WO1998034638A1

WO1998034638A1 - Maspin inhibition of tpa activity

Info

Publication number: WO1998034638A1
Application number: PCT/US1998/001657
Authority: WO
Inventors: Shijie Sheng; Ruth Sager
Original assignee: Dana-Farber Cancer Institute; Pardee, Arthur
Priority date: 1997-02-06
Filing date: 1998-01-29
Publication date: 1998-08-13
Also published as: AU6253998A

Abstract

A method for controlling the in vivo activity of tissue plasminogen activator (tPA) is described. The method includes steps of (a) administering a therapeutically effective amount of tPA to a mammalian subject and (b) administering a therapeutically effective amount of maspin to the mammal. The maspin is administered when excessive tPA activity is detected in vivo.

Description

MASPIN INHIBITION OF TPA ACTIVITY

Statement as to Federally Sponsored Research Work leading to this invention was supported, in part, by National Institutes of Health Grant No. CA 61253. Therefore, the government has certain rights in the invention.

Background of the Invention The invention relates to biochemistry, cell biology, and cancer treatment.

Maspin is a protein present in epithelial cells of normal mammalian breast tissue, but absent from such cells in tumors derived from breast tissue. The maspin protein exhibits tumor suppressor activity in breast and prostate cancers. It inhibits cell motility and invasion (Sheng et al . , 1996, Proc . Natl . Acad . Sci . USA 93:11669- 11674) . In nude mice, it inhibits growth and metastasis of breast carcinoma cells transfected with the maspin gene (Zou et al . , 1994, Science 263:526-529). See U.S. Patent No. 5,470,970.

Maspin is a serine protease inhibitor (serpin) . This classification was initially based on sequence homology to known serpins such as plasminogen activator inhibitor type 1 and type 2. It was not based on observed inhibition of proteases. Studies with recombinant maspin indicate that maspin acts at the cell membrane (Sheng et al . , 1996, supra) . Maspin's inhibition of cell migration and invasion across a reconstituted basement membrane (Matrigel matrix) depends on an intact reactive site loop (RSL) (Zou et al . , supra ; Sheng et al . , 1996, supra ; Sheng et al . , 1994, J. Biol . Chem . 269:30988-30993; Sager et al . , 1996, In Current Topics in Microbiology and Immunology, Vol. 213/1, pp. 51-64, Springer-Verlag, Berlin.

In 1995, Pemberton et al . concluded that the maspin RSL does not function as an inhibitor of tPA or any of several other proteases tested:

[T] he reactive site loop of maspin exists in an exposed conformation that does not require activation by cofactors . The reactive site loop of maspin, however, does not act as an inhibitor of proteinases such as chymotrypsin, elastase, plasmin, thrombin, and trypsin but rather as a substrate. Maspin is also unable to inhibit tissue and urokinase type plasminogen activators. ... [M] aspin cannot undergo the stressed-relaxed transition typical of proteinase- inhibitory serpins .... It is likely therefore, that maspin is structurally more closely related to ovalbumin and angiotensinogen, and its tumor suppressor activity is independent of a latent or intrinsic trypsin-like serine proteinase- inhibitory activity. (Pemberton et al . , 1995, J^". Biol . Chem . 270:15832-15837)

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin. The enzymatic activity of tPA is enhanced by the presence of fibrin. When introduced into the systemic circulation at pharmacologic concentration, tPA binds to fibrin in thrombi, where it converts thrombus-entrapped plasminogen to plasmin. The plasmin then degrades fibrin in the thrombus. Thus, tPA is a fibrinolytic agent used to treat acute myocardial infarction, pulmonary embolism, and stroke. See generally, Physicians ' Desk Reference (50th ed.), 1996, pp. 1058-1061; Wagstaff, et al . , 1995, Drugs 50:289-316. Recombinant tPA is marketed by Genentech, Inc. (South San Francisco, CA) under the name ACTIVASE^® . The most common complication of tPA administration is bleeding (Califf et al . , 1988, Am . J. Med . 85:353-359; Bovill et al., 1991, Ann . Int . Med . 115:256-265). Bleeding associated with tPA administration can be internal or superficial . Internal bleeding can occur in intracranial or retroperitoneal sites, or it can occur in the gastrointestinal, genitourinary, or respiratory tracts. Superficial bleeding associated with tPA adminstration can occur at locations such as surgical incisions, arterial puncture sites, and catheter insertion sites.

The activity of endogenous tPA is tightly regulated in vivo (Plow et al . , 1995, FASEB J. 9:939- 945) . Naturally occurring tPA is secreted from cells (primarily endothelial) as an active enzyme (Pennica et al., 1983, Nature 301:214-221). Natural regulation of tPA activity can occur at the level of protein synthesis (Kooistra et al . , 1994, J^". Hematol . 59:233-255), enzyme release (Gualandris et al . , 1996, J. Neurosci . 16:2220- 2225) , cell surface receptor-mediated endocytosis (Camani et al., 1994, Intl . J. Hematol . 60:97-109), and allosteric/biochemical modulations by both stimulatory and inhibitory molecules (Mullertz, 1956, Acta Physiol . Scand . 38 (Suppl. 130): 1-66; Astedt et al . , 1985, Scan . J. Clin . Lab . Invest . 45:429-435; Kruithof et al . , 1986, J. Biol . Chem . 261:11207-11213; Espana et al . , 1993,

Thrombosis and Haemostasis 70:989-994). The substrate of tPA, plasminogen, stimulates tPA activity at high concentrations (0.6-2.5 μM) by binding to a second substrate binding site on the N-terminal domain, i.e., the A-chain, of sctPA (Geppert et al . , 1992, Arch . Biochem . Biophys . 297:205-212). Inhibition of tPA can be exerted by PAI-1 (Kruithof et al . , 1986, J. Biol . Chem. 261:11207-11213), PAI-2 (Astedt et al . , 1985, Scan . J. Clin . Invest . 45:429-435), protein C inhibitor (Espana et al., 1993, Thrombosis and Haemostasis 70:989-994), and benzamidine-type small molecules (Hu et al . , 1996, Biochemistry 35:3270-3276) .

Summary of the Invention Tissue plasminogen activator has now been identified as a maspin target. It has been discovered that biologically active recombinant maspin specifically binds to single-chain tPA (sctPA) and inhibits fibrinogen-associated sctPA activity, i.e., cleavage of plasminogen to plasmin. The invention includes a method for controlling the in vivo activity of tPA. The method includes the steps of: (a) administering a therapeutically effective amount of tPA to a mammal, e.g., a human patient; and (b) administering a therapeutically effective amount of maspin to the mammal. The maspin can be administered when excessive tPA activity is detected.

Administration of maspin is parenteral . Preferably, it is intravenous or intra-arterial . The maspin can be administered by bolus injection or gradual infusion.

Excessive in vivo tPA activity can be detected by any suitable test or criterion. Typically, excessive tPA activity is indicated by internal bleeding or superficial bleeding. Internal bleeding can be detected at any anatomical site, e.g., intracranial, retroperitoneal, gastrointestinal, genitourinary, or respiratory locations. Superficial bleeding can be detected at any anatomical site, e.g., surgical incisions, arterial puncture sites, and catheter insertion sites. The maspin dose can be initiated at a low level and increased incrementally, until the desired reduction in tPA activity is achieved. Preferably, the dosage of maspin produces a serum maspin/tissue plasmin activator ratio between about 10:1 and about 0.1:1. More preferably, the dosage of maspin produces a serum maspin/tissue plasmin activator ratio between about 5:1 and about 1:1.

As used herein, "maspin" means the 375 amino acid protein described by Zou et al . (1994, Science 263:526- 529) or any mutein or fragment thereof that binds specifically to tPA and inhibits tPA-catalyzed conversion of plasminogen to plasmin. Preferably, the mutein will share at least 80% sequence identity with the maspin described by Zou et al . Preferably, the fragment will include at least the RSL peptide, i.e., CIEVPGARILQHKDEL (SEQ ID NO:l) .

As used herein, "sequence identity" means the percentage of identical subunits at corresponding positions in two sequences when the two sequences are aligned to maximize subunit matching, i.e., taking into account gaps and insertions. When a subunit position in both of the two sequences is occupied by the same monomeric subunit, e.g., if a given position is occupied by an adenine in each of two DNA molecules, then the molecules are identical at that position. For example, if 7 positions in a sequence 10 nucleotides in length are identical to the corresponding positions in a second 10- nucleotide sequence, then the two sequences have 70% sequence identity. Preferably, the length of the compared sequences is at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides. Sequence identity is typically measured using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) .

Unless otherwise defined, each technical and scientific term used herein has the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present application, including definitions, will control. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference.

Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are described below. The materials, methods, and examples are illustrative only and not intended to be limiting.

Various features and advantages of the invention will be apparent from the detailed description, and from the claims.

Brief Description of the Drawings

Fig. 1 is a chromatogram illustrating sctPA purification from MDA-MB-435 conditioned medium. The chromatogram shows protein concentration as a function of fraction number during elution of bound material from a maspin RSL peptide affinity column.

Fig. 2 is a histogram illustrating immunological identification of sctPA in fraction 5 using tPA ELISA detection (A₄₉₀) . Open bars, fraction 5 immunoreactivity; shaded bars, high molecular weight uPA immunoreactivity; black bar, sctPA immunoreactivity.

Fig. 3 is a histogram illustrating dose-dependent inhibition of plasmin-generating activity in fraction 5 by an anti-tPA monoclonal antibody. Black bars, tPA activity; open bars, fraction 5 activity; shaded bars, uPA activity. The plasmin-generating activity was assayed using a chromogenic plasmin substrate. Activities were normalized against those of the same samples in the absence of antibody. The error bars represent the standard deviations from two experiments. Fig. 4 is a graph illustrating dose-dependent binding of sctPA to immobilized rMaspin(i) . Background (10 ng of BSA was coated in place of rMaspin(i)) was subtracted from the absorbance of each reaction. Data represent an average of duplicates. Error bars are standard errors .

Fig. 5 is a graph illustrating dose-dependent reduction of detectable immobilized sctPA by rMaspin(i) . Background (10 ng of BSA was coated in place of sctPA) was subtracted from the absorbance of each reaction.

Data represent an average of duplicates. Error bars are standard errors .

Fig. 6 is a histogram illustrating the effect of microenvironment on the activity of rMaspin(i) toward sctPA. The final concentration of rMaspin(i) was 0.48 μM. The concentrations of other reagents are as described in the specification {infra) . Bar 1, control with sctPA activity determined under optimized assay condition; bar 2, rMaspin(i) added with plasminogen and plasmin substrate to preincubated mixture of setPA and fibrinogen/gelatin (37 C, 1 hr) ; bar 3, sctPA, fibrinogen/gelatin, rMaspin(i), plasminogen and plasmin substrate added simultaneously; bar 4, sctPA, plasminogen and plasmin substrate added to preincubated mixture of fibrinogen/gelatin and rMaspin(i) (37°C, 1 hr) ; bar 5, Fibrinogen/gelatin, plasminogen and plasmin substrate added to preincubated mixture of rMaspin(i) and sctPA (37 C, 1 hr) ; bar 6, plasminogen and plasmin substrate added to preincubated mixture of rMaspin(i), sctPA and fibrinogen/gelatin (37°C, 1 hr) . The initial velocities were obtained as described in Equation 1 ( infra) . The sctPA activities were normalized and presented as percentage of the positive control (2.5 pM plasmin/min) in bar 1. Error bars show standard deviations of duplicate determinations.

Fig. 7 is a double-reciprocal plot of 1/v vs. 1/s of the sctPA plasminogen-dependent activities in the presence of saturating fibrinogen/gelatin and various concentrations of rMaspin(i). The substrate, s, was plasminogen. The velocities were calculated as the production of plasmin per min. Each point represents the average of quadruplicate results.

Fig. 8 is a replot of the slopes of the double reciprocal plot vs. the concentration of rMaspin(i). The intercept on the horizontal axis indicates the Ki value as 0.13 μM.

Fig. 9 is a graph illustrating the dose-responsive effect of rMaspin(i) on the plasminogen-dependent sctPA activity in the presence of saturating fibrinogen/gelatin under the optimized assay conditions. The error bars represent the standard deviations of two parallel experiments.

Fig. 10 is a graph illustrating the dose- responsive effect of rMaspin(i) on the plasminogen- dependent sctPA activity when rMaspin(i) was used instead of fibrinogen/gelatin under the optimized assay conditions. The error bars represent the standard deviations of two parallel experiments.

Fig. 11 is a schematic diagram illustrating the kinetic model for the effect of maspin on tPA.

Detailed Description The invention provides a simple and rapid method for controlling the in vivo proteolytic activity of exogenous tPA after the tPA has been administered to a mammal, e.g., a human cardiac or stroke patient. According to this invention, maspin can be used in conjunction with conventional tPA formulations and conventional therapeutic or prophylactic treatment methods involving administration of tPA. Indications and contraindications for tPA, as well as dosage and administration, are known to those of skill in the art. See, e.g., Physicians ' Desk Reference (50th ed.), 1996, pp. 1058-1061. The most common adverse reaction to tPA therapy is bleeding, especially when the tPA is administered in conjuction with an antithrombotic agent such as heparin or aspirin. The bleeding can be serious, or even fatal, particularly when it occurs at a critical location, e.g., intracranial, gastrointestinal, retroperitoneal, or pericaridal (Gore, et al . , 1991, Circulation 83:448-459). For this reason, tPA therapy requires careful monitoring to detect bleeding at critical sites. The risk of serious bleeding limits tPA dosage in thrombolytic therapy.

According to this invention, when a physician detects excessive in vivo tPA activity, e.g., tPA- associated bleeding, the physician administers an effective amount of maspin. Substantially immediately upon contact with tPA, the maspin inhibits tPA enzymatic activity. Both the tPA and maspin are administered parenterally . Preferably, they are administered intravenously or intra-arterially.

Maspin production, purification, and formulation Maspin used in this invention is preferably obtained by recombinant DNA technology. Recombinant maspin can be obtained according to methods described in Sheng et al . , 1994, "Production, Purification, and Characterization of Recombinant Maspin Proteins," J^". Biol . Chem. 49:30998-30993.

Maspin used in this invention can be formulated using conventional methods to prepare pharmaceutically acceptable compositions. See, e.g., Remington ' s

Pharmaceutical Sciences (E.W. Martin) . The composition can include a pharmaceutically acceptable buffer, e.g., phosphate-buffered saline (PBS) . The composition can include one or more components for adjusting osmotic activity, e.g., sodium chloride, mannitol, or sorbitol .

Maspin dosage

Suitable maspin dosage will depend on various factors, including tPA dosage previously administered, time since last tPA dosage, degree of excess tPA activity, anatomical location of excess tPA activity, and overall condition of the patient. Maspin dosage can be increased incrementally, until the desired reduction in tPA activity is achieved. Preferably, the maspin dosage is sufficient to achieve a serum maspin/tPA ratio between about 10:1 and about 0.1:1. More preferably the maspin dosage is sufficient to achieve a serum maspin/tPA ratio between about 5 : 1 and about 1:1.

Experimental Examples Experiments were conducted to isolate and identify maspin binding protein (s). MDA-MB-435 cells were chosen for these experiments, because they displayed maspin- dependent inhibition of motility and invasion (Zou et al . , supra; Sheng et al . , 1996, supra ; Sheng et al . , 1994, supra ; Sager et al . , 1996, supra) . Using affinity columns incorporating rMaspin(i) or maspin RSL peptide, no maspin binding protein was identified from the total cell extract or the fractionated cell extract proteins of MDA-MB-435 cells. To search for an extracellular protein target, an affinity column of maspin RSL peptide was constructed, and conditioned DFCI-1 medium from MDA-MB-435 cell culture was loaded onto it. A single-peak (Fraction No 5 and 6) protein elution profile was obtained with 0.1 M glycine/pH 2.5 as shown in Fig. 1.

The affinity chromatography fractions were tested for plasminogen-dependent proteolytic activity by substrate incorporated zymographic gel electrophoresis . Other analyses, described below, were also performed.

Cell culture and materials

Human mammary carcinoma MDA-MB-435 cells from American Type Culture Collection (Rockville, MD) were cultured in C.MEM (GIBCO BRL, Grand Island, NY) supplemented with 5% fetal calf serum (HyClone

Laboratory, Logan, UT) (Band et al . , 1990, Cancer Res . 50:7351-7357). When the cells were approximately 70% confluent, the medium was switched to DFCI-1 medium (Band et al., 1989, Proc . Natl . Acad . Sci . USA 86:1249-1253). After 24 hours of cell culture, conditioned medium was collected.

The maspin reactive site loop (RSL) peptide, CIEVPGARILQHKDEL (SEQ ID NO:l), was synthesized and HPLC- purified at the Molecular Biology Core Facility of Dana- Farber Cancer Institute. Glutamate-type plasminogen, high molecular weight urokinase, chromogenic plasmin substrate Spectrozyme PL, chromogenic urokinase substrate Spectrozyme UK, inhibitory monoclonal antibody against sctPA and IMUBIND tPA Strip-well ELISA kits were obtained from a commercial source (American Diagnostica,

Greenwich, CT) . Unless otherwise specified, all other chemicals and reagents were purchased from Sigma Chem. Co. (St. Louis, MO) . Recombinant maspin from cultured insect cells (rMaspin (i) ) , and yeast cells (rMaspin(y)) was produced and purified as described by Sheng et al . , 1994, supra . Trypsin-cleaved rMaspin (y) was a gift from LXR BioTechnology, Inc. (Richmond, CA) .

Binding of tPA to immobilized maspin RSL

A maspin RSL peptide affinity column was prepared by conventional methods. When the column was treated with culture medium conditioned by mammary carcinoma cell line MDA-MB-435, a single peak of material bound to the column. The peak contained sctPA. Identification of sctPA was based on molecular mass, plasminogen-dependent proteolytic activity, an ELISA test, and specific inhibition of enzymatic activity by a monoclonal antibody directed against sctPA.

The affinity column was prepared using commercial gel (SULFOLINK™, Pierce Chem. Co., Rockford, IL) according to the vendor's recommendations. The maspin RSL peptide (5 mg) in PBS (pH 7.4) was mixed with 1 ml of SulfoLink gel. The gel mixture was incubated at room temperature for 1 hour and packed into a column (1 cm i.d.) . The column was washed with PBS prior to loading conditioned DFCI-1 medium from the MDA-MB-435 cell culture. Non-specifically bound proteins were removed from the column by washing with PBS until no protein was detected in the column through-put . Protein was measured by the Bradford method (Bradford, 1976, Anal . Biochem. 72:248-254). Protein specifically bound to the column was eluted with 0.1 M glycine (pH 2.5) . Chromatographic fractions were neutralized with 0.1 volume of 1.0 M Tris/HCl (pH 9.5) . Proteins from the fractions were analyzed by conventional 4-20% gradient SDS-PAGE and silver staining (Bio-Rad Silver Stain kit, Bio-Rad, Hercules, CA) . ELISA detection of sctPA in the affinity chromatography fractions was performed using a commercial kit (IMUBIND tPA Strip-well ELISA kit) according to the vendor' s recommendations .

Formation of tPA/Maspin complex

Recombinant Maspin (i) and tPA in different molar ratios were mixed in 50 mM Tris/HCl (pH 7.5) containing 0.1% Triton X-100 in a final volume of 25 μL. Samples were incubated at 37 C for 15 minutes and denatured at 85°C in SDS-PAGE sample buffer for 2 minutes. Protein samples separated by SDS-PAGE (7.5% gel) were transferred to a PVDF membrane. Maspin and maspin complexes were detected by Western blot analysis using 1000 -fold diluted polyclonal antiserum against maspin AbS3A and enhanced- chemiluminescent (ECL) reagents (Amersham, Arlington Heights, IL) . AbS3A was designed to elicit antibodies that recognize amino acids 169-182 near the middle of the maspin primary sequence (Zou et al . , supra) .

ELISA assay for sctPA/rMaspin (i) complex Each well of a 96-well microtiter plate was coated with rMaspin (i) (10 ng) or sctPA (10 ng) in 0.2 M sodium bicarbonate buffer (pH 9.3) overnight at 4°C. The negative control wells were each coated with 10 ng BSA in the same buffer. Subsequent steps were performed at room temperature. The plate was washed with PBS/0.1% Triton X-100 and blocked with 5% BSA/PBS (pH 7.2) for 1 hour. The sctPA was added to rMaspin (i) coated wells. Recombinant maspin (i) was added to sctPA-coated wells. Following an incubation for 1 hour, the plate was washed and treated with an anti-sctPA monoclonal antibody, at a final concentration of 5 μg/ml , with 1% BSA/PBS/0.1% Triton X-100, for 1 hour. Subsequently, the plate was treated with 1000-fold diluted horse radish peroxidase (HRP) -conjugated goat-anti-mouse IgG (Amersham,

Arlington Heights, IL) for 1 hour. Bound HRP was detected colorimetrically with 10 mM 2, 2'-azino-bis (3- ethylbenzthiazoline 6-sulfonic acid) diammonium

(substrate) in 0.1 M acetate/pH 4.2 and quantitated according to absorbance at 405 nm.

Assay for plasminogen-to-plasmin conversion

Chromogenic plasmin substrate Spectrozyme PL (0.2 mM final cone.) and glu-type plasminogen (83 nM final cone.) were added to a pre-incubated mixture of sctPA (0.4 NIH units), fibrinogen (0.4 μM) and gelatin (133 μg/ml) in 50 mM Tris/HCl (pH 7.5) containing 0.1% Triton X-100 (1 hour at 37°C) . No sctPA was added in the blank. The final volume of each reaction was 150 μl . The absorbance of the reaction mixtures at 405 nm was monitored at 37 °C for 2 hours, using a microplate reader (Bio-Rad Model 3550) . To determine the dissociation constant of tPA/rMaspin(i) intermediate, the concentration of plasminogen was varied for a series of concentrations of rMaspin(i), under the standard tPA assay conditions.

The velocity of plasmin production was determined

2 from the plots of A₄₀₅ vs. t based on Equation (1) : ^vplasmin production ^{= e A} 02 ^cat2 - where A₄₀₅ and e are absorbance and absorption coefficient at 402 nm of the product derived from spectrozyme PL, respectively, t is time in min, and k_cat is the rate constant for the catalysis.

The activity of uPA was assayed by two different methods. In one method, uPA was assayed using a uPA- specific chromogenic substrate, Spectrozyme UK, in 50 mM Tris/HCl (pH 8.0) at 37°C. Alternatively, uPA activity was determined under the optimized assay condition for sctPA. Plasmin activity was assayed in 50 mM Tris/HCl, 0.1% Triton X-100 (pH 7.5), using Spectrozyme PL as substrate. Elastase was assayed in 0.1 M HEPES/0.5 M NaCl/pH 7.5 with substrate N-methoxysuccinyl-ala-ala-pro- val p-nitroanilide. Chymotrypsin activity was assayed in 0.2 M phosphate buffer (pH 6.2), using N-succinyl-ala- ala-pro-phe p-nitroanilide as substrate. Trypsin was assayed in 50 mM Tris/HCl (pH 7.5) using the same substrate used for chymotrypsin.

Zvmography, SDS-PAGE, and ELISA

Zymography was performed essentially as described in Lorand et al . , 1993, Meth . Enzymol . 22:22-35. The following materials were loaded onto separate lanes of a gelatin substrate incorporated zymogram gel with 10 μg/ml plasminogen: an aliquot of culture medium conditioned by MDA-MB-435 cells; fraction 5 from maspin RSL peptide affinity column; high molecular weight uPA; and sctPA.

The zymogram gel was washed with two changes of 2% Triton X-100 in ddH₂0 in a 1 hour period. The washed gel was incubated at 37 °C in 0.1 M glycine (pH 8.3) for 2 1/2 hours at 37 C. After this incubation, the gel was stained with 0.25% Coomassie brilliant blue R-250 and destained with methanol/acetic acid/water (1:1:8). Human sctPA, human high molecular weight uPA and porcine pancreatic elastase were used as size references.

No plasminogen activator activity was detected in fresh DFCI-1 culture medium containing 1% FCS . The conditioned DFCI-1 medium had low detectable activity in converting plasminogen to plasmin, about 100-fold less than that of fractions 5 and 6. The clear zone resulting from fraction 5 appeared at a position corresponding to a molecular mass similar to that of sctPA. The proteolytic activity of fraction 5 was estimated to be 1 IU/ml by reflection densitometry. All the other fractions were negative in this assay. Zymogram assays for metalloprotease and plasminogen- independent protease activities were performed and all the elution fractions tested negative.

SDS-PAGE followed by silver staining revealed four major protein components in the original conditioned medium, with bovine albumin being the most abundant . Fraction 5 had additional components (unidentified) with apparent molecular weights ranging from 14 kD to 90 kD . Fractions 5 and 6 differed markedly from adjacent fractions. Fraction 3 had a pattern similar to the conditioned medium, while fraction 8 had little detectable protein.

When tested with a tPA-capturing ELISA kit, fraction 5 gave a dose-dependent positive signal (Fig. 2), estimated to be 15 ng/ml . At the same protein concentration, uPA gave a substantially negative result. When the plasminogen-dependent proteolytic activity was analyzed in solution using a chromogenic substrate, a commercial sctPA standard and an aliquot of elution fraction 5 lost their activities in a dose-dependent manner with increasing concentration of the anti-tPA antibody (Fig. 3) . In contrast, the uPA standard was not inhibited by this antibody. Thus, the immunological and enzymatic activity assays confirmed the presence of sctPA in fraction 5.

Detergent-resistant rMaspin (i) /tPA complex

The interaction of sctPA with maspin was demonstrated by detection of a detergent-resistant complex of recombinant maspin, rMaspin(i), and sctPA, on Western blots, using an anti-maspin antibody. In addition, sctPA in the complex was identified by reciprocal competitive ELISA assays.

A binding interaction between maspin and sctPA proteins was indicated by the purification of sctPA from the maspin RSL peptide affinity column. Two ELISA assays were used to confirm the binding interaction between sctPA and rMaspin (i) . Bound sctPA was detected by a monoclonal antibody. In the first ELISA, sctPA in the range of 0-20 ng was bound to rMaspin (i) coated wells in a dose-dependent manner (Fig. 4) . At sctPA levels greater than 10 ng, the binding of setPA appeared to approach saturation. These results indicated that the binding of se PA to rMaspin (i) was dependent on the amount of immobilized rMaspin (i). In the second ELISA assay, rMaspin (i) was added to sctPA coated wells. As shown in Fig. 5, rMaspin (i) reduced detectable setPA in a dose-dependent manner. At rMaspin (i) levels greater than 10 ng, sctPA decreased to almost background level. Because the binding of sctPA to rMaspin (i) did not result in sctPA degradation (Fig. 4), the decrease in detectable sctPA was probably due to masking of the antibody recognition site on sctPA by tight binding of rMaspin (i) . To determine whether maspin could form a stable complex with sctPA, acting as a typical inhibitory serpin, mixtures of rMaspin (i) and sctPA in various molar ratios were incubated at 37 C for 15 min and analyzed by SDS-PAGE and Western blotting using maspin antibody AbS3A. When the molar ratio of maspin/sctPA was between 1 and 5, a band whose apparent molecular weight corresponded to the combined molecular weights of sctPA and maspin, i.e., approximately 110 kDa, was detected. The 110 kDa band was not observed with the polymerized rMaspin (i) sample, or with setPA alone. The 110 kDa band represents a complex between maspin protein and sctPA. s the molar ratio between maspin and sctPA was further increased, the 110 kDa band was no longer detected. rMaspin (i) inhibition of tPA requires fibrinogen/gelatin Recombinant maspin (i) inhibited fibrinogen/gelatin activation of sctPA, but not urokinase-type plasminogen activator (uPA) , plasmin, chymotrypsin, trypsin, or elastase. The Ki for rMaspin (i) /setPA binding was found to be 0.13 μM. This inhibitory potency is comparable to that reported for PAI-2, i.e., 0.55 μM (Astedt et al . , 1985, Scan . J. Clin . Lab . Invest . 45: 429-435). Maspin's inhibitory potency, however, is less than that reported for PAI-1, i.e., 3.3 nM (Kruithof et al . , 1986, J^". Biol . Che . 261: 11207-11213) .

In the presence of saturating fibrinogen and gelatin, sctPA was inhibited competitively by up to 0.5 μM rMaspin (i) . As the concentration of rMaspin (i) was further increased, sctPA activity was stimulated, producing a bell-shaped dose-response curve. The maximal inhibition and inflection point depended upon the plasminogen concentration. A slight activity of sctPA, seen in the absence of fibrinogen/gelatin, was stimulated by rMaspin (i) . These data are consistent with a two step model in which maspin first competes with plasminogen to complex sctPA plus fibrinogen, and then an additional maspin molecule replaces fibrinogen as the activating component. Under the assay conditions used, rMaspin (i) had no effect on several other serine proteases, including uPA.

Optimal activity of sctPA in converting plasminogen to plasmin requires the presence of fibrin (ogen) (Mullertz, 1956, Acta Physiol . Scand . 38(suppl. 130):l-66) and of a nonspecific protein, e.g., casein or gelatin (Thorsen et al . , 1972, Throm . Diath . Haemorrh . 28:65-74). Fibrinogen at a saturating concentration of 0.4 μM, together with gelatin at a final concentration of 133 μg/ml, were used in our optimized sctPA activity assay. The order and time of mixing these components affected the results, as shown in Fig. 6. When sctPA and fibrinogen/gelatin were preincubated at 37°C for 1 hour prior to the addition of plasminogen, rMaspin (i) inhibited the sctPA activity by approximately 50% (bar 2) . When rMaspin (i) was added without the preincubation of sctPA and fibrinogen/gelatin (bars 3 and 4), it was not inhibitory. Thus, rMaspin (i) inhibited the fibrinogen/gelatin associated sctPA, but not the free sctPA.

Several known serpins have an arginine at the P₁ site and cross-inhibit several trypsin-like serine proteases (Lomas et al . , 1993, J^". Biol . Chem . 268:516- 521). The possible cross-reactivity of rMaspin(i), which also has an arginine residue at its P_x position, was tested with five trypsin-like serine proteases, i.e., setPA, high MW uPA, plasmin, trypin and chymotrypsin. Elastase was also tested. The assays were performed with and without inclusion of fibrinogen/gelatin. The results are summarized in Table 1. Recombinant Maspin (i) inhibited only sctPA.

Table 1 Effect of rMaspin (i) on Proteolytic Activities rMaspin (i) /

Protease rMaspin (i) Fibrinogen/gelatin sctPA NDE Ki = 0.13 μM uPA NDE NDE

Plasmin NDE NDE Chymotrypsin NDE NDE Trypsin NDE NDE Elastase NDE NDE

NDE indicates "no detectable effect."

Maspin competitively inhibited sctPA at low concentration and stimulated sctPA at high concentration

A kinetic analysis of rMaspin (i) inhibition of setPA was performed. rMaspin (i) added together with plasminogen and the chromogenic substrate of plasmin to the preincubated mixture of sctPA and fibrinogen/gelatin inhibited the proteolysis in a dose-dependent manner. As the concentration of rMaspin (i) was increased up to 0.48 μM, the production of plasmin decreased, as demonstrated in Fig. 7 by the increase in the slope of the l/v_initial vs. 1/ [plasminogen] plot (Km of plasminogen = 5.3 nM) . The activity was about 75% inhibited at 10 nM plasminogen. These double-reciprocal plots intercept nearly on the 1/v axis. The replot of the slopes of the double-reciprocal plots vs. rMaspin (i) concentration was linear (Fig. 8), consistent with maspin's role as a competitive inhibitor of the activation by sctPA of plasminogen. The deduced Ki value from this replot was 0.13 μM.

Under optimized sctPA assay conditions with high plasminogen (83 nM) , competitive inhibition by rMaspin(i) was low (Fig. 4, bar 2) . An inverse bell-shaped dose- response curve was obtained when rMaspin (i) was tested over a broad range of concentrations (Fig. 9) . Recombinant maspin (i) was a competitive inhibitor of sctPA, at concentrations lower than 0.48 μM. At rMaspin (i) concentrations above 0.48 μM, sctPA activity also increased. The spontaneous polymerization of rMaspin (i) at higher concentrations possibly could result in a reduced inhibitory efficiency. However, when the concentration of rMaspin (i) reached 2.4 μM, the sctPA activity was higher than the control with no maspin protein. This indicated that the increase of sctPA activity was not simply due to a decrease in inhibitory potency. In accordance with this stimulation of preincubated sctPA plus fibrinogen/gelatin, when rMaspin (i) (0.48 μM) and setPA were preincubated prior to the addition of fibrinogen/gelatin and plasminogen, the sctPA activity was slightly stimulated (Fig. 6, bar 5), and even more from a preincubation of rMaspin (i), sctPA and fibrinogen/gelatin prior to the addition of plasminogen (Fig. 6, bar 6).

To determine whether fibrinogen/gelatin was required for stimulation of sctPA by rMaspin (i), rMaspin (i) was used instead of fibrinogen/gelatin in the standard assay. Recombinant maspin (i) had only a stimulatory effect on sctPA activity, especially at higher concentrations (> 0.5 μM) . As shown in Fig. 10, sctPA activity was steadily elevated when the concentration of rMaspin (i) was increased. As with fibrinogen/gelatin, the stimulation of sctPA by rMaspin (i) appeared to approach saturation. Thus, rMaspin (i) can stimulate sctPA in the absence of fibrinogen/gelatin .

A kinetic model was developed, based on the assumption that a second maspin molecule binds to the regulatory site of sctPA to replace fibrinogen/gelatin (Fig. 11) . This model explained the stimulatory effect of rMaspin (i) coexisting with or in the absence of fibrinogen/gelatin. (K(s) are dissociation constants, whereas k(s) are rate constants.) With both plasminogen and the plasmin substrate at constant saturating concentrations, initial velocities of plasmin production were calculated using Equation 2 with the following kinetic parameter settings: KI = 0.1 μM, K2 = 2.75 μM, k_χ = 1.25 x 10^"4/min, k₂ = 4 x 10^"4/min. The plot of calculated initial velocities vs. maspin concentration coincides with the experimental results (Fig. 9) . According to this model, the effect of maspin alone on sctPA activity was calculated using the same set of kinetic parameters except that k_x was set at 0. This plot of the calculated initial velocities vs. maspin concentration also coincides with the experimental results (Fig. 10) . Thus, these data support the kinetic model .

Other embodiments are within the following claims.

SEQUENCE LISTING

(1) GENERAL INFORMATION

(i) APPLICANT: Dana Farber Cancer Institute, Inc.

(ii) TITLE OF THE INVENTION: MASPIN INHIBITION OF TPA ACTIVITY

(iii) NUMBER OF SEQUENCES: 1

(iv) CORRESPONDENCE ADDRESS:

(A) ADDRESSEE: Fish & Richardson P.C.

(B) STREET: 225 Franklin Street (C) CITY: Boston

(D) STATE: MA

(E) COUNTRY: US

(F) ZIP: 02110-2804

(v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Diskette

(B) COMPUTER: IBM Compatible

(C) OPERATING SYSTEM: Windows95

(D) SOFTWARE: FastSEQ for Windows Version 2.0

(vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER:

(B) FILING DATE:

(C) CLASSIFICATION:

(vii) PRIOR APPLICATION DATA:

(A) APPLICATION NUMBER: 60/039,304 (B) FILING DATE: 06-FEB-1997

(viii) ATTORNEY/AGENT INFORMATION:

(A) NAME: Creason, Gary L.

(B) REGISTRATION NUMBER: 34,310

(C) REFERENCE/DOCKET NUMBER: 00530/084WO1 (ix) TELECOMMUNICATION INFORMATION:

(A) TELEPHONE: 617/542-5070

(B) TELEFAX: 617/542-8906

(C) TELEX: 200154

(2) INFORMATION FOR SEQ ID NO : 1 : (i) SEQUENCE CHARACTERISTICS:

(A) LENGTH: 16 amino acids

(B) TYPE: amino acid

(D) TOPOLOGY: linear

(ii) MOLECULE TYPE: peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO : 1 :

Cys lie Glu Val Pro Gly Ala Arg lie Leu Gin His Lys Asp Glu Leu

1 5 10 15

Claims

1. The use of maspin for the manufacture of a medicament for the treatment or prevention of bleeding resulting from excessive tissue plasminogen activator (tPA) activity in a mammal.

2. The use of claim 1, wherein the treatment or prevention of bleeding includes administering a therapeutically effective amount of maspin to said mammal intravenously or intra-arterially.

3. The use of claim 1, wherein said mammal is a huma .

4. The method of claim 1, wherein said bleeding is intracranial,^' retroperitoneal, gastrointestinal, genitourinary, respiratory, or superficial .

5. The use of claim 1 or 2 , wherein therapeutically effective amount of maspin produces a serum maspin/tissue plasmin activator ratio between about 10:1 and 0.1:1.

6. The method of claim 4, wherein said therapeutically effective amount of maspin produces a serum maspin/tissue plasmin activator ratio between about 5 : 1 and 1:1.