting peptidyl, oligopeptidyl, protein or small organic molecule (e.g., medicinal compounds), that has the ability to selectively impart thrombogenic activity when positioned (functionally complemented) in a function-forming-context at a biologically susceptible site(s).
- thrombogenic potential refers to the ability of such substructures to selectively impart thrombogenic activity when localized and oriented in a complementary function-forming-context at a biologically susceptible site(s) so as to result in thrombogenic activity.
- substructures with thrombogenic potential include one or more domains or modules of coagulation factors.
- coagulation factors include fibrinogen, prothrombin, tissue factor (TF), factor V, factor VII through factor XIII (in addition to their activated states), von WiUebrand 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., granzymes), and the like.
- Preferred coagulation factors include those involved in the coagulation promoting pathways including TF, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, activated states of such factors, combinations of co-factors (i.e., TF:factor Vll/VIIa, factors VIIIa:IXa, factors Va:Xa), and the like.
- thrombogenic activity refers to the selective initiation, promotion, activation and/or propagation of occlusive thrombosis (either partial or complete, transient or prolonged) at biologically susceptible site(s).
- a preferred thrombogenic activity includes the function of a coagulation factor to activate 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 Vila, factor IX to factor IXa, factor X to factor Xa, prothrombin to thrombin, and the like.
- 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 susceptible site(s).
- Occlusive thrombosis would result in therapeutic activation of coagulation on biologically susceptible site(s) (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, unaffected vessel (Danekamp et al. (1984) Prog Appl Microcir 4:28-38).
- biologically susceptible site(s) i.e., selected endothelial cells
- a preferred substructure with thrombogenic potential is modified or wild-type tissue factor (TF), preferably of human derivation.
- modified or wild-type tissue factor (TF) 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.
- the modified or wild-type TF retains the capacity to induce factor Vll/VIIa-dependent coagulation.
- 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.
- 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:l) which anchors native TF to the cell membrane.
- the TF comprises substantially the ammo-terminal amino acids up to approximately residue 252 of SEQ ID NO:l.
- 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:l).
- the modified TF is modified or further modified to increase thrombogenic activity when placed or oriented in the fiinction-forming-context at a biologically susceptible site(s).
- modifications include substituting the amino acid residue at one or more positions, e.g., TF 167 or position 199 of SEQ ID NO:l, as well as residues within 15 Angstrom of TF 167 or residue 199 of SEQ ID NO:l, with a basic amino acid such as lysine, arginine, histidine, and the like.
- TF protein, or functional fragments 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 Enzymology Vol. 182, (Academic Press, 1990), which is incorporated herein by reference.
- TF, and biologically active fragments thereof, useful in the practice of the present invention can also be produced by chemical synthesis.
- Synthetic polypeptides can be produced, for example, using Applied Biosystems, Inc. Model 430A or 431 A automatic polypeptide synthesizer and chemistry provided by the manufacturer.
- TF, and biologically active fragments thereof can also be isolated directly from cells which have been transformed with the expression vectors described below in more detail.
- TF, or functional fragment thereof, useful in the practice of the present invention 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.
- nucleic acid encoding TF or functional fragment thereof
- 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.
- 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.
- one embodiment of the present invention includes nucleic acid consisting essentially of the nucleic acid sequence as set forth in SEQ ID NO:l 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.
- nucleic acid encoding the soluble form of TF is contemplated, in a preferred 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.
- the phrase “context-enhancing substructure” 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 biologically susceptible site(s).
- 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).
- the context-enhancing substructure will have a low affinity for the desired biologically susceptible site(s) so as to transiently activate 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).
- the function of the context-enhancing substructure is to provide transient orientation and transient localization of the thrombogenic substructure 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 thrombogenic potential of the context-dependent functional entity), and the like.
- Specific examples of context-enhancing substructures include uPAR binding antagonists (e.g., peptide/clone 20 (1994) Goodson et al.
- RN4S 91:7129-7133 anti-angiogenic proteins ((e.g., endostatin from collagen XVIII 20kD C-terminus, (1997) O'Reilly et al. Cell 88(2):277-285; nucleotide sequences 1502 to 2053 from genbank HUMCOL18AX ACCESSION L22548), peptides derived from TSP-1 (e.g., Mallll from (1993) J Cell Biol 22:497-511 ; peptide 246, (1992) PNAS 89:3040-3044; Laminin binding peptides (e.g., Peptide G, Guo et al.
- biologically susceptible site(s) refers to one or more molecules found at the cell surface, unique to, induced or up-regulated in, vascular 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 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).
- the invention context-dependent functional entity recognizes these differences as sites in which to selectively initiate, promote, activate and/or propagate occlusive thrombosis.
- biologically susceptible sites include vascular structures, specific cells or tissue types associated with cancer and/or angiogenesis, wounds caused by trauma, 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 substructure.
- the biologically susceptible site(s) recognized by the context-dependent functional entity should preferably be molecule(s) on the cell surface of the tumor-associated endothelial cells and one that is not secreted into body fluids.
- Preferred biologically susceptible site(s) for which the context-enhancing substructure has a functional preference include tumor-associated vascular endothelial cells.
- function-forming-context refers to the necessary 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.
- the context-enhancing substructure can be positioned at the carboxy terminus of the substructure with thrombogenic potential, the amino terminus of the substructure with thrombogenic potential, or between the amino terminus and the carboxy terminus of the substructure with thrombogenic potential (i.e., inserted within a hydrophilic surface loop of the substructure with thrombogenic potential).
- the context-dependent functional entity comprises two or more context-enhancing substructures to enhance proper or desired alignment of 5 the substructure with thrombogenic potential at the biologically susceptible site(s).
- the context-enhancing substructure(s) is(are) located at the carboxy terminus of the substructure with thrombogenic potential, the amino terminus of the substructure with thrombogenic potential, between the amino terminus and the carboxy terminus of the substructure with thrombogenic potential, inserted in a hydrophilic surface loop of l o the substructure with thrombogenic potential, or any combinations thereof.
- context-dependent functional entities further comprise activity-modulating substructure(s).
- activity-modulating substructure refers to one or more molecules which 15 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).
- activity modulating substructures include spacer substructures, protease sites, and the like.
- spacer substructure refers to one or more spacer molecules that serve to link substructures with thrombogenic potential with context-enhancing substructures. The nature of the linkage between the spacer substructure and either the substructure(s) with thrombogenic potential or the
- context-enhancing substructure(s) depends on the functionality employed in the substructures with thrombogenic potential and the context-enhancing substructures.
- substructures with thrombogenic potential are linked to context-enhancing substructures in a manner that retains the ability of the context-dependent functional entity to activate the substructure with thrombogenic potential.
- spacer is typically employed in the substructures with thrombogenic potential.
- 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.
- the spacer substructure increases degradation of the context-dependent functional entity by releasing the substructure with thrombogenic potential from the context-enhancing substructure.
- 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-context with cell surfaces at the biologically susceptible site, subjecting the spacer to degradation upon exposure to a specific enzyme.
- bonds within spacer molecules include esters, peptides, amides, phosphodiesters, and even glycosidic bonds, which are hydrolysed by exposure to an esterase, protease or 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 site(s) and/or irreversibly inactivating the context-dependent functional entity, thus preventing dissemination of native thrombogens or access of additional entities to biologically susceptible site(s).
- the spacer substructure comprises homo- or hetero-bifunctional crosslinking agents or chitin oligomers.
- Exemplary spacer substructures include combinations of Gly and Ser modules, such as ((Gly) 4 Ser) n , ((Ser) 4 Gly) n , and the like.
- Additional spacer substructures contemplated 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.
- cysteine residues of such hinge regions may be substituted by glycine, alanine, or serine in any combination to eliminate difficulties presented by the presence of particular cysteine thiol groups that may disrupt spacer structure or inhibit proper formation of tertiary structure of the substructure with thrombogenic potential.
- 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).
- context-dependent functional entities further comprise production substructure(s).
- 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 substructures include restriction sites, vectors, cys residues, His-tags, and the like.
- Restriction enzyme sites can be optionally introduced to the 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 substructure(s) and/or cloning cassette) by additions, deletions, substitutions or modifications made at nucleic acid sequences encoding the amino-terminal, the carboxyl terminal and/or sequences in between to produce the function-forming context.
- Unique restriction sites may be placed for the convenience of constructing a context-dependent functional entity(ies) with different orientations and configurations, i.e., different combinations of the individual substructures.
- Unique restriction sites at the junctions of each of these 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:l, 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 substructures without restriction sites.
- restriction sites examples include Xmal (CCCGGG), BamHI (GGATCC), Kpnl (GGTACC), Hindlll (AAGCTT), Aval (CCCGGG), EcoRI (GAATTC), Avrll (CCTAGG), or Pmll (CACGTG), and the like.
- Specific preferred examples of context-dependent functional entity(ies) constructs are provided (e.g., SEQ ID NOs: 6, 12, 15, 24 and 31).
- the individual substructures can be conjugated chemically via production substructures to produce the context-dependent functional entity with the preferred function-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 substructure(s) and/or activity-modulating substructure(s) that contain a cysteine residue production substructure can be coupled to substructure(s) with thrombogenic potential through the cysteine.
- a peptide that may act as a context enhancing substructure(s) and/or activity-modulating substructure(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 a disulfide bond constrained peptide (1) are illustrated schematically in Figure 1. The preferred modification does not reduce the ability of the context enhancing substructure(s) and/or activity modulating substructure(s) to facilitate the thrombogenic activity of the final construct.
- An activity-modulating substructure containing a cysteine production substructure at or near the amino terminus, or at any site within an activity-modulating substructure can be constructed synthetically and fused to the substracture with thrombogenic potential.
- an activity-modulating substructure of the following sequence may be constructed between a Kpnl and Xmal site:
- the cysteine in this example is placed 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.
- a thrombin-sensitive sequence such as PRG must be retained in order to efficiently cleave the resulting protein with thrombin.
- Other peptides inserted between the hexahistidine sequence and the beginning of the substracture with thrombogenic potential may be used in the construct, which would necessitate the use of a different enzyme(s), known to those of skill in the art, to cleave and release the substracture with thrombogenic potential during its purification.
- the cysteine thiol of the substructure with thrombogenic substracture and the cysteine thiol of cysteine-containing activity-modulating substracture may also be linked by thioether bonds using bismaleimido groups such as that of BMH, bismaleimidohexane.
- 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 substracture with thrombogenic potential are known to those of skill in the art (see Figure 2).
- NHS N-hydroxysuccinimide
- 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 herein by reference.
- Context-dependent functional entity as well as the individual substructures that comprise the context-dependent functional entity (i.e., substracture(s) with thrombogemc potential, context enhancing substracture(s), activity-modulating substructure(s) and/or production substracture) 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 affinity chromatography, and the like. Other well-known methods are described in Deutscher et al., Guide to Protein Purification: Methods in Enzymology Vol. 182, (Academic Press, 1990), which is incorporated herein by reference.
- context-dependent functional 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 substructures, is to express nucleic acid encoding such entity and/or substracture(s) 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.
- 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.
- 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 430 A or 431 A automatic polypeptide 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 functional entity, as well as the individual substructures thereof, can also be isolated directly from cells which have been transformed with the expression vectors described below in more detail.
- noncovalent links can be established to link the individual substructures together to form an assembly-dependent functional entity.
- leucine zippers are preferably used to hold together, by noncovalent interaction, the substracture with thrombogenic potential with the context-enhancing substracture to form an entity with thrombogenic potential.
- the context-dependent functional entity is modified to reduce immunogenicity and/or modify biological half-life.
- modifications include employing polyethelene glycol (PEG), and the like.
- the context-dependent functional entity further comprises a cloning cassette.
- cloning cassette refers to one or more additional substractural elements which facilitate orientation of the context-dependent functional entity on the biologically susceptible site(s) or to add synergistic functions.
- cloning cassette(s) contemplated for inclusion into the context-dependent functional entity will be inserted into a permissive hydrophilic loop of the context-dependent functional 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.
- amino acid residues which can be replaced include amino acid residues from about 112 to about 123, preferably from about 115-123, of human TF as set forth in SEQ ID NO:l.
- 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.
- the phrase "synergistic function" refers to functions which enhance the thrombogenic function of the substructure with thrombogenic potential or enhance the function of the context-enhancing substracture to orient the context-dependent functional entity in a function-forming-context at the biologically susceptible site(s).
- cloning cassettes which can be employed in the invention entity, including cloning cassettes identified from combinatorial libraries.
- compositions comprising context-dependent functional entities in combination with coagulation factor Vila. Binding of factor Vila to TF enhances the enzymatic activation of substrate factors IX and X as much as 5,000 fold (Rao et al (1988) PNAS 85:6687).
- Factor VII can be prepared as described by Fair (1983) Blood 62:784-791.
- Recombinant Factor Vila can be purchased from Novo Biolabs (Danbury, Conn.).
- nucleic acid constructs encoding the invention context-dependent functional entity are provided.
- 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 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 site(s) including intraperitoneal, subcutaneous, intravascular, intramuscular, intranasal or intravenous injection or infusion, implant modes of administration, and the like.
- the context-dependent functional entity is supplied indirectly by administering a nucleic acid segment encoding same to the subject.
- the phrase "the vasculature of solid tumors” refers to endothelial lined vascular channels of the tumor and existing vasculature adopted by invading tumor cells.
- tumors also referred to herein as vascular tumors (tumors of the vasculature as well as vascular malformations), which are contemplated to be treated include solid tumors such as breast, prostrate, lung, liver, colon, rectal, melanoma, kidney, stomach, pancreas, ovarian, bladder, cervical, oral, uteri, brain, and the like.
- 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, drags, local hyperthermia, and the like.
- the phrase "obliterating vasculature malformations" refers to the partial or complete occlusive thrombosis of malformations derived from or influenced by blood vessels and/or structures.
- vasculature malformations include hemangiomas, preferably inoperable hemangiomas, aneurisms, granulomas, and the like.
- compositions of the present invention are administered as therapeutic agents, it may be necessary to combine the 5 context-dependent functional entities with a suitable pharmaceutical carrier.
- suitable 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 context-dependent o functional entity amenable to oral delivery, intravenous delivery, intramuscular delivery, topical delivery, nasal delivery, and the like.
- the context-dependent functional entity can be delivered in a variety of pharmaceutically acceptable forms.
- the context-dependent functional entity can be delivered in the form of a solid, solution, emulsion, dispersion, micelle, liposome, and the like.
- 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, 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, 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, corn 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.
- auxiliary, stabilizing, thickening and o coloring agents and perfumes may be used.
- 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
- 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.
- Sterile, fixed oils are conventionally employed as a solvent or suspending medium.
- 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.
- the injection rate will depend on a 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.
- the context-dependent functional entity diffuses rapidly into the blood and initiates 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 therapeutic agent used.
- a 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 lie within the range of from about 1 ⁇ g up to about 100 mg per kg body weight, and, preferably within the range of from 50 ⁇ g to 10 mg per kg body weight and can be administered up to four times daily.
- the daily IV 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.
- 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 function-forming-context at the biologically susceptible site(s).
- the complex transiently becomes functional upon 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.
- PCR is performed to amplify a DNA fragment of 639 base pairs from a 5 Marathon-Ready cDNA of human placenta origin (Clontech:(97/98 cat.#7411-l).
- a 100 ⁇ L reaction contains: 2 ⁇ L cDNA mixture, 100 pmoles each of oligos BM21 and BM33 (SEQ ID NO:3 and SEQ ID NO:4 (Oligos Etc.)), buffer, BSA, MgSO 4 according to manufacturer, 1 ⁇ L lOmM dNTPs and 2 units of Vent DNA polymerase (New England Biolabs 96/97 cat.#254S) which is added during the first cycle after the o temperature reaches 94°C.
- thermocy cling 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 fragment is purified by 5 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 fragment concentration is estimated by 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:5, Oligos Etc.) and BM33.
- a 100 ⁇ L PCR reaction contains: 10 ng 640 base pair DNA fragment, 100 pmoles each of 5 oligos BM51 and BM33, buffer, BSA, MgSO 4 according to manufacturer, 100 ⁇ M dNTPs 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 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 670 base pairs in length.
- This 0 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 diluted to 1 mL volume with Low-Salt buffer (0.2M NaCl, 20mM Tris-HCl, 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 NaCl, 20mM Tris-HCl, 1 mM EDTA pH7.4). The DNA is desalted and concentrated by ethanol precipitation according to Maniatis. The result is a 640 bp cDNA fragment encoding human TF residues 3 to 211 (i.e., amino acid residues 35-243 of SEQ ID NO:l.
- Different substructures may be joined by employing the thiol group of a cysteine production substracture.
- the conjugation is by the cysteine thiol groups of substracture(s) with thrombogenic potential, context enhancing substracture(s) and/or activity-modulating substracture(s), thereby forming a disulfide bond that links the cysteine-containing spacer - TF moiety to the context enhancing substructure(s).
- a thiol group may be artificially introduced into an amino group of context enhancing substructure(s), for example, using 2-iminothiolane (Traut's Reagent).
- cysteine thiol of the context enhancing substracture(s) (or the cysteine thiol of the cysteine-containing activity-modulating substracture) is formed into a mixed disulfide bond through the use of a 10- to 100-fold molar excess of 5,5'-bis(2-nitrobenzoic acid), DTNB.
- the amount of reaction is monitored by ultraviolet absorption of DTNB and measured using 14,150 for the molar absorption coefficient at 412 nm. After 1 hour incubation at room temperature in 0.1 M sodium phosphate, pH 7.5, 0.1 M NaCl, 1 mM EDTA, excess DTNB is removed by gel filtration.
- the mixed disulfide context enhancing substructure is mixed with the cysteine-containing activity-modulating substracture - TF moiety or the cysteine-containing context enhancing substracture(s) (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 in order to form the desired product (i.e., context-enhancing substracture-SS-spacer substracture - 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.
- a water-soluble, monitorable peptide and protein crosslinking agent A water-soluble, monitorable peptide and protein crosslinking agent
- N-maleimido-6-aminocaproyl ester of l-hydroxy-2-nitro-4-benzenesulfonic acid (mal-sac-HNSA) is reacted with an amino group of a peptide.
- the preferred peptide does not contain lysine at any position other than the amino terminus (Aldwin and Nitecki (1987) Anal Biochem 164:494-501). Reaction with amino groups releases the dianion phenolate, HNS A, 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.
- N-maleimido-6-aminocaproyl amide derivative is reacted with the thiol of the cysteine-containing activity-modulating substracture - TF to form a thioether bond that conjugates the context enhancing substracture(s) to TF through a spacer of variable length.
- heterobifunctional cross linkers may be used to introduce a maleimido 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- N-hydroxysulfosuccinimide ester; SMPB, succinimidyl 4-(p-maleimido-phenyl) -butyrate; sulfo-SMPB, succinimidyl-4- (p-maleimidophenyl)- butyrate; GMBS, N-(-maleimidobutyryloxy) succinimi
- 670 bp TF cDNA fragment from PCR is prepared (described above in Example 1, in PCR of TF cDNA).
- the purified 670 base pair DNA fragment is digested with 5 units restriction endonucleases BamHI and Hindlll (New England Biolabs cat#l 36S and #104S respectively) per ⁇ g DNA in IX BamHI buffer (New England Biolabs) for 4 h at 37°C.
- the 659 base pair fragment is purified from an agarose gel electrophoresis band using the QIAEX II kit as previously described for the 640 base pair fragment.
- the -659 bp band is cloned into the BamHI and Hindlll sites of the vector pTrcHisC (Invitrogen) by standard methods. Briefly, the pTrcHisC DNA is digested with BamHI and Hindlll and then the ⁇ 5 kb band is purified on an agarose gel. Ligation is performed with about 10 ng/ ⁇ L of the purified vector fragment mixed with a three fold molar excess of the purified 654 bp fragment and T4 DNA ligase reaction conditions according to the manufacturer (New England Biolabs). The resulting clone (NuV120: SEQ ID NO:6) places the TF coding sequence in a translational reading frame with the translational start of the vector.
- the clone NuV120 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.
- oligonucleotides NuV005 through NuV009 (SEQ ID NOs:7-l l, respectively (Oligos Etc.)) at 10 pmol/ ⁇ L in 20 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, pH 7.5. Heat mixture to 80°C for 2 min then cool to 25°C over a period of 30 l o min to allow annealing of oligonucleotides. Clone the resulting synthetic spacer into the Bam HI and Aval sites of NuV120.
- the vector DNA is prepared by digesting Clone NuV 120 with BamHI and Aval and isolation of the ⁇ 5kb fragment from an agarose gel and purification by QIAEX II (QIAGEN).
- the 10 ⁇ L ligation reaction contains 100 ng of NuV120-BamHI/AvaI fragment, 0.5 ⁇ L of the annealed oligonucleotide mixture, and
- Oligonucleotide mutagenesis is performed on Clone NuV121 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 primer Stul, SEQ ID NO: 13, (from Oligos Etc.)) changes the unique Seal site in NuV121 to a Stul site, thus allowing selection against the parental plasmid by restriction with Seal.
- the mutagenic primer (TF+K, SEQ ID NO: 14 (Oligos Etc.)) changes a Thr codon (ACA) to a Lys codon (AAA). Screening for the mutation is performed by DNA sequencing.
- the modified clone is NuV122 (SEQ ID NO: 15), 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, with residue 167 changed to Lys.
- Oligonucleotides NuV20-l through NuV20-8 are mixed at 10 pmol ⁇ L in 20 mM Tris-HCl, 10 mM MgCl 2 , 50 mM NaCl, pH 7.5. The mixture 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 Aval sites of NuV122.
- the vector DNA is prepared by digesting Clone NuV 122 with BamHI and Aval and isolation of the ⁇ 5kb fragment from an agarose gel and purification by QIAEX II (QIAGEN).
- the 10 microL ligation reaction contains 100 ng of NuV122-BamHI/AvaI 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, NuV124 (SEQ ID NO:24), is a plasmid for expression of a recombinant protein containing a His-tag, a thrombin cleavage site, a peptide sequence referred to as clone20, a spacer segment, and TF3-211 with K167.
- NV124 The protein, NV124, is expressed from the E. coli expression plasmid NuV124. This protein is insoluble when expressed in E. coli and was recovered from inclusion bodies by solubilizing the proteins with guanidine hydrochloride (GuHCl). NV124 was partially purified by IMAC (immobilized metal anion chromatography) on a Ni-NTA column (Qiagen) in guanidine HC1. After extensive washing, low pH (4.0) was used to release the NV124 from the column. Folding of the protein was performed by dilution of the denatured protein into 20 mM TrisCl containing reduced and oxidized glutathione. The final concentration of the protein after dilution in folding buffer was 50 ⁇ glm .
- Thrombin was added under precisely controlled conditions to remove the (His) 6 tag-thrombin cleavage peptide from the NV124. 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 removed from the NV124. In some studies, the (His) 6 tag was not removed, yet this protein (referred to here as His-NV124) displayed similar biological activity to the mature protein. The final step of purification was MonoQ-HR ion-exchange chromatography using FPLC and a Pharmacia 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, containing 1 M NaCl.
- the estimated purity at this stage is typically greater than 90%. Yields of 20 mg NV124 per liter of E. coli culture are typical.
- NV124 and His-NV124 were physically characterized by SDS-PAGE and mass spectrometry (MALDI).
- MALDI mass spectrometry
- NV124 was subjected to rigorous in vitro characterization of the function of its
- TF moiety This included analysis of NV124 dependent enhancement of factor Vila amidolytic activity, measurements of the affinity of NV124 for factor Vila, and measurements of NV124 dependent enhancement of factor Vila's proteolytic activity and plasma clotting activity.
- Table 1 summarizes some of the results obtained from the analyses of NV124, in comparison with other STVTs characterized. Amidolytic activity of factor Vila complexed with NV124 compared favorably with the complex of factor Vila 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 Vila that are responsible for allosteric enhancement of factor Vila amidolytic function.
- NV124 for factor Vila as measured by surface plasmon resonance or titration of factor Vila amidolytic activity was also similar to that of TF 1-218. Thus, the NV124 modification does not interfere with extensive interactions between TF and factor Vila.
- Table 1 Characterization of STVTs produced according to the invention.
- 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 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 Vila complex. 4. Apparent dissociation constant for factor Vila determined from amidolytic activity plots at 5 nM factor Vila concentration and varying the STVT concentration. 5. Dissociation constant determined by surface plasmon resonance in a BIAcore 2000. 6. Normalized factor Xa generation activity of STVT:factor Vila complexes.
- each STVT has amidolytic (measured by in vitro activity on a synthetic substrate) and factor Vila binding activity (measured by 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. Toxicity 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.
- a dose of 2.5 mg per mouse (-125 mg/kg) is administered, about half of the mice die.
- native TF is inserted in a phospholipid vesicle and administered, death of all the mice results at a dose of about 10 ng/mouse (-500 ng/kg).
- Fully constituted native tissue factor is more than 250,000 times more toxic than the soluble, truncated TF used in construction of NV124.
- soluble, truncated TF or full length, reconstituted TF 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 mg/kg). Therefore, NV124 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.
- FIG. 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.
- incubating an antibody (10H10) that recognizes, but does not neutralize, TF before injecting the complex into the mouse produces a much longer half-life.
- an antibody (10H10) that recognizes, but does not neutralize, TF
- the mice were sacrificed soon after the last time point and their organs collected and counted, most of the radiolabelled proteins were found in the lung.
- soluble TF accumulated in the lung, little thrombosis in the lung was observed.
- the lack of phosphatidylserine expression in lung vesicles may account for the lack of thrombogenic activity in this tissue.
- mice (strain A/J) were given C1300 neuroblastoma tumor cells subcutaneously on their flank, the tumors were allowed to grow to substantial size (up
- NV124 contained a facilitator, clone 20 described by Goodson et al (Proc. Natl. Acad. Sci. USA (1994) 91:7129-7133), which is reputed to interact with human uPAR and much less efficiently with murine uPAR.
- NV124 on tumor growth C1300 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 NV124 that were spaced by 4 days. NV124 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 ⁇ g (closed squares) and the other 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.
- a hard black cap was formed over the tumors.
- 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.
- 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 15 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 through the window.
- NV124 Before the introduction of NV124, 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. NV124 produced a very rapid 25 response; it caused rapid occlusion of arterioles, which prevented blood flow in the tumor vessels. In some instances, thromboses were visible in the vasculature.
- Example 8 PCR of human plasminogen cDNA fragments
- PCR is performed to amplify a DNA fragment of 965 base pairs from the Clontech cDNA mixture (Marathon-Ready cDNA of Human placenta origin from Clontech (97/98cat.#7411-l)) which encodes kringle domains 1 and 2.
- a 100 ⁇ L reaction should contain: 2 ⁇ L cDNA mixture, 100 pmoles each of oligos Plg+63 and Pig- 1005 (SEQ ID NO:25 and SEQ ID NO:26, respectively), buffer, BSA, MgSO 4 according to manufacturer; 1 ⁇ L lOmM dNTPs and 2 units of Vent DNA polymerase which is added during the first cycle after the temperature reaches 94°C.
- 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 fragment 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 965 base pair DNA fragment 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 NO:27 and SEQ ID NO: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 fragment; 100 pmoles each of oligos Plg+289 and Plg-523, buffer, BSA, MgSO 4 according to manufacturer, 100 ⁇ M dNTPs 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 denaturation for 1 min.
- the PCR product is about 267 bp in length.
- This 267 bp DNA fragment is purified by passage over an elutip-D column according to the manufacturer's instractions. Briefly, the DNA is diluted to 1 mL volume with Low-Salt buffer (0.2M NaCl, 20mM Tris-HCl, 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 (IM NaCl, 20mM Tris-HCl, 1 mM EDTA pH7.4).
- the DNA is desalted and concentrated by ethanol precipitation according to Maniatis. PCR is performed to amplify a DNA fragment of 1677 bp from the Clontech cDNA mixture which encodes kringle domains 3, 4 and 5.
- a 100 ⁇ L reaction should contain: 2 ⁇ L cDNA mixture, 100 pmoles each of oligos Plg+737 and Plg-2393 (SEQ ID NO:29 and SEQ ID NO:30, respectively), buffer, BSA, MgSO 4 according to the manufacturer; 1 ⁇ L lOmM dNTPs 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 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 bp in length.
- This DNA fragment 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 manufacturers instractions for DNA Extraction from Agarose Gels (QIAEX II Handbook 08/96).
- the 1677 bp DNA fragment concentration is estimated by agarose gel electrophoresis according to Maniatis.
- the result is a 965 bp cDNA fragment encoding plasminogen kringle domains 1 and 2, a 1677bp cDNA fragment encoding plasminogen kringle domains 3 through 5, and a 267bp cDNA fragment encoding plasminogen kringle domain 1.
- the plasminogen kringle 1 fragment is cloned into the BamHI and Kpnl sites of
- NuV 124 replacing the clone 20 sequence with the plasminogen sequence.
- the 267 bp Plasminogen cDNA fragment from PCR of human plasminogen cDNA fragments is digested with BamHI and Kpnl, separated on an agarose gel, purified with QIAEX II resin, and ligated with NuV 124 that was prepared as follows: digestion with BamHI and Kpnl, separation on an agarose gel, and purification of the ⁇ 6kb band with QIAEX II resin.
- the resulting clone is designated NuV125 (SEQ ID NO: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.
- TF K and variants of TF K are purified by the same basic protocol with modifications appropriate to the particular characteristics of the TF K fusion protein such as differences in elution from ion exchange chromatography and migration upon SDS-PAGE.
- TF fusions are produced in yeast (see Stone et al. (1995) Biochem. J. 310:605-614) or mammalian cells (see Rufet al. (1991) J Biol. Chem.
- a human plasminogen fragment encoding the fifth kringle domain was amplified by PCR from a human EST clone (Genome Systems, genbank accession H61584) using Vent DNA polymerase (New England Biolabs) and the following oligonucleotides:
- Plasmid NuV 127 is a vector derived from pTrcHisC (Invitrogen) for cloning of amino terminally linked TF- fusion proteins.
- NuV129 encodes a protein, NV129, 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.
- NV129 Purification of NV129 was performed as described for purification of NV124 (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 determination of 39,305 Da in close agreement with the predicted mass of 39,303.
- NV129 TF activity was performed as previously described for NV124. As shown in Table 1 although amidolytic activity of NV129:factor Vila was slightly lower than that of TF 1-218, affinity for factor Vila was similar to that of TF1-218. NV129:factor Vila proteolytic activity for factor X activation was also similar to that of TFl-218:factor Vila, as assayed both in the presence and absence of phospholipids. The results demonstrate that the TF entity of NV129 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 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 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 2 *b)ll, where a is the smallest dimension of the tumor and b is the dimension at right angles to a.
- A/J mice bearing C1300 tumors of an initial size of about 6 X 6 mm were infused with 125 micrograms of NV129 for 60 minutes. This treatment produced a statistically significant effect on the growth of C1300 neuroblastoma tumors, as shown in Fig. 6. Saline treated control tumors grew to a volume of near 4 mis in 10 days.
- Oligonucleotides encoding a peptide facillitator were synthesized:
- KLYD-1 5' GATCCCCGCGTAAACTGTACGACGGTAC 3' (SEQ IDNO:35)
- KLYD-2 5' CGTCGTACAGTTTACGCGGG 3' (SEQ IDNO:36)
- NuV144 encodes a protein, NV144, with a His-tag near the N-terminus, a thrombin cleavage site, a six residue peptide , a 15 residue flexible spacer, and human TF residues 3 to 211 at the C terminus.
- 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 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 infusion was carried out for 60 minutes without anesthesia. Two hundred microliters was infused. Saline was infused into tumor bearing mice as a control.
- FIG. 7 shows the effect of multiple infusions of NV144 on C1300 tumor growth.
- A/J mice bearing C1300 tumors were infused over a period of 1 hour with 125 micrograms NV144 (also referred to as K5p-TF) on Day 0 and 50 micrograms NV144 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 NV144 produced better efficacy than the lower dose.
- TP test protein
- Dose ranging study to identify the acute effects of test protein (TP) is performed in test animals by intravenous infusion of the drag.
- the maximum anticipated dosage of a therapeutic candidate molecule(s) in vivo is determined in adult rats, rabbits, dogs, nonhuman primates, etc. by an intravenous infusion schedule that mimics the anticipated potential therapeutic protocol in order to observe the acute effects of the drug.
- 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 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 rate of 20 L/min lOOgBW. 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 SPS or LRS proceeds for 10 min. The test material shall be labeled by confidential test number and have been adjusted to five concentrations.
- SPS sterile physiologic saline
- LRS lactated Ringer's solution
- 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 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 200 L/min/lOOgBW. The duration of infusion is 0.5 to 120 min.
- the rats are observed for changes in behavior, convulsions, increase of respiratory rate, or death and each shall be scored.
- 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 rat is euthanized 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 10% neutral formalin. After processing and embedding in paraffin blocks, cutting at 5 microns, sections are stained with Hematoxylin and easin, as well as by Carstair's method for histological examination.
- Example 14 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 10% neutral formalin. After processing and embedding in paraffin blocks, cutting at 5 microns, sections are stained with Hematoxylin and easin, as well as by Carstair's method for histological examination.
- 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 (SCID) 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 RPMI tissue culture medium containing 10% FCS until surgery (next day).
- 100:1 of diluted Ketamine HC1 (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.
- a sample of foreskin is placed within the wound and secured by sutures.
- the 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 with test materials.
- 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.
- mice and their host strains of rodents include colon adenocarcinoma CT-26 cells into Balb/c mice, C1300 neuroblastoma cells into A/J 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 Cancer Treatment, Diagnosis and Centers (DCTDC) Tumor Repository Catalogue of Transplantable Animal and Human 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.
- DCTDC Diagnosis and Centers
- Test materials are injected intravenously after the tumors 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 a course of treatment ranging between 1 to 6 treatment cycles. The tumors are measured with calipers and the rate of growth of the tumors are followed before and after treatment to distinguish effects of the test materials on growth. The tumor sites are removed from the mice for histological examination.
- pellets which are approximately 100 microns in diameter and containing one or more angiogemc 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.
- angiogemc factors such as vascular endothelial growth factor (vascular permeability factor), basic fibroblast growth factor, etc.
- vascular endothelial growth factor vascular permeability factor
- basic fibroblast growth factor etc.
- 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 structure of the vessels and compared with the control biopsy.
Abstract
Description
Claims
Priority Applications (3)
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CA002318434A CA2318434A1 (en) | 1997-12-23 | 1998-12-22 | Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium |
EP98965484A EP1041999A1 (en) | 1997-12-23 | 1998-12-22 | Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium |
AU20941/99A AU2094199A (en) | 1997-12-23 | 1998-12-22 | Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium |
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US99674497A | 1997-12-23 | 1997-12-23 | |
US08/996,744 | 1997-12-23 |
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WO1999032143A1 true WO1999032143A1 (en) | 1999-07-01 |
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PCT/US1998/027498 WO1999032143A1 (en) | 1997-12-23 | 1998-12-22 | Thrombogenic polypeptide chimeras and conjugates having activity dependent upon association with tumor vascular endothelium |
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EP (1) | EP1041999A1 (en) |
AU (1) | AU2094199A (en) |
CA (1) | CA2318434A1 (en) |
WO (1) | WO1999032143A1 (en) |
Cited By (5)
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WO2001085198A1 (en) * | 2000-05-10 | 2001-11-15 | Novo Nordisk A/S | tHARMACEUTICAL COMPOSITION COMPRISING A FACTOR VIIA AND A FACTOR XIII |
WO2003035688A2 (en) * | 2001-10-26 | 2003-05-01 | Novartis Pharma Gmbh | Targeted thrombosis by tissue factor polypeptides |
US6576610B1 (en) | 1999-10-04 | 2003-06-10 | Nuvas, Llc | Use of a context-dependent functional entity to enhance the efficacy of an agent |
US7008921B2 (en) | 2000-09-05 | 2006-03-07 | Karolinska Innovations Ab | Materials and methods relating to endothelial cell growth inhibitors |
US7393833B2 (en) * | 2005-03-09 | 2008-07-01 | The Board Of Regents Of The University Of Oklahoma | Chimeric proteins with phosphatidylserine binding domains |
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1998
- 1998-12-22 AU AU20941/99A patent/AU2094199A/en not_active Abandoned
- 1998-12-22 WO PCT/US1998/027498 patent/WO1999032143A1/en not_active Application Discontinuation
- 1998-12-22 EP EP98965484A patent/EP1041999A1/en not_active Withdrawn
- 1998-12-22 CA CA002318434A patent/CA2318434A1/en not_active Abandoned
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Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6576610B1 (en) | 1999-10-04 | 2003-06-10 | Nuvas, Llc | Use of a context-dependent functional entity to enhance the efficacy of an agent |
WO2001085198A1 (en) * | 2000-05-10 | 2001-11-15 | Novo Nordisk A/S | tHARMACEUTICAL COMPOSITION COMPRISING A FACTOR VIIA AND A FACTOR XIII |
EP1593389A1 (en) * | 2000-05-10 | 2005-11-09 | Novo Nordisk Health Care AG | Pharmaceutical composition comprising a factor VIIa and a factor XIII |
CN1304049C (en) * | 2000-05-10 | 2007-03-14 | 诺沃挪第克健康护理股份公司 | Pharmaceutical composition comprising factor VIIa and factor XIII |
US7008921B2 (en) | 2000-09-05 | 2006-03-07 | Karolinska Innovations Ab | Materials and methods relating to endothelial cell growth inhibitors |
WO2003035688A2 (en) * | 2001-10-26 | 2003-05-01 | Novartis Pharma Gmbh | Targeted thrombosis by tissue factor polypeptides |
WO2003035688A3 (en) * | 2001-10-26 | 2004-03-18 | Novartis Pharma Gmbh | Targeted thrombosis by tissue factor polypeptides |
US7393833B2 (en) * | 2005-03-09 | 2008-07-01 | The Board Of Regents Of The University Of Oklahoma | Chimeric proteins with phosphatidylserine binding domains |
WO2006096828A3 (en) * | 2005-03-09 | 2008-11-20 | Stuart E Lind | Chimeric proteins with phosphatidylserine binding domains |
Also Published As
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CA2318434A1 (en) | 1999-07-01 |
AU2094199A (en) | 1999-07-12 |
EP1041999A1 (en) | 2000-10-11 |
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