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  • C12N9/14—Hydrolases (3)
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  • 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)
  • C12N9/6456—Plasminogen activators
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  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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  • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
  • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
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  • C07K14/4721—Lipocortins
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  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide
  • C07K2319/33—Fusion polypeptide fusions for targeting to specific cell types, e.g. tissue specific targeting, targeting of a bacterial subspecies

Definitions

• the invention relates to conjugates and pharmaceutic compositions with affinity for phospholipids and capability of lysing fibrin clots.
• the invention also relates to methods and therapeutic regimens for treating disorders relating to fibrin clots.
• Fibrin deposits form at sites of vascular injury including ruptured atherosclerotic plagues.
• the initial event leading to fibrin formation is the activation of the extrinsic coagulation pathway which is triggered by the contact of tissue factor with circulating factor Vll/VIIa.
• Tissue factor is a membrane-bound regulatory protein present outside blood vessels as well as in atherosclerotic plaques. When tissue factor is exposed to blood by vascular injury or plaque rupture, it binds to factor Vll/VIIa in the presence of Ca ++ .
• tissue factor and factor Vll/VIIa activates factor X to factor Xa, which in turn, activates prothrombin to thrombin in the presence of factor Va, phospholipid and Ca**.
• the resulting thrombin converts soluble fibrinogen to insoluble fibrin which deposits as clots.
• tissue factor and factor Vll/VIIa not only activates factor X but also activates factor IX to form factor IXa.
• Factor IXa activates factor X in the presence of phospholipid, factor Villa and Ca**.
• the reactions, except for the conversion of fibrinogen to fibrin, require negatively charged phospholipid, such as phosphatidylserine (PS) , for optimal catalysis and proceed at the surface of insoluble phospholipid to localize fibrin clot formation.
• PS phosphatidylserine
• major components of fibrin clots are insoluble fibrin, phospholipid and the activated coagulation factors.
• Phosphatidylserine which is highly thro bogenic in vitro , generally is absent from the external face of the plasma membrane in both erythrocytes and platelets. The asymmetry is maintained by an active transport mechanism. It is believed that the asymmetrical distribution of PS is altered by platelet activation and PS becomes exposed on the external face of the plasma membrane. That reorientation provides negatively charged phospholipids for the formation of the prothro binase complex and also enhances other phospholipid-dependent reactions in the coagulation cascade.
• factor X The coagulation factors that contain gamma-carboxyglutamic acid residues (factors X, IX, VII and prothrombin) bind to negatively charged phospholipids with binding constants in the 10 "6 -10 "7 M range in the presence of Ca**.
• the major source of negatively charged phospholipids for blood coagulation in vivo is thought to be the platelet plasma membrane.
• factor Xa binds to the surface of activated platelets where it forms a complex (prothrombinase) with platelet-bound factor Va to activate prothrombin.
• Plasmin a serine protease, is the sole plasma enzyme responsible for fibrin dissolution. It circulates in blood as a precursor, plasminogen.
• Plas inogen is a single-chain polypeptide that is converted to the two-chain active form, plasmin, by plasminogen activators.
• Plasmin is composed of an N ⁇ -terminal A-chain and a COOH-terminal B-chain held together by a disulfide bond.
• the A-chain contains five characteristic repeating units (kringle domains) while the B-chain contains the serine-protease catalytic unit.
• the region from the first kringle through fourth kringle binds to fibrinogen and some of the circulating plasminogen coprecipitates with fibrin when a clot is formed.
• Plasminogen that is proteolytically cleaved has a higher affinity for fibrin clots than intact plasminogen (Glu-plasminogen) and thus accelerates lysis of fibrin clots.
• the fibrin binding sites of plasminogen are located in the first and fourth kringle domains.
• Tissue-type plasminogen activator (tPA) and urokinase (uPA) are two physiologic activators of plasminogen. Both activators are synthesized as single-chain zymogens and are converted into two-chain active forms.
• tPA is a membrane bound protein synthesized mainly in endothelial cells and released into the blood stream in response to certain stimuli. The secretion of tPA into the blood stream triggers extrinsic fibrinolysis.
• the NH 2 -terminal chain of tPA contains a finger domain, a growth factor-like domain and two kringle domains.
• the second kringle domain has binding affinity (K d , also known as binding constant or affinity constant) for the fibrin clot of 1.6 x 10 *7 M.
• K d binding affinity
• the catalytic efficiency of tPA is about 1,000 times higher toward fibrin-bound plasminogen than circulating plasminogen.
• Prourokinase single-chain urokinase or scuPA
• scuPA single-chain urokinase
• Prourokinase is activated by plasma kallikrein and plasmin to stimulate intrinsic fibrinolysis.
• scuPA has a kringle domain, it shows little binding affinity for fibrin clots. However, it hydrolyzes fibrin-bound plasminogen more efficiently than free plasminogen.
• a bacterial protein, streptokinase (SK) forms a stoichiometric complex with plasminogen which converts plasminogen to plasmin.
• Fibrinolytic agents tPA, uPA, scuPA and SK are being used as therapeutic agents to treat patients suffering from thrombosis.
• the agents represent a major advance, problems remain due to short half-life in circulation and a propensity to cause systemic fibrinogenolysis.
• the relatively insufficient binding affinity for fibrin and probable cross-binding to circulating fibrinogen force a high-dose administration of tPA, which causes a significant degree of fibrinogenolysis.
• the above therapeutic proteins have been modified in attempts to overcome the above-noted problems.
• the strategies used for improvements include: making the molecules resistant to circulating inhibitors; strengthening binding affinity for fibrin clots; and targeting fibrin deposits by conjugating plasminogen activator with antibody specific to fibrin clots.
• Acylated plasminogen/SK has a higher fibrin selectivity than SK.
• the APS conjugate is not inactivated by a ⁇ 2 -plasmin inhibitor because the hydroxy group of the active site serine residue is blocked.
• APS binds to fibrin clots and its acyl group is cleaved slowly to produce the active form.
• the half-life of APS is significantly longer than that of the unmodified parent molecule.
• a truncated scuPA (residues 1-143 deleted) which lacks the growth factor and kringle domains in the heavy chain was expressed.
• the molecule has a fibrin selectivity identical to the intact form but is not inhibited by plasminogen activator inhibitor-1 (PAI-1) .
• PAI-1 and other serpins in circulating blood PAI-1 and other serpins in circulating blood.
• a chimeric molecule that combined the fibrin binding domain of plasminogen (A-chain) with the catalytic domain of urokinase had a eight-fold higher binding affinity for fibrin clots than urokinase. It also had a higher catalytic activity toward the fibrin monomer.
• a chimeric molecule composed of the A chain of plasminogen and the catalytic domain of tPA had the same binding affinity for fibrin as plasminogen and the same catalytic activity as native tPA.
• Murine monoclonal antibodies specific to the beta-chain of fibrin were conjugated with the catalytic chain of tPA or scuPA using a disulfide cross-linking agent.
• the antibodies have dissociation constants on the order of 2 x 10 "7 M.
• the antibody/tPA conjugate was ten times more active than native tPA in lysis of the fibrin monomer.
• An antibody/urokinase conjugate showed 1,000 times higher activity than urokinase.
• proteins with anticoagulatory activity have been isolated from human placenta. The proteins were found to be members of the lipocortin/annexin family and to date, eight members of the family have been isolated from various tissues and cultured cells with many different functions proposed. The proteins are given the common name, "annexin”. All annexins share the property of calcium-dependent binding to anionic phospholipids. Funakoshi et al., Biochem.
• Annexin V (also known as PAP-I) is a major component of the family and is isolated from placenta. It contains one free sulfhydryl group and does not have any attached carbohydrate chains.
• the primary structure of annexin V deduced from the cDNA sequence shows that annexin V comprises four internal repeating units (each unit has 60-80 amino acid residues).
• annexin V has the strongest binding affinity (K d ⁇ 10 "10 M) for phospholipid vesicles containing 80% phosphatidylcholine (PC) and 20% PS under conditions that are comparable to plasma and extracellular fluid (1.2 mM ionized calcium, 0.15 M ionic strength) .
• Annexin shows high affinity for membranes containing PS and phosphatidic acid (PA) , phospholipids carrying two negative charges.
• Annexin V binds to human platelets. Unstimulated platelets express a small number of binding sites, but the number of binding sites is increased greatly by certain platelet agonists (for example, approximately 15-20 fold by a combination of thrombin and collagen) . There are approximately 100,000 binding sites per platelet after stimulation with thrombin and collagen. The binding sites have an apparent dissociation constant (K d ) of 7 nM. The binding is calcium-dependent, reversible and can be inhibited completely by PS-containing vesicles. Annexin V also can displace previously bound factor Xa from the platelet surface. Thiagarajan & Tait, J. Biol. Chem. (1990) 265, 17420-17423.
• Annexin V inhibits all of the activation reactions in the coagulation cascade where phospholipid is involved.
• gam a-carboxyglutamic acid-containing coagulation factors bind to negatively charged phospholipids in the presence of Ca**.
• the dissociation constants of the ⁇ -carboxyglutamic acid-containing coagulation factors for phospholipid are in the 10 "6 -10 *7 M range, which is three to four orders of magnitude weaker than that of annexin V.
• greater dissociation constants are those with greater numerical molarity values, thus a constant of 10 '8 M is greater than a dissociation constant of 10 "10 M.
• a dissociation constant of 10" 10 M represents greater binding propensity than a constant of 10 "8 M) .
• the inhibition mechanism of annexin V is to compete with the coagulation factors for binding to anionic phospholipids.
• Annexin V levels of annexin V in human plasma and cells in contact with blood were measured by ELISA using an affinity purified rabbit antiseru .
• Annexin V is present intracellularly in platelets, endothelial cells and leukocytes but is absent in erythrocytes.
• Annexin V essentially is not present in normal human plasma and can be released by cell damage or death. Thus the protein that appears to be intracellular under most normal conditions can be released into the extracellular milieu with cell damage or death. Flaherty et al., J. Lab. Clin. Med. (1990) 115, 174-181.
• annexin V for negatively charged phospholipids is approximately 50 times stronger than that of tPA for fibrin and 10-100 times stronger than that of fibrin-specific antibodies.
• An object of the invention is to provide annexin-plasminogen activator conjugates useful for thrombolysis.
• Another object of the instant invention is to provide methods for making annexin-plasminogen activator conjugates.
• Another object of the instant invention is to provide therapeutic compositions and therapeutic methods comprising annexin-plasminogen activator conjugates for treating disorders resulting from thrombosis.
• Figure 1 depicts a map of recombinant plasmid pMT017.
• the trp promoter region is denoted as trp p/o, the prourokinase coding region as ProUK, the hirudin linker as HIRUDIN, the annexin V coding region as AnV and the trp attenuator as trpA. Commonly known and useful restriction sites are denoted.
• FIG. 2 depicts a map of recombinant plasmid pMT024.
• the Pichia AOX promoter region is denoted as 5'Aoxl
• the scuPA-hirudin-annexin V coding region is denoted as PPA/HIRUDIN/AV
• the AOX terminator region is denoted as 3'Aoxl
• the yeast selectable marker is denoted as HIS4.
• the solid black box just downstream from the Xhol site of the 5'Aoxl sequence represents the Mucor rennin signal sequence.
• the 2.86 kb sequence between the 3'Aoxl and 5'Aoxl sequences are pBR322-derived sequences. Sizes are noted in kilobases (kb) and commonly known and useful restriction sites are denoted.
• Figure 3 depicts a map of recombinant plasmid pMR391. Beginning with the Hindlll site, the first narrow right slanting cross hatched region is the SV40 enhancer and promoter, the second narrow left slanting cross hatched region is the SV40 splice junction, the third clear region is the PPA (scuPA) coding region, the fourth wide right slanting cross hatched region is the hirudin linker, the fifth wide left slanting cross hatched region is the annexin V coding region and the sixth region downstream from the Xhol site is the SV40 polyA region.
• PPA scuPA
• the annexins may be isolated from a variety of tissue extracts. Funakoshi et al. (1987a) supra; (1987b) supra; Tait et al. (1988) supra; U.S. Pat.
• Suitable tissues include liver, lung and placenta.
• a particularly suitable tissue is human placenta. Briefly, the tissue is cut into small pieces and washed with chilled physiologic buffer, such as phosphate buffered saline or 50 mM
• tissue chunks are homogenized in a blender generally in a physiologic buffer containing 5 M EDTA and 5 mM benzamidine. The homogenate is filtered to obtain a filtrate.
• the filtrate is next exposed to a precipitation procedure, for example ammonium sulfate.
• Ammonium sulfate is added to the filtrate to a saturation from about 30% to about 50%.
• Precipitates formed therefrom are removed by centrifugation.
• Additional ammonium sulfate is then added to the supernatant to a concentration for about 70% to about 90% and any precipitates formed therefrom are collected by centrifugation.
• the precipitates are pooled and dissolved in a physiologic buffer, such as those noted above, and the resulting solution is dialyzed overnight against large volumes of physiologic buffer to remove the ammonium sulfate.
• the buffer is changed at regular intervals.
• the dialysate then is passed over an anion exchange medium such as DEAE coupled to, for example, Sephadex, cellulose and Sepharose.
• Adsorbed proteins are eluted with a linear gradient of increasing salt concentration, for example from about 50 mM to about 500 mM NaCl.
• Annexin-containing fractions are pooled.
• Suitable gel filtration column comprises a matrix of Sephadex G-75. Active fractions were collected, pooled and dialyzed to reduce the salt concentration. The dialysate then is exposed to a cation exchange medium. Suitable cation exchange media include CM-Sephadex, SP-Sephadex, CM-cellulose or Mono S (Pharmacia) . The adsorbed proteins are eluted with a buffer gradient of increasing salt concentration. Active fractions are pooled and concentrated as described above.
• placenta is used as a tissue source
• several different species of annexin can be obtained.
• the species are distinguishable following the ion exchange chromatography and thus the species are distinguishable by elution at differing salt concentration.
• Degree of purification can be determined by, for example, SDS-PAGE under reducing or non-reducing conditions using procedures that are recognized in the art.
• the annexins obtained from placenta so far identified have molecular weights in the range of 30,000 to 35,000. Accordingly, gels of appropriate polyacrylamide concentration are selected.
• annexin shows an anti-coagulant activity.
• One such anti-coagulant assay method incorporates rabbit brain cephalin.
• One vial of cephalin (Sigma) is suspended uniformly in 100 ml of saline. Equal volumes of cephalin and 0.033 M CaCl 2 are mixed.
• acid washed kaolin is suspended in saline at a concentration of 5 mg/ml.
• Clotting time is determined, for example by the method described above except that the kaolin suspension is replaced by thromboplastin. Human brain thromboplastin is diluted with 50 mM Tris-HCl, pH 7.4, containing 0.15 M NaCl to obtain a controlled clotting time of approximately 60 seconds.
• Annexins also show an affinity for phospholipids.
• One method for quantifying the degree of annexin binding to phospholipids is founded on fluorescence quenching. (Tait et al., J. Biol. Chem. (1989) 264, 7944-7949) Annexin (50 ⁇ M) is labeled by incubating with fluorescein isothiocyanate (50 ⁇ M) for one hour at 37°C in a buffer of 0.05 M borate, pH 9.0, 0.15 M NaCl, 1 mM EDTA. The reaction mixture is dialyzed against 20 mM HEPES pH 8.0 buffer. The dialysate is applied to a cation exchange column and eluted with a linear salt gradient.
• phospholipid vesicles are prepared by adding approximately, 20% PS, 20% diheptanoyl-phosphatidylcholine and 60% long chain PC resulting in the spontaneous formation of unilamellar vesicles. Aliquots of phospholipid stock solutions in chloroform are mixed to yield the desired molar ratios and the chloroform is removed by evaporation under nitrogen. The phospholipids are then dissolved in HEPES buffer by sonication for 3 minutes on ice followed by overnight equilibration at 4*C.
• Fluorescence measurements can be performed in a fluorometer (for example, SLM 8000 C/Aminco, Urbana, Illinois) .
• the appropriate wavelengths are selected, for example, for fluorescein the excitation wavelength is 495 ⁇ 16 nM and emissions are monitored at 520 ⁇ 10 nM.
• Binding assays are performed in standard quartz fluorescence cells containing a buffer and the fluorescence labeled annexin. The solution is mixed once by inversion and then varying amounts of phospholipids are added to the cuvette. The contents are mixed again by inversion and fluorescence intensity is recorded. Following addition of 5 mM EDTA to the cuvette, fluorescence intensity is recorded again. The degree of quenching is calculated from the ratio of the final fluorescence intensity to fluorescence intensity in the presence of EDTA.
• Binding of annexin to phospholipid vesicles occurs with high affinity (K d ⁇ 10 "10 M) under conditions that are comparable to that of plasma and extracellular fluid (1.2 mM ionized calcium, 0.15 M ionic strength). Binding is reversible and completely calcium dependent.
• Other assays that are suitable for monitoring the presence of annexin include for example antibody-based methods such as ELISA and Western blot.
• annexin can be produced by recombinant methods.
• cDNA clones can be obtained using antibody screening of expression libraries or using oligonucleotide probes deduced from annexin peptides.
• a full length cDNA clone of annexin V has been obtained and subcloned in expression vectors.
• Funakoshi et al. Biochem. (1987a) 26, 5572-5578; (1987b) 8087-8092) used an affinity-purified antibody to screen a cDNA bank to obtain an annexin V clone.
• a 1.3 kb Ncol/Hindlll fragment containing the coding sequence of annexin V was cloned into the expression vector pKK233.2 (Pharmacia) to form the expression plasmid pPAP-I-wt.
• the recombinant annexin is expressed cytoplasmically at a level of about 2% of cellular protein.
• the recombinant annexin is obtained from host cells using procedures recognized in the art.
• Annexin V contains four tandem, imperfect repeats of about 75 amino acid residues (Funakoshi et al. Biochem. (1987b) 26, 8087-8092).
• the repeats contain conserved amino acid residues at 4 sites, 15 positions comprise hydrophobic amino acid residues, 4 positions comprise hydroxyamino acids and 2 sites comprise acidic amino acids.
• Each of the four repeating sequences contains two regions that are commonly present in phospholipid binding proteins.
• the first region, the NH g -terminal 17 residues, conforms to a consensus sequence (Geisow et al., Nature (1986) 320, 636-638) ofLys-Gly-X-Gly-Thr-Asp-Glu-X-X-h-h- X-h-h-X-Ser-Arg (SEQ ID N0:1), where h represents hydrophobic amino acids and X can be any amino acid.
• the sequence has been found in Ca** regulated membrane binding proteins such as endonexin and calelectrin.
• Phospholipase A j and a viper venom phospholipase A g inhibitor also have closely related sequences.
• the second homologous region in the proteins is a stretch of six residues of hydrophobic amino acids at the C-terminal portion of each repeat. The two regions are thought to be involved directly in binding to phospholipid (Geisow, FEBS Lett. (1986) 203, 99-103). The strong anticoagulant activity can be attributed to the presence of the phospholipid binding regions.
• annexin V The crystallographic analysis of annexin V showed that the four domains of annexin V have a similar folded structure, each consisting of five helices (Huber et al., EMBO J. (1990) 9, 3867-3874). The same group also found three strong calcium binding sites of annexin V located on the convex face of the I, II and IV repeats. Huber et al. suggest the calcium binding sites mediate the phospholipid binding to the annexin V molecules (Huber et al. FEBS Lett. (1990) 275, 15-21).
• the annexin molecule can be subdivided or altered at one or more amino acid residues so long as the phospholipid binding capability is not reduced substantially.
• annexin can be truncated, for example, to include one or more domains or contain fewer amino acid residues than the native protein, or can contain substituted amino acids. Any changes are acceptable within the scope of the invention so long as the mutein or second generation annexin molecule does not contain substantially lower affinity for phospholipid. Substantially lower affinity is a binding constant for phospholipid that is greater than about 10 "7 M.
• the fibrinolytic agent can be modified or altered within the scope of the invention so long as the resulting agent retains the capability of lysing fibrin clots.
• point mutated, point deleted, point substituted and/or truncated fibrinolytic agents such as tPA, urokinase or prourokinase in which one or more domain(s) has (have) been deleted
• fibrinolytic agents such as tPA, urokinase or prourokinase in which one or more domain(s) has (have) been deleted
• tPA urokinase or prourokinase in which one or more domain(s) has (have) been deleted
• US 4,752,581, US 4,908,204, US 4,992,274, US 4,916,071 and EP 2311883A describe hybrid molecules such as urokinase/tPA molecules.
• modified fibrinolytic agents can be assessed in known assays such as using the chromogenic substrates, S-2288, S-2444 or S-2251 (Helena Laboratories) as taught in Schnee et al. (Proc. Natl. Acad. Sci. (1987) 84, 6904-6908) or as taught in Bode et al. (J. Biol. Chem. (1987) 262, 10819-10823).
• a reduction of fibrinolysis activity of up to about 50% in a modified fibrinolytic agent relative to the native protein is contemplated to fall within the scope of the instant invention.
• Annexin V can target a thrombus in vivo.
• Radiolabeled annexin V was administered intravenously and the localization of radio activity was determined. A substantial accumulation was noted at the thrombus with a thrombus:blood ratio of about 17:1 at 100 minutes after injection. The radiolabeled annexin V was rapidly cleared from the blood.
• annexin can be used to target a thrombus wherein it may exert its anticoagulant activities or annexin can serve as a means for targeting a second molecule conjugated thereto.
• Suitable second molecules include thrombolytic agents such as tissue plasminogen activator, streptokinase, urokinase and prourokinase.
• Annexin conjugates are particularly useful for thrombolytic agents that are not fibrinophilic such as urokinase.
• Many thrombolytic agents such as streptokinase, urokinase, prourokinase and tissue plasminogen activator, are available commercially, either produced reco binantly or purified from natural sources.
• thrombolytic agents Through selective proteolytic digestion or subcloning, second generation forms of thrombolytic agents have been produced with the advantages of having enhanced fibrin binding and enhanced half-life, for example, while minimizing adverse side effects.
• tPA lacking the epidermal growth factor and finger domains or containing just the second kringle and serine protease domains have been made.
• chimeric thrombolytic agents for example comprising portions of urokinase and tPA, are active in activating plasminogen (Pierard et al., J. Biol. Chem. (1987) 262, 11771).
• Annexins can be cross-linked chemically with thrombolytic agents.
• U.S. Pat. No. 4,564,596 teaches the use of an aliphatic diamine to conjugate urokinase with fibrinogen.
• U.S. Pat. No. 4,536,391 teaches a plasmin urokinase complex using a coupling reagent of the succinimide ester variety. Sevilla et al. (Biochem. Biophys. Res. Comm.
• Hashida et al. (J. Appl. Biochem. (1984) 6, 56-63) teach a number of maleimide compounds useful for conjugating proteins. Briefly scuPA, SK or plasminogen is incubated with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC) in a phosphate buffer, pH 7.0. SMCC binds covalently to free amino groups preferably to the NH 2 -terminal amino group under these conditions. The reaction mixture then is applied to a gel filtration column to remove side products.
• SMCC sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate
• SMCC-protein for example SMCC-scuPA, SMCC-SK or SMCC-plas inogen, is incubated with annexin V in a phosphate buffer, pH 6.0, containing 6 M urea or 6 M guanidine.
• SMCC-proteins bind specifically to the single free sulfhydryl group present at the C-terminal end (fifth residue from the c-terminus) of the annexin V molecule.
• the reaction product is applied to a gel filtration column (for example, Sephadex G-100) to isolate the monomeric form, which is expected to be the major product under those conditions.
• a gel filtration column for example, Sephadex G-100
• the annexin V/plasminogen hybrid is activated to form annexin V/plasmin by urokinase. Annexin V is resistant to mild digestion by most proteases under native conditions.
• the compounds for conjugating annexin and thrombolytic agent can be varied to provide a spacer should steric effects compromise either annexin binding affinity or thrombolytic agent activity or both.
• Hashida et al. teaches compounds with variable numbers of methylene group between the maleimide and succinimide functions and the diamine bridging agent of Maximenko et al. (supra) comprises an aliphatic diamine with 1-12 methylene groups.
• spacer regions can be configured into recombinant conjugates by inserting appropriate coding sequences between the annexin and thrombolytic agent coding sequences.
• Conjugates can also be prepared recombinantly as many of the above recited thrombolytic agents have been cloned.
• Schnee et al. Proc. Natl. Acad. Sci. (1987) 84, 6904-6908 teach the expression of a fibrin monoclonal antibody-tPA conjugate by recombinant means.
• the heavy chain gene of the anti-fibrin antibody was inserted adjacent to the tPA ,9-chain gene in an expression vector and recombinant hybrid protein was produced.
• Pierard et al. J. Biol. Chem.
• E. coli, B. subtilis, yeast and mammalian cells can be used.
• the cassette containing the coding sequence is placed into the operable site of an appropriate expression vector comprising the necessary host cell recognizable 5' non-coding sequences including promoters and downstream 3• non-coding sequences.
• a suitable E. coli expression vector is pKK233-2 (Amann & Brosius, Gene (1985) 40, 183-190) ;
• a suitable yeast expression vector is DPOT (Thim et al., Proc. Natl. Acad. Sci.
• telomeres may contribute jointly or severally to phospholipid binding and the anticoagulant activity. Accordingly, nucleotide coding sequences of one or more domains or duplicates of one domain can be subcloned adjacent to the coding sequence of the thrombolytic agent to produce conjugates with novel or enhanced properties.
• the conjugates can comprise the active component of pharmaceutic compositions.
• Such compositions would contain pharmaceutically acceptable carriers, diluents and excipients.
• suitable carriers include buffers, physiologic saline, tissue culture medium and water.
• the conjugates can be administered using art recognized methods such as intravenous infusion.
• the treatment regimen is determined empirically from animal studies and clinical trials, and is keyed to the severity of the disease, physical condition of the patient and the like. The artisan can obtain suitable guidance from many of the treatises in pharmacology such as,
• the targeting capability of annexin can be used to deliver pharmaceutics to local sites.
• a cytotoxic agent, itogen, antibiotic and the like can be used.
• delivery route and dosage can be ascertained by the artisan practicing methods known in the art.
• Iodinated annexin V is prepared using standard procedures such as methods using the lodogen reagent. Thiagarajan and Tait, J. Biol. Chem. (1990) 265, 17420-17423.
• the animal model of arterial thrombosis is essentially that described earlier for dogs but adapted to rabbits (Ritchie et al.. Circulation (1986) 73, 1006-1012) as described above.
• a rabbit is intubated, anesthetized with 1-2% halothane and monitored continuously by electrocardiogram.
• the artery of interest carotid or femoral
• a 2-5 cm section is isolated between two ligatures.
• the section is crushed repeatedly with forceps, the proximal ligature is released for 1 minute to allow fresh blood to enter and then retightened.
• radiolabeled I 12S -annexin V is injected as a bolus via a peripheral vein and the ligatures released thirty seconds later.
• the artery is removed, fixed in formalin, sectioned and measured for radioactivity.
• PCR U.S. Pat. Nos. 4,683,195; 4,683,202
• a scuPA cDNA is amplified by PCR with a 5' Xbal site and a 3' Hindlll site.
• Primers for scuPA are designed from the published cDNA (Holmes et al., Bio/Tech (1985) 3, 923-929) and amplification can be from a cDNA library.
• the two PCR products are digested with Ncol and Xbal; and Xbal and Hindlll, respectively, and are then ligated into an E. coli expression vector such as pKK233-2 (Amann & Brosius, Gene (1985) 40, 183-190) previously digested with Ncol and Hindlll.
• the plasmid is placed into an appropriate host such as DH5 ⁇ . Recombinants are identified by colony hybridization, and the construction verified by restriction mapping and DNA sequencing.
• the expression level of the hybrid protein in E. coli is then optimized by varying incubation temperature and the length of the induction period with IPTG.
• hybrid protein will be packaged in inclusion bodies as occurs with annexin V and scuPA individually (Winkler & Blaber, Biochem. (1986) 25, 4041-4045) .
• the inclusion bodies are isolated by centrifugation (10 minutes at 10,000 x g) , solubilized in 6 M urea and renatured by dialysis.
• the hybrid protein then is purified by conventional low-pressure and FPLC chromatographic methods, with fractions monitored by fluorescence polarization immunoassay using anti-annexin antibody.
• the hybrid protein can be characterized in the same manner as described for chemically produced hybrids.
• Cultures of E. coli containing the desired plasmid are grown overnight at 37"C in L broth containing 100 ⁇ g/ml ampicillin. Cultures are diluted 1:10 in 1 liter of the same medium and grown at 37"C with shaking until the A 600 is > 0.3. Synthesis then is induced by adding IPTG to 3 mM and growth continued for 4 hours. Bacteria are harvested by centrifugation, washed once with 100 ml PBS containing 10 mM EDTA and stored at -20"C.
• Pellets are lysed by sonication for 2-3 minutes on ice in 25 ml of a buffer consisting of PBS, 10 M EDTA, 6 M urea, 0.5 ⁇ g/ml leupeptin, 0.5 ⁇ g/ml pepstatin, 1% Triton X-100 and 0.2 mM phenylmethanesulfonyl fluoride.
• the extract is centrifuged (20 minutes at 25000 x g) .
• the supernatant is dialyzed at 4*C against a buffer comprising 50 mM Tris-HCl, pH 8.0, 0.5 ⁇ g/ml leupeptin, 0.5 ⁇ g/ml pepstatin, 1 mM EDTA, 3 mM NaN 3 and 100 mM NaCl, with repeated buffer changes.
• the dialysate is membrane filtered and is applied to an affinity column, for example, an anti-annexin column, at 4*C.
• the column is washed with at least 100 ml of the same buffer used for dialysis and bound proteins are eluted with 0.1 M glycine, pH 2.5 buffer.
• the eluate is collected in one-fourth volume of 1 M Tris-HCl, pH 8.0 for immediate neutralization of pH and then subjected to dialysis and concentration.
• Human annexin binds to rabbit platelets in a manner similar to when annexin binds to human platelets, indicating that the rabbit can be used as an experimental model to test the activity of annexin V hybrids.
• Thrombi were induced experimentally by mechanical trauma and stasis in the carotid artery of a rabbit.
• Radiolabeled annexin V 12S l-annexin V, 150 uCi
• Serial blood samples were then taken at 5, 10, 15, 30 and 60 minutes to determine blood clearance rate; the animal then was sacrificed at 100 minutes and samples of blood, urine and tissue were taken for measurement of weight and 125 I content. No signs of acute systemic toxicity were noted.
• the 125 I-annexin V accumulated substantially at the thrombus, with a thrombus:blood ratio of 17:1 at 100 minutes after injection.
• Example II Essentially identical results, as that presented in Example I, were obtained when thrombosis was induced in the femoral rather than the carotid artery.
• Annexin/scuPA and annexin/plasminogen are activated by plasma kallikrein (or plasmin) and urokinase, respectively.
• Activated annexin/urokinase, annexin/plasmin and annexin/SK are incubated with plasminogen and the resulting plasmin is assayed using the synthetic substrate, Boc-Glu-Lys-Lys-MCA (Peptides International) .
• the plasminogen activating activities of the hybrid molecules can be compared with that of the parent proteins.
• the fibrinolytic activity of hybrids can be assayed by the method of Rijnen et al. (Thromb. Haemostas. (1984) 52, 308-310), except that platelet-rich plasma is used instead of platelet-poor plasma.
• Fresh human platelet-rich plasma is added to thrombin (or tissue factor) , CaCl 2 and 125 I-fibrinogen, immediately placed in silastic tubing (4 mm I.D.) and incubated at 37 ⁇ C for 30 minutes. The tubing is cut into pieces of defined length and clots are removed from the tube and washed with buffer.
• the clots are incubated with the hybrids or the native proteins (for example, scuPA, SK or plasmin) . Fibrinolytic activity is assessed by determining the solubilized radioactivity. The concentrations of fibrinogen and plasminogen in the sample plasma are assayed by ELISA to determine fibrinogenolysis.
• the native proteins for example, scuPA, SK or plasmin
• scuPA Human single-chain urokinase-type plasminogen activator
• ScuPA was dialyzed overnight against 0.1 M sodium phosphate, pH 7.3 containing 6 M urea. After dialysis, the concentration of scuPA was adjusted to 32.5 ⁇ M. Sulfo-SMCC was added to dialyzed scuPA aliquots at two final concentrations of 65 ⁇ M and 325 ⁇ M. The samples were incubated for 30 minutes at room temperature. At the end of incubation, glycine (10 mM) was added. The samples were applied to a gel filtration column (PD-10, Pharmacia) to remove excess unreacted reagents. The column then was eluted with 0.1 M sodium phosphate buffer, pH 6.0 containing 6 M urea and 10 mM EDTA.
• PD-10 gel filtration column
• the protein fractions were pooled (scuPA-SMCC) .
• Annexin V was dialyzed overnight at room temperature against 0.1 M sodium phosphate buffer, pH 7.3 containing 6 M guanidine (or 8 M urea) .
• the sample was applied to a gel filtration column (PD-10) and eluted with 0.1 M phosphate buffer, pH 6.0 containing 6 M urea and 10 mM EDTA.
• the protein fractions were pooled (unfolded annexin V) .
• SMCC-scuPA was conjugated with unfolded annexin V by incubation overnight at room temperature. Aliquots of scuPA/annexin V conjugate were applied to SDS-polyacrylamide gels and were separated electrophoretically. SMCC-scuPA alone, annexin V alone and molecular weight markers served as controls. Formation of the conjugate was confirmed as a species of molecular weight equal to the sum of the molecular weights of the two components.
• Example VII was activated by plasmin. Fifty microliters of scuPA/annexin V conjugate
• scuPA cDNA can be derived from any of a number of available scuPA clones, for example, plasmid psV-GI-preUK (JP-A-60-180591, EP-A-253241 or EP-A-154272) .
• a promoter such as at the Clal site of the trp promoter (EP 0152830) and available in vectors pDR720 from PL Biochemicals and pGX112 from Genex, Gaithersburg, MD
• SEQ ID NO: 2 a synthetic DNA having the following base sequence (SEQ ID NO: 2 ) can be used:
• the synthetic DNA comprises the nontranslated region from the Clal site of scuPA to the ATG codon and the region ranging from the ATG codon to the TthHB ⁇ l site, which corresponds to the 10th amino acid residue, with an Xhol site upstream from the Clal site for joining to an expression vector capable of functioning in yeast as well.
• the synthetic DNA was ligated to the 5' terminus of scuPA cDNA to enable operably linking the scuPA cDNA to a trp promoter.
• Annexin V cDNA can be derived from, for example, the clone of Funakoshi et al., (1987b) supra, Fujikawa et al., U.S. Pat. No. 4,937,324 or plasmid pPAP-I-1.6, see Example II.
• the synthetic DNA having the following base sequence was used as a hirudin linker (Biol. Chem. Hoppe-Seyler (1986) 367, 731-740) for joining the C-terminus of scuPA to the N-terminus of annexin V.
• the hirudin linker (synthesized using known techniques) comprises a base sequence encoding the 50th to 64th amino acid residues of the amino acid sequence of hirudin (SEQ ID NO:3) .
• UK*-1 l ⁇ hirudin Arg lie Arg Ser His Thr Lys Glu Glu Asn Gly Leu Ala Leu Ser His Asn Asp Gly Asp
• plasmid pMT017 ( Figure 1) was constructed using plasmid pYN5, which was prepared by inserting a trp promoter/operator (p/o) into pBR322 (JP-A-60-160887 or EP-A-158230) ; and a second plasmid comprising a sequence prepared by ligating Bglll linkers at both ends of trpA (trp attenuator which is a terminator having a polyT tail, commercially available from PL Biochemicals) having the following base sequence:
• Plasmid pMT017 comprises under the control of the trp promoter, scuPA cDNA, a base sequence encoding 15 amino acid residues corresponding to the 50th to 64th amino acid residues of hirudin, annexin V cDNA and the trp attenuator. Plasmid pMT017 was introduced into an appropriate E. coli host, such as E.
• Plasmid pMT024 ( Figure 2) , a Pichia expression vector, was constructed using plasmids pMT017 and pAO807NX, which was prepared by converting the EcoRI cloning site of plasmid pAO807N (JP-A-2-104290 or EP-A-344459) to an Xhol site using conventional methods described in, for example, Molecular Cloning, supra. Plasmid pMT024 comprises under the control of the A0X1 promoter, a base sequence corresponding to a signal sequence of Mucor rennin (Hiramatsu et al., J. Biol. Chem.
• Plasmid pMT024 was introduced into an appropriate Pichia yeast host, such as P * Lchia pastoris GTS115 (NRRL Y-15851) , following NotI digestion (The fragment was EtOH precipitated and suspended in TE buffer at a concentration of about 2 mg/ml. A lO ⁇ l portion was added to lOO ⁇ l of competent cells.
• His* transformants were selected.) and the transformed cells were incubated at 30 ⁇ C for 3 days in a medium containing 1% yeast extract, 2% Bacto Peptone and 1% MeOH to allow the cells to secrete gene expression products.
• the recombinant products were prourokinase/annexin V conjugates (hybrid protein) having a molecular weight of about 85k and reactive with both anti-urokinase antibody and anti-annexin V antibody. The products possessed thrombolytic activity in a fibrin plate assay.
• Plasmid pMR391 ( Figure 3), a CHO cell expression vector, was constructed using plasmids pMT017 and psV-Gl-preUK, described hereinabove, in accordance with conventional methods, for example, described in Molecular Cloning, supra. Plasmid pMR391 comprises under the control of the SV40 enhancer/promoter, a SV40 splice junction, scuPA cDNA, a base sequence encoding 15 amino acid residues corresponding to the 50th to 64th amino acid residues of hirudin, annexin V cDNA and SV40 poly A signal sequences.
• CHO cells were transformed with plasmid pMR391 and the transformed cells were cultivated at 37 ⁇ C for 3 days in MEM supplemented with 10% FCS to allow the cells to secrete gene expression products.
• the products were prourokinase/annexin V conjugates (hybrid protein) having a molecular weight of about 90k and reactive with both anti-urokinase antibody and anti-annexin V antibody.
• the products possessed thrombolytic activity in a fibrin plate assay.
• SMCC-scuPA is conjugated with modified annexin V with an additional cysteine residue at the N-terminus by incubation overnight at room temperature.
• the modified annexin V was made by ligating to the 5' end of the annexin V coding sequence an oligonucleotide encoding about ten amino acids including a cysteine residue at the amino terminal end.
• Aliquots of scuPA/modified annexin V conjugate are applied to SDS-polyacrylamide gels and are separated electrophoretically.
• SMCC-scuPA alone, modified annexin V alone and molecular weight markers serve as controls. Formation of the conjugate is confirmed as a species of molecular weight equal to the sum of the molecular weights of the two components.
• Annexin V (8 nmoles) in 250 ⁇ l of 50 mM triethanolamine (TEA)-HCl buffer, pH 8.0 containing 50 mM KC1 and 1 mM MgCl 2 , was mixed with 2.5 ⁇ l of 0-mercaptoethanol (final concentration of 1%) and cooled to 0"C. Then varying concentrations of iminothiolane (10 ⁇ l) were added to individual samples and allowed to stand for 20 min at 0°C.
• TEA triethanolamine
• the samples were desalted on Fast Desalting columns (Pharmacia, HRlO/10) with 50 mM Tris-HCl, pH 7.4 containing 50 mM NaCl, 1 mM EDTA and 0.05% NaN 3 (the buffer was flushed with N 2 before use) and protein-containing fractions (1.1 ml) were collected.
• the amount of newly introduced sulfhydryl group was determined using Ell an's reagent (Ellman, Arch. Biochem. Biophys. (1958) 74, 443).
• the clotting activity was determined as described above and the percent inhibitory activity was calculated from a standard curve constructed using authentic annexin V.
• the moles of sulfhydryl group (SH) per mole of annexin V and the percent clotting activity are shown in Table II. The inhibitory activity of annexin V was retained after modification.
• the amount of iodoacetyl group (IA) introduced into scuPA was determined as follows: iodoacetyl-scuPA was mixed with known amounts of reduced glutathione and the remaining glutathione was assayed using Ellman's reagent. The amidolytic activity of iodoacetyl-scuPA was assayed using the peptide substrate, S-2444, after it was activated by plasmin. Table III shows the moles of iodoacetyl group per annexin V and the amidolytic activity of iodoacetyl-scuPA. The results indicate that iodoacetyl-scuPA has the same amidolytic activity as intact scuPA.
• Sulfhydryl-annexin V and iodoacetyl-scuPA that were prepared as described above were mixed and allowed to stand for 1 hour at 37 ⁇ C in dark.
• the products were applied to and separated in an SDS-polyacrylamide gel and examined by immunoblot analysis using anti-scuPA and anti-annexin V antibodies (6.7 nmol of three different preparations of sulfhydryl-annexin V, i.e., moles of sulfhydryl group/annexin, 1.31, 0.82 and 0.57, were conjugated with 6.7 nmol of iodoacetyl-scuPA) .
• Annexin V-Nl is designed to be an amino-terminally extended molecule in which the sequence of Met-Ala-Cys- Pro-Ser-Gly-Gly-Pro-Ser-Gly-Gly-Pro-Met (SEQ ID NO:5) is connected to the amino-terminal Ala residue of mature annexin V.
• the SH group of the third Cys residue may be used for conjugates with iodoacetyl-scuPA.
• annexin V-Nl DNA For the construction of annexin V-Nl DNA, two complementary oligonucleotides containing Ncol sites. (5'-CATGGCATGCCCGTCTGGTGGTCCGTCTGGTGGTCC-3• (SEQ IDNO:6) and 5•-CATGGGACCACCAGACCGACCACCAGACGGGCATGC-3' (SEQ ID NO:7) were synthesized, purified, phosphorylated and annealed by standard methods. The plasmid pPAP-1-319 (Tait & Smith, Arch. Biochem. Biophys. (1991) 288, 141) was digested with Ncol.
• the double-stranded oligonucleotide then was ligated into the plasmid to create plasmid, pPAP-I-Nl.
• DNA sequencing confirmed that the recombinant plasmid contained the intended sequence.
• the recombinant protein was expressed and purified as described (Tait & Smith, supra) .
• Transformed E. coli cells were collected from 10 liters of culture medium and washed once with 50 mM Tris-HCl buffer, pH 7.4 containing 50 mM NaCl. Cells were suspended in 250 ml of phosphate buffer, pH 7.4 containing 6 M urea, 1% Triton X-100, 0.1 mM diisopropyl fluorophosphate and 10 mM EDTA and sonicated for 3 x 1 min. The cell lysate was centrifuged for 20 min at 15,000 rpm and the supernatant was collected followed by dialysis against 50 mM Tris-HCl buffer, pH 7.4 containing 50 mM NaCl, 1 mM EDTA.
• the sample then was applied to a Mono Q column (Pharmacia) with the same buffer and eluted with an NaCl gradient (0.05-1 M) .
• Annexin V-Nl was detected by dot-blot and the fractions containing annexin V-Nl were pooled and concentrated by ultrafiltration.
• the concentrated sample was applied to a TSKgel G3000 SWXL (20 x 500 mm) column and the fractions containing annexin V-Nl (detected by dot-blot) were pooled and concentrated.
• annexin V-Nl The inhibitory activity of annexin V-Nl was determined by clotting activity as described above and the results shown that annexin V-Nl and the activity as described above and the results showed that annexin V-Nl and the wild type annexin have similar activities.
• the protein sequence analysis confirmed that annexin V-Nl had the intended amino terminal oligopeptide with deletion of the amino terminal methionine.

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