WO2001038560A2

WO2001038560A2 - Active enzyme detection using immobilized enzyme inhibitors

Info

Publication number: WO2001038560A2
Application number: PCT/US2000/032315
Authority: WO
Inventors: Daniel A. Lawrence; Duane Day
Original assignee: American Red Cross
Priority date: 1999-11-22
Filing date: 2000-11-22
Publication date: 2001-05-31
Also published as: WO2001038560A3; AU1801101A

Abstract

The present method utilizes the capture of the functionally active form of an enzyme that covalently binds or binds with a dissociation constant of 1 x 10-9M or less to an enzyme inhibitor or mutant thereof. The present invention is directed to a method for detecting a functionally active form of an enzyme in a biological sample, comprising contacting an enzyme inhibitor or mutant thereof immobilized on a solid substrate with the biological sample, and measuring the binding of the enzyme inhibitor or mutant thereof to the active form of the enzyme by a detectable label. Specific enzyme inhibitors or mutants thereof are designed to covalently bind to specific clinically important enzymes. These enzyme inhibitors contain modifications that affect the binding to a target enzyme or affect the stability or the inhibitor. The nethod is particularly useful in measuring the presence of enzymes, such as tPA, elastase, cathepsin G and prostate specific antigen.

Description

NOVEL DETECTION METHOD FOR A FUNCTIONALLY ACTIVE FORM OF AN ENZYME IN BIOLOGICAL SAMPLES AND A KIT

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for detecting the presence of a functionally active form of a target enzyme in a biological sample by contacting an immobilized enzyme inhibitor or a mutant thereof with a biological sample and determining the extent of binding of the functionally active form of the target enzyme in the biological sample to the immobilized enzyme inhibitor or mutant thereof. The present invention particularly relates to a method of detecting the presence of a functionally active form of a proteinase in a biological sample. The present invention also relates to an analytical element comprising the immobilized enzyme inhibitor and a kit containing the analytical element for use in performing the method of the present invention.

2. Description of the Related Art

There are many enzymes that are considered to be of great clinical importance because they play a role in a variety of biological processes, and therefore, it is important to determine the presence of and to measure the amount of these enzymes present in body fluids and tissues. Of these important enzymes, the measurement of proteinases, and particularly, serine proteinases, are useful for determining the role of these enzymes in specific clinical conditions, and for following the presence and levels of these enzymes during treatment regimens.

The enzymes of the plasminogen activator (PA)-system are examples of such clinically important enzymes and are members of the serine proteinase family of proteins (Kraut, 1977). The serine proteinases are so named because they have a serine residue in the active site that is involved in the catalytic cleavage of peptide bonds. This family of proteins contain two subgroups, the trypsin-like enzymes and the subtilisin-like enzymes (Neurath, 1984). All of the enzymes in the PA-system belong to the trypsin-like subset of serine proteinases.

It is believed that the serine proteinases, and most other enzymes, bring about catalysis by providing a template that can recognize and stabilize an activated transition state intermediate of the substrate. This hypothesis was first put forward by Linus Pauling over 50 years ago (Pauling, 1946; Pauling, 1948). Figure 1 shows one proposed mechanism of peptide bond cleavage by the serine proteinases. In the first step the enzyme recognizes its substrate. Substrate recognition, or specificity, of the serine proteinase is for the most part determined by the binding pocket, or S specificity crevice, which binds the side chain of the amino acid after which cleavage occurs (Kraut, 1977). Following binding of the substrate, a stabilization of transition state geometry is achieved and a nucleophilic attack on the peptide bond to be cleaved takes place. The nucleophilic attack is from the oxygen atom on the serine side chain and is facilitated by the acceptance of a hydrogen ion by a histidine residue which in turn is stabilized by an aspartic acid residue. The three invariable residues, Ser, Asp, His in all serine proteinases, are called the catalytic triad. Next, an acyl-enzyme intermediate is formed between the new carboxy-terminal of the substrate and the serine residue of the enzyme. The freed carboxy-terminal peptide of the substrate is then replaced by a water molecule and the process proceeds in reverse, a new transition state intermediate is formed as a molecule of water is split. The transition state intermediate then breaks down and the active site serine residue is regenerated along with a free carboxy-terminal on the substrate ((Kraut, 1977), Warshel et al., 1989).

There is clinical interest in measuring the active form of the specific serine proteinases of the PA-system, such as: plasminogen, a single chain glycoprotein with a molecular weight of approximately 92 kDa (Wallen, 1980); tissue type plasminogen activator (tPA), a single chain glycoprotein of 527 amino acids with a molecular weight of approximately 65 kDa and containing 7-13% carbohydrate (Rijken, 1988); and urokinase- type plasminogen activator (uPA), a single chain glycoprotein with a molecular weight of 54 kDa containing approximately 7 % carbohydrate with the mature protein consisting of 411 amino acids (Blasi et al. , 1987).

Additionally, there is interest in measuring coagulation enzymes, such as protein C, a vitamin K dependent serine proteinase involved in the regulation of the coagulation cascade, as well as Factor XII and prekallikrein, both zymogens that are part of the intrinsic coagulation system (Lammle & Griffin, 1985).

In addition to proteinases of the PA-system there also are a number of other proteinases that are important to measure accurately. For example, the inflammatory proteinases, such as neutrophil elastase and cathepsin G. Elastase is a serine proteinase released by activated neutrophils and macrophages and monocytes. During inflammatory responses, neutrophils are activated and release elastase leading to tissue destruction through proteolysis. In the lung, elastase degrades elastic tissues and leads to emphysema. Elastase is also a compounding factor in cystic flbrosis (CF) and in both adult and infant acute respiratory distress syndrome (ARDS). Elastase has also been implicated in TNF- mediated inflammation (Massague et al., 1993) and HIN infection (Bristow, et al., 1995). Elastase has a broader spectrum of reactivity than plasminogen activators each of which acts preferentially on a precursor substrate to activate it.

The natural defense to elastase is a protein called αi anti-trypsin (αiAT), also called αi-proteinase inhibitor (αiPI). Patients who are deficient in αiAT are prone to emphysema, especially smokers. Furthermore, smoking provokes inflammation. In such αiAT deficiencies, the enzyme is present (CRM⁺ - cross reacting material) (Dorland's Illustrated Medical Dictionary, 1974) but is functionally impaired. In addition, even in individuals with normal enzyme, smoking directly inactivates αiAT. Therefore, the determination of the presence and measurement of the levels of elastase in susceptible subjects would be highly desirable to monitor the disease state during treatment.

Considerable information is known about enzyme inhibitors, particularly proteinase inhibitors. The wide distribution of proteinase inhibitors and their ability to regulate many different proteinases suggest that there are a large number of inhibitors that are useful in the present invention to measure the presence of many different proteinases. Particularly useful as enzyme inhibitors are the serpins which is the designation used for a family of serine proteinase inhibitors.

The serpins are a gene family that encompasses a wide variety of protein products, including many of the proteinase inhibitors in plasma (Huber and Carrell, 1989). However, in spite of their name, not all serpins are proteinase inhibitors. They include steroid binding globulins, the prohormone angiotensinogen, the egg white protein ovalbumin, and barley protein Z, a major constituent of beer. The serpins are thought to share a common tertiary structure (Doolittle, 1983) and to have evolved from a common ancestor (Hunt and Dayhoff, 1980). Proteins with recognizable sequence homology have been identified in vertebrates, plants, insects and viruses but not, thus far, in prokaryotes (Huber and Carrell, 1989; Sasaki, 1991; Komiyama et al., 1994). Current models of serpin structure are based largely on seminal X-ray crystallographic studies of one member of the family, αi-antitrypsin (o^AT) (Huber and Carrell, 1989). The structure of a modified form of αiAT, cleaved in its reactive center, was solved by Loebermann and co workers (Loebermann et al., 1984). An interesting feature of this structure was that the two residues normally comprising the reactive center (Met-Ser), were found on opposite ends of the molecule, separated by almost 70 A. Loebermann and co workers proposed that a relaxation of a strained configuration takes place upon cleavage of the reactive center peptide bond, rather than a major rearrangement of the inhibitor structure. In this model, the native reactive center is part of an exposed loop, also called the strained loop (Loebermann et al., 1984; Carrell and Bos well, 1986; Sprang, 1992). Upon cleavage, this loop moves or "snaps back", becoming one of the central strands in a major β-sheet structure (β-sheet A). This transformation is accompanied by a large increase in thermal stability, presumably due to reorganization of the six stranded β-sheet A (Carrell and Owen, 1985; Gettins and Harten, 1988; Bruch et al., 1988; Lawrence et al., 1994a).

Recent crystallographic structures of several native serpins, with intact reactive center loops, have confirmed Loebermann's hypothesis that the overall native serpin structure is very similar to cleaved αjAT, but that the reactive center loop is exposed above the plane of the molecule (Schreuder et al., 1994; Carrell et al., 1994; Stein et al., 1990; Wei et al., 1994); Sharp et al, 1999). Additional evidence has come from studies where synthetic peptides, homologous to the reactive center loops of at AT, antithrombin III (ATIII), or plasminogen activator inhibitor- 1 (PAI-1) when added in trans, incorporate into their respective molecules, presumably as a central strand of β-sheet A (Bjδrk et al., 1992a; Bjδrk et al., 1992b; Schulze et al., 1990; Carrell et al., 1991; Kvassman et al., 1995). This leads to an increase in thermal stability similar to that observed following cleavage of a serpin at its reactive center, and converts the serpin from an inhibitor to a substrate for its target proteinase. A third serpin structural form has also been identified, the so-called latent conformation. In this structure the reactive center loop is intact, but instead of being exposed, the entire amino-terminal side of the reactive center loop is inserted as the central strand into β-sheet A (Mottonen et al. , 1992). This accounts for the increased stability of latent PAI-1 (Lawrence et al. , 1994b) as well as its lack of inhibitory activity (Hekman and Loskutoff, 1985). The ability to adopt this conformation is not unique to PAI-1, and has also now been shown for ATIII and c^AT (Carrell et al., 1994; Lomas et al., 1995). Together, these data have led to the hypothesis that active serpins have mobile reactive center loops, and that this mobility is essential for inhibitor function (Carrell et al., 1991; Carrell and Evans, 1992; Lawrence et al., 1994a; Shore et al., 1994; Lawrence et al., 1995; Fa et al. , 1995; Olson et al., 1995; Lawrence et al., 1990).

The serpins act as "suicide inhibitors" that react only once with a target proteinase to form an SDS-stable complex. They interact by presenting a "bait" amino acid residue, in their reactive center, to the enzyme. This bait residue is thought to mimic the normal substrate of the enzyme and to associate with the specificity crevice, or SI site, of the enzyme (Carrell and Boswell, 1986; Huber and Carrell, 1989; Bode and Huber, 1994). The bait amino acid is called the PI residue, with the amino acids toward the N-terminal side of the scissile reactive center bond labeled in order PI P2 P3 etc. and the amino acids on the carboxyl side labeled PI' P2' etc. (Carrell and Boswell, 1986). The reactive center Pl-Pl' residues, appear to play a major role in determining target specificity. This point was dramatically illustrated by the identification of a unique human mutation, αjAT "Pittsburgh", in which a single amino acid substitution of Arg for Met at the PI residue converted at AT from an inhibitor of elastase to an efficient inhibitor of thrombin, resulting in a unique and ultimately fatal bleeding disorder (Owen et al., 1983). Numerous mutant serpins have been constructed, demonstrating a wide range of changes in target specificity, particularly with substitutions at PI (York et al., 1991; Strandberg et al., 1991; Shubeita et al. , 1990; Lawrence et al. , 1990; Sherman et al., 1992).

The exact structure of the complex between serpins and their target proteinases is not known. It has been suggested that the complex is a tight non-covalent association similar to proteinases in complex with the small peptide inhibitors of the Kazal and Kunitz family, or that it is frozen in a tetrahedral transition state configuration (Matheson et al., 1991; Longstaff and Gaffney, 1991; Shieh et al., 1989; Potempa et al. , 1994). However, several more recent studies have indicated that the complex is covalently linked via an ester bond between the active site serine residue of the proteinase and the new carboxyl-terminal end of the PI residue, forming an acyl-enzyme complex. See Fig. 2. (Nilsson and Wiman, 1982; Cohen et al., 1977; Fa et al., 1995; Wilczynska et al., 1995); Lawrence et al , 1995) In this model a serpin binds to its target proteinase forming a reversiblecomplex analogous to a Michaelis complex between an enzyme and substrate. Next, the proteinase cleaves the PI -PI' reactive center peptide bond resulting in formation of a covalent acyl- enzyme intermediate (Lawrence et al., 1995; Wilczynska et al., 1995). This cleavage is coupled to a rapid insertion of the reactive center loop into b-sheet A at least up to the P9 position (Shore et al., 1994). Since the reactive center loop is covalently linked to the enzyme via the active-site serine, this shift must also affect the proteinase, significantly altering its position relative to the inhibitor. If, the reactive center loop is prevented from attaining full insertion because of its attachment to the enzyme, and the complex becomes locked, with the reactive center loop only partially inserted, then the resulting stress might be sufficient to distort the active site of the enzyme. This distortion could then prevent efficient deacylation of the acyl-enzyme intermediate, thus trapping the complex. However, if reactive center loop insertion is blocked (Bjork et al., 1992; Lawrence et al. , 1994), or if deacylation occurs before reactive center loop insertion, then the cleaved serpin is turned over like any other substrate and the active enzyme released. This suggests that what determines whether a serpin is an inhibitor or a substrate is the ratio of the rate of deacylation to the rate of loop insertion. If deacylation is faster than reactive center loop insertion then the substrate reaction predominates. However, if reactive center loop insertion and distortion of the active site can occur before deacylation, then the complex is frozen in a covalent acyl-enzyme form. Such a scheme for stabilization of the serpin complex was first proposed in 1990 (Lawrence et al., 1990), and is consistent with studies demonstrating that reactive center loop insertion is not required for proteinase binding but is necessary for stable inhibition (Lawrence et al., 1994) and with the observation that only an active enzyme can induce reactive center loop insertion (Olson et al., 1995). More recently, direct evidence for this model was elegantly provided by Plotnick et al., who by NMR observed an apparent distortion of an enzyme's catalytic site in a serpin-enzyme complex (Plotnick et al. , 1996). Together, these data suggest that serpins act as molecular springs where the native structure is kinetically trapped in a high energy metastable state. Upon association with an enzyme some of the energy liberated by reactive center loop insertion is used to distort the active site of the enzyme, preventing deacylation and trapping the complex. PAI-1 is the major plasminogen activator (PA) inhibitor in plasma and platelets (Booth et al., 1988; Fay et al., 1992; Fay et al., 1994). The PAI-1 gene is 12.3 kb in length, and yields two mRNA species of 2 kb and 3 kb that both encode the same 50 kDa single-chain glycoprotein (Ny et al., 1986; Strandberg et al., 1988; van Mourik et al., 1984). PAI-1 is the most efficient inhibitor known of both uPA and tPA (Lawrence et al., 1989; Sherman et al. , 1992). It is a member of the serpin family. PAI-1 can exist in multiple conformational states, including an active and latent form (Hekman and Loskutoff, 1985). Active PAI-1 decays to the latent form with a half-life of approximately 1 hour at 37 °C. With exposure to denaturants (guanidine HCl or SDS), latent PAI-1 can be returned partially to the active form. Though recent X-ray crystallographic findings suggest a structural basis for these two conformations (Mottonen et al., 1992), their biological significance remains unknown. Negatively-charged phospholipids can convert latent PAI-1 to the active form, suggesting that cell surfaces may modulate PAI-1 activity (Lambers et al., 1987). The observation that latent PAI-1 infused into rabbits is apparently converted to the active form is consistent with this hypothesis (Vaughan et al., 1990). Kinetic and other evidence has also been presented for a second site of interaction between PAI-1 and tPA, outside of the PAI-1 reactive center (Chmielewska et al. , 1988; Lawrence et al., 1990; Hekman and Loskutoff, 1988). PAI-1 also binds to other non-proteins ligands such as vitronectin and the lipoprotein receptor related protein (LRP) (Lawrence et al , 1997; Stefansson et al , 1998). In addition, it has been previously demonstrated that PAI-1 shows a marked sensitivity to inactivation by oxidants, apparently involving a critical Met residue (Lawrence and Loskutoff, 1986; Strandberg et al., 1991). A similar sensitivity to oxidation has also been observed for other serpins and may represent a common mechanism for regulation of serpin activity in vivo.

The present invention utilizes enzyme inhibitors and mutants thereof, such as proteinase inhibitors, particularly PAI-1 and altered or mutated forms of PAI-1, to capture the functionally active form of an enzyme in a biological sample and then detect its presence via measuring the presence of a label. Although methods of measuring the presence and/or levels of an enzyme in a biological sample by employing an inhibitor of the enzyme and/or antibodies to the enzyme are known in the art, none of these prior art methods utilize an immobilized enzyme inhibitor or mutant thereof, to capture a functionally active form of an enzyme in a biological sample and then detect the presence of this enzyme as disclosed in the present invention. It is believed that no one has been able to immobilize the inhibitor without destroying the inhibitor's ability to bind to its binding partners (Deng et al. , 1995).

U.S. 4,458,013 discloses a competitive immunoassay in solution for measuring elastase- 1 in body fluids in which none of the reagents are immobilized. An inhibitor of elastase- 1 is contacted with the sample containing elastase- 1 and with a labeled form of elastase- 1. The amount of elastase- 1 in the sample is determined by the addition of an antibody specific for the elastase- 1. The quantity of the elastase- 1 is calculated by measuring the activity of the labelling agent in the antibody-bound labelled antigen on the basis of standard values plotted previously with respect to the standard antigen. This method differs from the present invention by not utilizing any immobilized reagents. This method also differs from the method of the present invention because it does not utilize the elastase- 1 inhibitor as the enzyme capture step, but rather utilizes an antibody that binds to the elastase- 1- elastase- 1 inhibitor complex. This assay provides an assay with a lower specificity for the target enzyme than the present method.

U.S. 4,753,875 discloses a method for determining a proteinase by contacting the sample containing the proteinase with a tagged proteinase inhibitor to form a mixture of proteinase and tagged proteinase inhibitor complexes. The mixture of complexes is then incubated with an antibody specific for the proteinase to form complexes of antibody- proteinase-tagged proteinase inhibitor complexes that are separated from the free tagged proteinase inhibitor and then either the antibody-proteinase-tagged proteinase inhibitor complexes or the free tagged proteinase inhibitor is measured. The anti-proteinase antibody may be immobilized on a solid support to facilitate separation of the antibody- proteinase-proteinase inhibitor complex that binds to the antibody . This method requires that the proteinase inhibitor be tagged and the present invention does not. This method does not immobilize the proteinase inhibitor to a solid support but rather immobilizes the anti-proteinase antibody. Even though there may be specificity between the proteinase and proteinase inhibitor, the antibody is the reagent used in the capture step to detect the proteinase. This method suffers from the same lack of specificity as methods using anti- proteinase antibody as the initial step to capture the antibody. The present invention differs from this method by utilizing strong covalent binding or a low disocciation constant that equates with a high binding affinity between the proteinase and proteinase inhibitor as the capture step. In doing so the present invention provides a method with increased specificity that is particularly useful to detect funcitonally active proteinases in a biological sample in which the active proteinase is a small percentage of the total proteinase. The present invention provides an advance over U.S. 4,753,875 by utilizing a high affinity capture step and less manipulative steps to provide a sensitve, commercial assay for the detection of enzymes in biological samples.

U.S. 4,868,106 discloses an analytical element and method of using the element to determine the presence of a specific component A in a sample based upon a specific reaction between the specific component A and a substance B that binds to A by the use of a label that provides a signal. The element contains a porous reaction layer composed of a carrier with immobilized B and a carrier having an immobilized adsorbing substance that binds to the label that has not bound to B. The analytical element of the present invention does not require a porous reaction layer with the immobilized binding partner of A and an adsorbing substance that binds to the label of the labelled material that has not bound to substance B, thereby modulates the signal. The analytical element of the present invention differs from this prior art analytical element by immobilized directly to a derivatized plastic support or other comparable supports via a method that allows the immobilized enzyme inhibitor to retain the property of specifically forming a covalent bond or binding with a dissociation constant of 1 x 10 ^"9 M or less with the functionally active form of the enzyme in the biological sample.

U.S. 5,096,811 discloses a method for in vitro measuring of in vivo levels of human tissue-type plasminogen activator (t-PA) from plasma that comprises contacting plasma with a thrombin inhibitor (D-Phe-Pro-Arg-Chloromethyl ketone) and thereby inactivating the t-PA and measuring its presence in the plasma via an antibody to t-PA. This method does not disclose immobilizing the thrombin inhibitor to a solid support and does not disclose that the thrombin inhibitor can distinguish between the active and inactive form of t-PA. This method is only useful for determining the total amount of t-PA present in the sample and cannot detect the amount of active free t-PA present in the sample.

U.S. 5,288,612 discloses a method for determining the amount of proteinase, such as protein C, in a body fluid sample, through the use of an antibody specific to the proteinase, immobilized to a solid support. The method includes contacting an irreversible inhibitor of interfering proteinases with the immobilized antibody to prepare a proteinase- free immobilized antibody support. The method further includes mixing a reversible inhibitor of the proteinase to be detected with a biological sample and then contacting this mixture with the proteinase free immobilized antibody support. The reversible proteinase inhibitor is removed and the proteinase that binds to the immobilized antibody is measured. This elaborate method is used to increase the sensitivity of the enzyme assay by removing interfering proteinases. This method also measures total proteinase in the body sample and not just active proteinase. This method further differs from the present method by not disclosing an immobilized proteinase inhibitor and also relies on an antibody to capture the proteinase for measurement of the enzyme.

U.S. 5,416,003 discloses a method of detecting an enzymatically active hydrolase, commonly associated with candidiasis, in a sample by assaying an immobilized reporter enzyme susceptible to hydrolytic cleavage in the presence of a hydrolase. Then the sample which remains in contact with the immobilized reporter enzyme is contacted with an immobilized indicator. If the indicator undergoes a change, then the hydrolase is present in the sample. The method does not immoblize a proteinase inhibitor on a solid support that specifically binds with the target enzyme. U.S. 5,585,273 has a similar disclosure to U.S. 5,416,003.

WO 90/05309 discloses a method of assaying for functional enzymes, specifically serine proteinase. This method immobilizes an antibody to a solid support matrix which is contacted with a sample containing the target enzyme in the presence of a labeled inhibitor, preferably a low-molecular synthetic inhibitor which reacts rapidly with the active free enzymes, to prevent competing physiological inhibitors present in the sample from complexing with the free enzymes . The amount of functional enzyme is measured by the amount of detectable label. Similar to the other prior art patents, the antibody is immobilized not the proteinase inhibitor.

The present invention provides an improved method of detecting the functionally active form of an enzyme in a biological sample utilizing the high affinity of an enzyme inhibitor to an enzyme as the capture step for detecting the enzyme in a biological sample.

This capture step takes advantage of the strong and more specific binding between the immobilized enzyme inhibitor and the active form of the enzyme. An immobilized antibody capture step does not provide this strong binding affinity nor is it able to distinguish between an active and inacative form of any enzyme. The present method's capture step provides a means for detecting low concentrations of functionally active enzymes in a biological sample utilizing a simple, commercial method with few manipulative steps, and represents an advance over the prior art methods and reagents.

The prior art antibody that is specific to the captured enzyme is reacted and detected by a label either directly or indirectly to determine the presence of the enzyme. Several of the patents discussed above use an immobilized antibody which does not provide the specificity that an immobilized enzyme inhibitor provides for the initial capture of the active form of the enzyme from the biological sample. None of these prior art patent documents disclose the present invention that is directed to determining the active form of an enzyme in a complex biological system, such as blood, plasma, serum or tissues in low concentrations.

SUMMARY OF THE INVENTION

The present invention is based upon the discovery of the present inventors that an enzyme inhibitor could be immobilized on a solid substrate yet still retain the capability of forming a covalent bond or binding with a dissociation constant of 1 x 10^"9M or less with the functionally active form of an enzyme. This accomplishment then allowed this immobilized enzyme inhibitor to be used in a method for the detection of a functionally active form of an enzyme in a biological sample which comprises contacting an enzyme inhibitor immobilized on a solid substrate with the biological sample and measuring the binding of the enzyme inhibitor to the active form of the enzyme by a detectable label.

Additionally, the invention is directed to an analytical element for use in the detection of a functionally active form of an enzyme in a biological sample comprising an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein the enzyme inhibitor forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with the active form of the enzyme.

Further, the present invention is directed to a kit useful in performing a method of detecting a functionally active form of an enzyme in a biological sample, comprising an analytical element comprising an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein the enzyme inhibitor specifically covalently binds or binds with a dissociation constant of 1 x 10^"°M or less to the active form the enzyme; and an analytical reagent conjugated to a detectable label or conjugated to a reactive molecule that generates a detectable label, wherein the analtyical element specifically binds to the functionally active form of the enzyme bound to the enzyme inhibitor.

In a further embodiment, the present invention is directed to a method of immobilizing an enzyme inhibitor or mutant thereof to a solid substrate comprising modifying the enzyme inhibitor sufficiently so that the modified enzyme inhibitor binds to the solid substrate while retaining the property of covalently binding to a fiinctionally active form of an enzyme; and contacting the modified enzyme inhibitor with the solid substrate.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows one proposed mechanism of peptide bond cleavage by the serine proteinases.

Figure 2 depicts a schematic representation of the serpin mechanism.

Figure 3a-3d show the nucleotide sequence encoding human PAI-1 with 5' and 3' untranslated regions from a specific clone. Also shown is the amino acid sequence of the full length human PAI-1 including the signal sequence.

Figure 4 is a graph showing the inhibition of neutrophil or pancreatic elastase by wtPAI-1, αiAT or PI Ala-PAI-1. The ordinate represents the residual enzymatic activity following 30 minute incubation with increasing concentrations of the inhibitor.

Figure 5 shows the amino acid sequence of the PAI-1 protein including the signal peptide which is shown in italics. The reactive center loop (RCL) regions is marked by asterisks. Preferred residues for substitution to generate mutants are indicated as the PI (346) and P4 (343) amino acids. Also highlighted are four additional sites which can be substituted to yield a more stable protein: Asnl50, Lysl54, Gln319, Met354 and Val343.

Figure 6 depicts a schematic of the method of the present invention. Panel A represents a polyhistidine tagged PAI-1 bound to a metal-coated microtiter plate. Panel B represents a mixture of active and inactive enzyme added to the metal-coated microtiter plate. Only the active enzyme is captured. Panel C shows the bound active enzyme detected by an antibody specific for the enzyme. The antibody is conjugated to a detectable label. Figure 7 is a graph showing the results of an ELISA in which 6X-His 14 IB PAI-1 is immobilized to a solid support and used as the analytical element to capture tPA in a biological sample.

Figure 8 is a comparison of the method of the present invention and the Biopool method using human tPA. Panel A shows the tPA concentration measured in a linear range between 0.5 and greater than 15 IU/ml of enzyme. The insert shows tPA concentration determined at the lower concentrations. Panel B shows the Biopool Chromolize™ tPA assay that is only linear between 0 and 2 IU/ml.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

The present invention provides a sensitive method for the detection of a functionally active form of an enzyme in a biological sample by contacting an enzyme inhibitor immobilized on a solid substrate with a biological sample, and measuring the binding of the enzyme inhibitor to the active form of the enzyme by a detectable label, wherein the enzyme inhibitor specifically forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with the active form of the enzyme.

The present method provides a superior analytical assay for detecting low levels of the functionally active form of an enzyme in a biological sample because the present inventors utilize an enzyme inhibitor that binds very tightly and with high affinity to the active form of the enzyme. The inventors use this high binding affinity between the enzyme inhibitor and the active form of the enzyme to design a sensitive, simple, easy to use, commercial method of detecting enzymes in biologically samples. The present invention utilizes an immobilized enzyme inhibitor that is contacted with the biological sample as the capture step for pulling the functionally active form of the enzyme from the biological sample milieu. The combination of the specificity of binding to only the active form of the enzyme in combination with using an immobilized enzyme inhibitor to do so, results in an extremely specific and high affinity capture step which is easy to use. Prior to the present invention, enzyme inhibitors of the type described herein could not retain the capability of covalently binding with a corresponding target enzyme after being immobilized on a solid support (Deng et al. , 1995). This high affinity binding is responsible for the increased specificity of the initial capture method of the present invention. As a result of the strength of this covalent binding property, this capture step makes the present method more sensitive than a method employing immobilized antibodies, and is particularly useful in the detection of low levels of functionally active enzymes in a sample.

Comparison of the present capture step with the prior art capture step utilizing an immobilized antibody directed to an epitope on the target enzyme shows that the present capture step is more efficient and sensitive because the immobilized enzyme inhibitors of the present invention covalently bind or bind with a dissociation constant of 1 x 10^"9M or less to a functionally active form of the target enzyme, whereas the antibody - enzyme binding is non-covalent and possesses a dissociation constant of generally greater than 1 x 10^"9M, indicating a lower binding affinity than the enzyme inhibitors encompassed by the present invention and described herein. A common antibody binding affinity is approximately 1 x 10^"9 M or greater calculated in terms of the dissociation constant. The affinity of an antibody can be formulated in terms of the dissociation constant (Kd) of the reaction:

AbH 5 Ab + H

Ab = concentration of the free antibody combining sites H = concentration of free hapten at equilibrium Expressing concentrations in moles per liter:

Kd = rAb moles/11 fH moles/11 [AbH moles/1] Kd is the reciprocal of Ka (the binding affinity defined through the equililibrium constant (Ka) of the association reaction of Ab + H → AbH. Kd has the units moles/1 or Molar. Conversely, Ka has the units 1/mole or M^"1. Thus, the lower the dissociation constant the stronger the binding and the lower the binding affinity (Roitt's Essential Immunology, 1997).

The enzyme inhibitors useful in the present invention are defined by their ability to either bind covalently or bind with a dissociation constant of 1 x 10^"9M or less to a corresponding target enzyme. The corresponding target enzyme in the context of the present invention is defined to encompass: (1) an enzyme to which the enzyme inhibitor would have bound to prior to immobilization to the solid support, (2) an enzyme to which the enzyme inhibitor would have bound to prior to immobilization to the solid support but for which the native or natural binding is not sufficiently high to provide the sensitivity and specificity required by the present invention; i.e., covalent binding or binding with a dissociation constant of 1 x 10^"9M or less or (3) an enzyme to which the enzyme inhibitor would not have bound to prior to immobilization to the solid support but for which the enzyme inhibitor has been modified in its reactive center loop (RCL) resulting in covalent binding or binding with a dissociation constant of 1 x 10^"9M or less to the enzyme. The covalent binding and disssociation constants of enzyme inhibitors that fall within the scope of the present invention can be determined by using the methods known by persons skilled in the art. See Lawrence 1995; Lawrence, et al. 1997; Stefansson, et al. 1998.

The enzyme inhibitor of the present invention can be any substance or molecule that has the property of high specific binding to its corresponding target enzyme, as defined herein, i.e., covalent binding or binding with a dissociation constant of 1 x 10^"9M or less. An enzyme inhibitor within the scope of the present invention is preferably is a proteinase inhibitor or a mutant thereof and preferably the enzyme to be detected is proteinase. More preferably the proteinase inhibitor is selected from the group consisting of serine, cysteine, aspartic, thiol, carboxyl, and metallo-proteinase inhibitors. The enzyme preferably is selected from the group consisting of serine, cysteine, aspartic, thiol, carboxyl, and metallo-proteinases. More preferably the proteinase inhibitor is a serine proteinase inhibitor or serpin, and the enzyme is a serine proteinase.

The serine proteinase detectable by the present method preferably is selected from the group consisting of tPA, uPA, thrombin, plasmin, neutrophil elastase, pancreatic elastase, trypsin, chymotrypsin, cathepsin G and prostate specific antigen.

The serine proteinase inhibitor preferably is a plasminogen activator inhibitor, and more preferably is the plasminogen activator inhibitor- 1 (PAI-1). Most preferably, the PAI-1 is the human PAI-1. In one embodiment, the present invention utilizes enzyme inhibitors or mutants thereof, preferably PAI-1 or mutants thereof, that react with high specificity with a number of enzymes selected from the group consisting of tissue plasminogen activator (tPA), urokinase, thrombin, plasmin, neutrophil elastase, pancreatic elastase, trypsin, chymotrypsin, cathepsin G and prostate specific antigen, with tPA being the most preferred enzyme to be detected by the present method.

The enzyme inhibitor or mutants thereof of the present invention are modified from the natural or native enzyme inhibitor in a particular manner to allow the immobilization of the enzyme inhibitor or mutant thereof to the solid support without losing its ability to specifically binds to the target enzyme either covalently or with a dissociation constant of 1 x 10^"9M or less. These methods of immobilization will be discussed below in detail. Thus, an immobilized native enzyme inhibitor is modified from the native enzyme inhibitor by the changes made to the inhibitor that facilitates immobilization on the solid support. Other enzyme inhibitor mutants useful in the present invention include enzyme inhibitors with additional modifications to the portion of the enzyme inhibitor that is involved in the specific binding to its corresponding target enzyme, such as the RCL, that will enhance the binding specificity and affinity to an enzyme to which it already bound or that will result in a new binding specificity to an enzyme to which the enzyme inhibitor did not previously bind. To be useful in the present invention, these enzyme inhibitor mutants would bind to a target enzyme either covalently or with a dissociation constant of 1 x 10^"9M or less. Additionally, enzyme inhibitor mutants of the present invention also encompass native enzyme inhibitors and those modified in their capacity to bind to a target enzymes as discussed above with additional modifications that increase the stability and shelf life of the inhibitor. Thus, in summary, the enzyme inhibitors of the present invention are modified to allow immobilization to a solid support with at least one of the following modifications: (1) one or more modifications in the portion of the inhibitor that binds to an enzyme that results in enhanced binding specificity or a new binding specificity to an enzyme, such that these enzyme inhibitor mutant bind to a target enzyme either covalently or with a dissociation constant of 1 x 10^"9M or less; and (2) one or more modifications that enhance the stability of the enzyme inhibitor so that it has a longer half life than an enzyme inhibitor that is not so modified. Such modifications may include deleting, substituting or adding amino acid residues to one or more sites within the enzyme inhibitor.

The present method also encompasses immobilizing small synthetic inhibitors of the chloromethylketone (CMK) family to a solid support for use in capturing a functionally active form of the enzyme and detecting it with a detectable label. An immobilized CMK capable of inhibiting any proteinase, particularly serine proteinases and cysteine proteinases, are encompassed by the present invention. The chloromethyl ketone particularly is a tri to dodeca- peptide chloromethyl ketone containing between three to twelve peptides. Preferably, the chloromethyl ketone is a tripeptide chloromethyl ketone with a formula of X-Y-Z-chloromethyl ketone, where X, Y and Z are independently selected amino acids. More preferably, Z is Arg and the tripeptide chloromethyl ketone is selected from the group consisting of: D-Phe-Pro-Arg-chloromethyl ketone; D-Glu-Gly- Arg-chloromethyl ketone; D-Val-Gly-Arg-chloromethyl ketone and D-Ile-Pro-Arg- chloromethyl ketone.

An important aspect of the present invention is the immobilization of an enzyme inhibitor as identified above, to a solid support or substrate, wherein the immobilized enzyme inhibitor still retains the ability to covalently bind to the target enzyme in the biological sample or binding with a dissociation constant of 1 x 10^'9M or less. Appropriate solid supports or substrates are selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate, polybutylene and any other fixed solid substrate to which the enzyme inhibitor binds. A microtiter plate is a preferred solid substrate to which the enyme inhibitor is immobilized. Additionally, the claimed method is suitable for using a dip stick as the solid support to which the enzyme inhibitor is immobilized to allow rapid quantitation of the taget enzyme in the biological sample.

The enzyme inhibitor is modified prior to immobilization so that the enzyme inhibitor binds to the solid substrate while retaining the property of covalently binding or binding with a dissociation constant of 1 x 10^"9M or less to the functioanlly active form of the target enzyme in the biological sample. To achieve immobilization, the enzyme inhibitor or mutant thereof is modified to facilitate immobilization to the solid support and then the so modified enzyme inhibitor is contacted with the solid support under conditions that the enzyme inhibitor or mutant thereof binds and still retains the property of covalently binding or binding with a dissociation constant of 1 x 10^"9M or less. The modification to the enzyme inhibitor or mutant thereof comprises the modification to at least one of the following: the addition of, the substitution of or the deletion of one or more amino acid residues, that facilitates the immobilization to a solid support. The enzyme inhibitor is immobilized to an appropriate solid substrate by specific ineraction with at least one amino acid residue of the enzyme inhibitor. The amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrates; at least one cysteine residue bindable to a maleimide derivatized solid substrate and at least one lysine residue bindable to a N- hydroxysucci iimide derivatized solid substrate. Histidine tagged recombinant proteins are made by methods known in the art. Metal-chelate affinity chromatography has been used for more than twenty years as a means of purifying proteins that possess natural metal binding sites in their structure. Appropriate metals are nickel, cobalt and other comparable metals that would function as these metals. The use of proteins engineered with added metal binding sites (6-X Histidine) are known ( Linder et al.1992, Porath 1992). Petty (1996) also references this technique. It is known in the art to manufacture plasmids designed to produce the His tagged proteins (either at the N or C terminus), and metal chelate resins are available to bind these tagged proteins (Hermanson G., 1996).

The detectable label used to determine the presence of the captured enzyme is selected from the group consisting of a radioactive label, a chromophore, a fluorophore, and any other detectable label. Preferably, the detectable label is conjugated to an analytical reagent, such an antibody that specifically binds to the enzyme, but any analytical reagent that binds to the captured enzyme and is conjugated to a detectable label is useful in the present invention. Alternatively, the analytical reagent is conjugated to a reactive molecule, such as an enzyme, that is capable of generating a detectable label, when contacted with a substrate of the enzyme that results in a color change, such as reagents used in an ELISA. A biotin label on the analytical reagent can be detected by a streptavidin molecule that is attached to a color-genreating agent also is useful in the present invention as a detectable label. Further, there are many labels and methods of labeling known in the analytical art that are useful in the present invention. Those of ordinary skill in the art will know of other suitable labels and methods of conjugating these labels to an analytical reagent or conjugating a reactive molecule to the analytical reagent that generates a label, or is capable of ascertaining such methods using routine experimentation. Furthermore, the binding of these labels to the analytical reagent, such as an antibody, is done using standard techniques such as cross-linking, covalent attachment, non-covalent attachment, or complexing and the like.

Any moiety that binds to the captured enzyme and is conjugated to a label or a reactive molecule is useful in the present invention for detecting the presence of the enzyme. For example, an antibody to the enzyme is preferred but the analytical reagent may also be a peptide, nucleic acid, proteins, co-factors, or any molecule that is known to bind specifically to the enzyme that is bound to the enzyme inhibitor.

The present invention provides an analytical element for use in the detection of a functionally active form of an enzyme in a biological sample comprising an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein the enzyme inhibitor or mutant thereof forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with the active form the target enzyme to be detected. Preferably, the enzyme inhibitor or mutant thereof as defined above. More preferably, the enzyme inhibitor is a proteinase inhibitor or a mutant thereof and the target enzyme is a proteinase. Further, the specific proteinase inhibitors also have been set forth above. The analytical element is a solid substrate and selected from glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene or any other material that allows the immobilization of enzyme inhibitors by a technique in which the enzyme inhibitor maintains its capability to covalently bind or bind with a dissociation constant of 1 x 10^"9M or less. The analytical element preferably is a microtiter plate or a dipstick, but can be a bead, and thus any material of which these particular types of solid support are composed.

The enzyme inhibitor or mutant thereof that is immobilized on the solid support is modified to facilitate such immobilization while retaining the capability to covalently bind or bind with a dissociation constant of 1 x 10^"9M or less with the functionally active form of the enzyme. The enzyme inhibitor is immobilized to the solid substrate by at least one of the following: the addition of, the substituion of or the deletion of one or more amino acid residues. The means and types of immobilization techniques are discussed above.

Examples of preferred immobilization complexes are a polyhistidine residue bindable to a metal chelate solid support; at least one cysteine residue bindable to maleimide derivatized solid support, at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid support or any other amino acid residue bindable complex and solid support combination that results in the immobilization of the enzyme inhibitor that retains the ability to covalently bind or bind with a dissociation constant of 1 x 10^"9M or less to an enzyme. Preferably, the metal chelate is nickel or cobalt and other metals that would function similarly as a chelate to bind histidine.

The present invention also is directed to a kit for the detection of a functionally active form of an enzyme in a biological sample, comprising an analytical element comprising an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein the enzyme inhibitor specifically forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less to the functionally active form of the enzyme; and an analytical reagent that is conjugated to a detectable label or is conjugated to a reactive molecule that is capable of generating a detectable label, where the analytical reagent specifically binds to the active form of the enzyme that is bound to the enzyme inhibitor. The preferred enzyme inhibitor is proteinase inhibitor or mutant thereof and the enzyme is a proteinase. Most preferably, the enzyme inhibitor is PAI-1 and the enzyme is t-PA. Additional enzyme inhibitors useful in the present kit are disclosed above.

The preferred enzyme inhibitor or mutant thereof that is immobilized to the solid support in the present invention is the native or natural PAI-1 has been modified as discussed above to bind it to the solid support. Any modification to the PAI-1 that allows it to be immobilized to a solid substrate while retaining the property to covalently bind or bind with a dissociation constant of 1 x 10^"9M or less to a target enzyme is intended to be encompassed by the present invention and included as an element in the present kit. Specific methods of immobilizing the enzyme inhibitor also have been discussed above.

In addition to the modifications to allow binding to the solid substrate, native or natural PAI-1 may be modified further to obtain enzyme inhibitors mutants, such as mutants of PAI-1, that also are useful in the present invention. Generally, modifications to the enzyme inhibitor PAI-1 are made by persons skilled in the art to change the properties of the native PAI-1 to allow it to be immobilized while retaining its specific binding properties to a corresponding enzyme; i.e. , covalent binding or binding with a dissociation constant of 1 x 10^'9M or less; and may optionally include modifications to change the specificity to bind to a different enzyme or to the same enzyme with enhanced binding specificity; i.e., covalent binding or binding with a dissociation constant of 1 x 10^"9M or less; and/or to increase stability of the enzyme inhibitor over the native one.

The present invention is intended to encompass the immobilization and use of known enzyme inhibitors or mutants thereof, for which the DNA sequences and/or amino acid sequences are known so that modification of the inhibitor can be performed. The skilled person is capable of modifying the sequences of these proteins and testing the modified enzyme inhibitors or mutants for their ability to be immobilized yet still capable of covalently binding or binding with a dissociation constant of 1 x 10^"9M or less to a targeted enzyme.

The analytical reagent useful in the present invention for detecting the extent of enzyme binding to the immobilized enzyme inhibitor has been described above. Additionally, the kit optionally contains a second analytical element comprising a second analytical reagent immobilized on a solid substrate, wherein the second analytical reagent specifically binds to both the active and inactive form of said enzyme. Preferably the second analytical reagent is antibody. The inclusion of the second analytical element permits the determination of the total target enzyme (both active and inactive forms) present in the biological sample. This second analytical element is used in as a standard sandwich ELISA. This information together with the determination of the amount of active enzyme provides the accurate determination of the percentage of enzyme that is active in a biological sample. The standard sandwich ELISA includes the second analytical element comprising an immobilized capture antibody specific for the enzyme of interest as disclosed in the prior art. The biological sample is contacted with the second analytical element under the same conditions as the sample is with the first analytical element comprising the immobilized enzyme inhibitor or mutant thereof. The amount of bound enzyme to the second analytical element will be determined with the same analtyical reagent as used to determine the functionally active form of the enzyme that was captured by the enzyme inhibitor.

PAI-1 cDNA encodes a protein of 402 amino acids that includes a typical secretion signal sequence (Ny et al , 1986; Ginsburg et al , 1986. Mature human PAI-1 isolated from cell culture is composed of two variants of 381 and 379 amino acids in approximately equal proportions. These two forms, likely arising from alternative cleavage of the secretion signal sequence, provide proteins with overlapping amino-terminal sequences of Ser-Ala-Nal-His-His and Val-His-His-Pro-Pro (see Figure 3 and disclosed in Lawrence et al , 1989). This latter sequence is generally referred to as mature PAI-1. The complete amino acid and nucleotide sequence of PAI-1 also is disclosed in Figure 3.

PAI-1 is a glycoprotein with three potential Ν-linked gly cosy lation sites containing between 15 and 20% carbohydrate (Van Mourik et al, 1984). Mature PAI-1 contains no cysteine residues, facilitating efficient expression and isolation of recombinant PAI-1 from E. coli. PAI-1 produced in E. coli, although nonglycosylated, is functionally very similar to native PAI-1. Recombinant PAI-1 can be isolated from E. coli in an inherently active form, which contrasts with PAI-1 purified from mammalian cell culture (Lawrence et al. , 1989; Hekman et al , 1988).

PAI-1 exists in an active form as it is produced by cells and secreted into the culture medium and an inactive or latent form that accumulates in the culture medium over time (Hekman et al, 1985; Levin et al , 1987). The active form spontaneously converts to the latent form with a half-life of about 1 h at 37°C (Lawrence et al. , 1994b; Hekman et al. , 1985; Levin et al, 1987).

The latent form can be converted into the active form by treatment with denaturants, negatively charged phospholipids or vitronectin (VΝ) (Lambers et al , 1987, Hekman et al, 1985; Wun et al, 1989). Latent PAI-1 infused into rabbits became reactivated in vivo by an unknown mechanism. The reversible interconversion between the active and latent structures, presumably due to a conformational change, is a unique feature of PAI-1 as compared to other serpins. The latent form appears to be more energetically favored.

In the three-dimensional structure of the latent form of PAI-1, the entire Ν-terminal side of the reactive center loop is inserted as the central strand into β sheet (Mottonen et al, 1992) which explains the increased stability (Lawrence, et al, 1994b) as well as the lack of inhibitory activity.

The RCL region of PAI-1 has been the subject of extensive mutational analysis which demonstrated the importance of the PI bait residue in inhibitor function, whereas the surrounding amino acids play a less critical role. The amino acid residues in the reactive center loop of PAI-1 (residues 332-351) are shown below labeled according to the foregoing naming convention:

332 333 334 335 336 337 338 339 340 341 342 343 344 345 346 347 348 349350 351 P15 P14 P13 P12 P11 P10 P9 P8 P7 P6 P5 P4 P3 P2 PI PI' P2' P3' P4' P5'

Gly Thr Val Ala Ser Ser Ser Thr Ala Val He Val Ser Ala Arg Met Ala Pro Glu Glu

Random mutagenesis of the P3, P2, and PI residues and the PI and PI' residues, respectively, clearly demonstrate that either arginine or lysine at PI is essential for PAI-1 to function as an effective inhibitor of uPA (York et al , 1991; Sherman et al. , 1992). Residues surrounding PI modulate PAI-1 inhibitor activity by up to two orders of magnitude and alter target proteinase specificity. The PI' site is surprisingly tolerant of amino acid substitutions with the exception of proline which caused almost total loss of function. When an 18 amino acid segment of PAI-1 encompassing most of the RCL was replaced with the same region from PAI-2, antithrombin III, or a serpin consensus sequence, most of the requirements for PAI-1 specificity (apart from the PI residue), were found to lie outside the RCL sequence. All three chimeras remained efficient inhibitors of tPA and uPa, and the antithrombin III chimera was not a signigicantly improved inhibitor of thrombim. Furthermore, the specific sequence of the RCL, the region inserted into β sheet A in the latent PAI-1 structure was not critical for the conversion between the active and latent conformations of PAI-1. Hence, loop insertion depends more on the flexibility of β sheet A than on the specific amino acid residues in the loop. Finally, binding to VN was not affected by these substitutions in the RCL. The P4' and P5' residues on the C- terminal side of the reactive-center bond have also been replaced with only a small effect on PAI-1 activity.

Previously it has been demonstrated that replacement of the wild-type Pι Thr residue in PAI-1 with a charged amino acid resulted in loss of inhibitory activity toward uPA due to conversion of PAI-1 to a substrate (Lawrence, 1994a). Replacement of the Pu

RCL residue in other serpins with charged amino acids have similarly shown a loss of inhibitor function due to transformation to a substrate (Hood et al. 1994; Davis et al,

1992). These findings in conjunction with X-ray crystallographic evidence (Loebermann et al, 1984) have suggested that a conformational change involving insertion of the serpin Pι residue into β-sheet A is an obligate step in the formation of a stable serpin-enzyme complex. It is shown that the association of a catalytically inactive proteinase with PAI-1

, EL ka 7!

Scheme 1: E+I E EL k E + I >

was not affected when the Pι residue was mutated to Arg, has indicated that the triggering of this RCL conformational change requires the action of the proteinase catalytic residues (Olson et al. , 1995). Together, these and other studies (Patston et al. , 1991; Schechter et al , 1993; Cooperman et al , 1993; Gettins et al , 1993; Lawrence et al , 1995; Kvassman et al. 1998) have suggested the hypothesis that serpins inhibit proteinases by the branched pathway mechanism outlined in scheme 1.

According to this mechanism a proteolytic enzyme, E, first binds reversibly to the RCL of the serpin I, to form a Michaelis-like encounter complex, E» I. This complex can either dissociate regenerating free enzyme and active inhibitor, or peptide bond cleavage can be initiated, with formation of a covalent acyl-enzyme intermediate, EL. This is similar to a proteinase reaction with a regular substrate except that the intermediate may be formed reversibly (Kvassman et al , 1998). In this complex L is cleaved inhibitor with its RCL still exposed, and covalently tethered to the active site serine of the enzyme. In the ensuing steps in this pathway there are two potential outcomes for the EL complex. In the first, the serpin RCL rapidly inserts into β-sheet A generating the EL complex, wherein the inhibitor is cleaved, and the RCL is fully inserted, with the inhibitor still tethered to the enzyme as a covalent acyl-enzyme intermediate. This results in stabilization and trapping of the covalent complex presumably due to distortion of the enzyme's active site (Plotnick et al. , 1996; Kaslik et al. 1997). Alternatively, the EL complex can undergo deacylation with release of the active enzyme before serpin RCL insertion. The free cleaved inhibitor with an uninserted RCL (L) can then insert into β-sheet A in a non-productive conformational rearrangement that generates an irreversibly inactivated serpin (L). The present method preferably utilizes PAI-1 or a mutant thereof as the immobilized enzyme inhibitor that covalently binds or binds with a dissociation constant of 1 x 10^"9M or less to the functionally active form of the target enzyme. In addition to modification to allow immobilization of PAI-1, it also is modified or mutated in the RCL so that a mutated PAI-1 is designed that covalently binds to functionally active forms of a specific target enzyme. U.S. Serial No. 08/840,204 filed on April 12, 1996 entitled "Mutant Plasminogen Activator-Inhibitor Type I (PAI-1) and Uses Thereof", herein incorporated in its entirety by reference, discloses modified PAI-ls or PAI-1 mutants that would be suitable for use in the present invention as well as disclosing how such altered PAI-ls are made. Optionally, PAI-1 also is mutatated at selected amino acids to increase the half life of the PAI-1 mutant and increase its stability. Site-directed mutagenesis and other methods are used to produce and characterize a large number of mutations in the PAI-1 reactive center loop (RCL) (Sherman et al., 1992, Sherman et al , 1995). This previous patent application discloses how to make and identify new mutants in the RCL of PAI-1 which confer on PAI-1 new and useful properties, in particular (a) the ability to interact with and inhibit elastase, an activity which is lacking in native PAI-1 and (b) the ability to inhibit vitronectin-associated cell migration.

Two functional classes of mutant PAI-1 molecules are disclosed in U.S. Serial No. 08/840,204 and these PAI-1 mutants are useful as immobilized enzyme inhibitors for the capture of neutrophil elastase (or other elastases) and enzymes involved in VN-dependent cell migration. Preferred mutants possess both of these characteristics.

A preferred elastase-inhibiting PAI-1 mutant has the following characteristics:

(1) a PAI-1 molecule of full length or having between 1 and 14 or its N-terminal amino acid truncated;

(2) has an amino acid substitution at the PI site, the P4 site or both, as further delineated below;

(3) inhibits neutrophil elastase with a second order rate constant of at least 10⁵ M^"1 sec^"1 with a stoichiometry of at least 2: 1 at physiological salt concentrations and pH in the color imetric assay described below.

A preferred substitution at PI is Ala (R346A) or Val (R346V), although another substitutions, e.g., Met (αiAT has Met at this position) or Asp, is acceptable. The amino acid substitutions described herein are designated interchangeably as, for example, "PI Ala", which indicates the position in the PAI-1 reactive center as being the PI site, or R346A which indicates Arg is replaced by Ala at position 346 of PM-1, (See Figure 3), using the single letter amino acid code.

Data show that pancreatic elastase is not inhibited by wtPAI-1 (wild-type PAI-1), and therefore does not covalently bind to pancreatic elastase, but pancreatic elastase is inhibited and does bind to the PI Ala PAI-1 mutant. See the results shown in Figure 4. All of the wtPAI-1 is cleaved and thus inactivated by interacting with elastase, which explains its lack of inhibitory action. Such inactivation of PAI-1 by elastase was also shown by Lawrence, et al, (1994a). In fact, others have published that PAI-1 is not an inhibitor of elastase (Levin et al, 1987). Shubeita et al. (1990) actually tried to modify PAI-1 to become an inhibitor of elastase and failed.

A mutant which included a replacement of PI Arg by Ala, in combination with the wild type Met at PI' was described earlier by the present inventors and their colleagues (Sherman et al, 1992). In Figure 4 of that reference, it was shown that such a mutant lost the ability to inhibit uPA. This same mutant was later found to lack inhibitory activity toward tPA and thrombin (Sherman et al, 1995). It is important to note that these described mutants were different from the mutants of the present invention in that the PAI- 1 protein contained seven additional amino acids added to its N-terminus Met-Thr-Met-Ile- Thr-Asn-Ser (Sherman et al, 1992). Furthermore, Shubeita et al, 1990, tried to change the PI site in PM-1 to inhibit elastase by using the αiAT amino acid sequence into PAI-1 but found no inhibition.

The reason that the PI Ala mutant inhibits elastase but not tPA or uPA is thought to be a function of the interaction with the specificity site (SI) of the proteinase (though the inventors do not wish to be bound by any particular mechanistic interpretation). This specificity site of the proteinase is the primary determinant of substrate specificity.

Depending on the size and hydrophobicity of the specificity site, it prefers to accommodate one type of amino acid or another. tPA and other PAs prefer basic residues at PI (Arg or

Lys). Elastase prefers small hydrophobic resides like Ala and Val. Hence, by a judicious choice of amino acids in the reactive center of the PAI-1 mutant, it is possible to select a substitution or combination of substitutions that optimize interaction with the elastase specificity site while preventing inactivation of the inhibitor and thereby maximizing it inhibitory capacity and ability to promote clearance of the elastase.

The substitution at the P4 site must be one which results in a protein which is not cleaved after the P4 residue by elastase. For this, it is useful to substitute for the Val at this position in the wtPAI- 1. This resistance to inactivation permits the mutant to successfully inhibit elastase. Thus, a preferred amino acid substituent (a) is resistant to cleavage by elastase at this site, i.e., does not act as a substrate site for elastase and (b) at the same time does not present side chains which interfere with the interaction and binding of PAI-1 to elastase to form a complex such that elastase activity is inhibited and the complex is efficiently cleared. Stated otherwise, the substituting amino acid at P4 should present a poor fit as a primary (substrate) site for elastase without distorting other subsite contacts which are needed for interaction and successful inhibition.

Preferred amino acids at P4 are small, such as Ala and Gly, though somewhat larger residues such as Leu and He are also contemplated. The amino acid may be charged, such as Asp which should make that site less amenable to cleavage by elastase.

If the P4 site is substituted, for example, with Ala, a larger number of possible substitutions at PI are expected to result in a molecule with the desired inhibitory properties. The efficiency or rate of inhibition (second order rate constant) is expected to will be highest with Val at PI .

As stated above, a fragment of PAI-1 which has the requisite elastase-inhibiting or migration-inhibiting activity is within the scope of this invention. Such a fragment generally has most of the amino acids of full length PAI-1, and preferably does not have more than the 14 N-terminal amino acids cleaved. However, if it is later discovered that other fragments of PAI-1 maintain the requisite biochemical functions, then mutants of those fragments in accordance with the description above are within the scope of this invention.

Also included is a mutant of a longer polypeptide which has the delineated properties of PAI-1 along with the particular characteristics of the mutants described herein. Thus, for example, the N-terminal 30 amino acids of PAI-1 have been replaced with the N-terminal 50 amino acids of αiAT, resulting in a polypeptide that is longer by 20 amino acids than PAI-1 but retains biochemical properties of PAI-1. Substitution mutants of such a longer molecule of the type described above are also intended, provided that such mutants covalently bind to elastase or other target enzymes specifically.

In addition to the aforementioned amino acid substitutions which bestow on PAI-1 the desirable characteristics for utility in accordance with the present invention, additional amino acid substitutions are known which stabilize PAI-1 (Berkenpas, et al, 1995). Preferred compositions will optionally include, in addition to substitutions at PI and P4 sites, four additional substitutions at positions 150, 154, 319 and 354 of Figure 5, as in the mutant designated 14-1B by Berkenpas et al, 1995. These substitutions are NI50H, K154T, Q319L, M354I.

The following is a non-exclusive list summarizing preferred PAI-1 mutants. The amino acid residues shown are at positions P4-P4' in the RCL (corresponding to residues 343 to 350 of Figure 5).

343 344 345 346 347 348 349 350 wtPAI-1 Val Ser Ala Arg Met Ala Pro Glu

Mutants

1. PlAla (R346A) Val Ser Ala Ala Met Ala Pro Glu

2. PlVal (R346V) Val Ser Ala Val Met Ala Pro Glu

3. PlGly (R346G) Val Ser Ala Glv Met Ala Pro Glu

4. PlAsp (R346D) Val Ser Ala Asp Met Ala Pro

Glu

5. P4Ala (V343A) Ala Ser Ala Arg Met Ala Pro Glu

6. P4Asp (V343D) Asp Ser Ala Arg Met Ala Pro

Glu

7. P4GIy (V343G) Gly Ser Ala Arg Met Ala Pro Glu

8. P4Leu (V343L) Leu Ser Ala Arg Met Ala Pro Glu

9. P4Ile (V343I) lie Ser Ala Arg Met Ala Pro Glu

10. P4AlaPlVal

(V343A, R346V) Ala Ser Ala Val Met Ala Pro Glu

11. P4AlaPlAla

(V343A, R346A) Ala Ser Ala Ala Met Ala Pro Glu 12. P4AlaPlAsp

(V343A, R346D) Ala Ser Ala Asp Met Ala Pro

Glu

13. R346A plus N150H,K154T,Q319L,M354I

14. R346V plus N150H,KI54T,Q319L, M354I

15. V343A,R346V plus N150H, K154T, Q319L, M3541

16. V343A.R346D plus N150H, K154T, Q319L, M3541

While the present disclosure is directed primarily to human PAI-1 or mutants thereof, it is to be understood that homologues of PAI-1 from other species, and mutants thereof, that possess the characteristics disclosed above are intended within the scope of this invention. In particular, the PAI-1 protein (or DNA) from other mammalian species may be used for the same purposes as human PAI-1 in the method of the present invention.

As noted above, the present invention also includes peptides which include at least that portion of the sequence which contains the substitution or substitutions, and which possess the requisite biochemical and biological activity such as elastase inhibition or the inhibition of other specific targeted enzymes. Such peptides are produced using well- known synthetic methods for the synthesis of polypeptides of desired sequence on solid phase supports and their subsequent separation from the support. Methods for solid phase peptide synthesis are well-described in the following references, hereby incorporated by reference: Merrifield, (1963); Merrifield, (1986); Wade, et al, (1986); Fields, (1990); MilliGen Report Nos. 2 and 2a, (1987). For example, the more classical method, "tBoc method," or the more recent improved "F-moc" technique may be used (Atherton, et al.,1981).

EXAMPLES

Production of PAI-1 Mutants bv Expression and Purification of Recombinant PAI-1 in E. coli

The following methods are preferred and do not represent the exclusive means for producing enzyme inhibitor mutants of the invention. Techniques for synthesizing oligonucleotides probes are well known in the art and disclosed by, for example, Wu, et al, 1978 or Gait, ed., (Current Edition). Procedures for constructing and expressing recombinant molecules in accordance with this invention, including appropriate promoters and other control elements, selection markers, etc., are disclosed by Sambrook, J. et al, 1989; Ausubel, F.M. et al, 1987, which references are herein incorporated by reference.

Included in this invention is the DNA encoding the PAI-1 mutant, which is preferably a cDNA having the appropriate nucleotide sequence subsitutions to encode the mutant proteins as disclosed herein. Such molecules are prepared using conventional methods. Also included herein are prokaryotic or eukaryotic host cells transformed or transfected with a vector comprising the above DNA molecule. Again, the method used for transferring the DNA, expressing the DNA and growing the host cells are well-known in the art and described in the references cited above. Eukaryotic host cells are preferably mammalian cells of an established cell line, although insect cells or plant cells are also contemplated. Appropriate vectors such as viruses, vector sequences, control sequences, such as promoters appropriate for the species of host cells, are conventional and well- known to those skilled in the art and are therefore not described in particular detail herein In addition to sense DNA, antisense DNA anad antisense RNA molecules to the mutant PAI-1 coding sequence are provided herein. Also included is an RNA molecule encoding the PAI-1 mutant.

Example 1: Site directed Mutagenesis of PAI-1

A preferred method for producing PAI-1 mutants utilizes a commercially available kit and was described by one of the present inventors and his colleagues in a reference which is hereby incorporated by reference in its entirety (Lawrence, et al, 1994b).

Site-specific or site-directed mutagenesis allows the production of peptide variants through the use of specific oligonucleotide sequences that encode the DNA sequence of the desired mutation plus a sufficient number of adjacent nucleotides, to provide a primer sequence of sufficient size and sequence complexity to form a stable duplex on both sides of the deletion junction being traversed. Typically, a primer of about 20 to 30 nucleotides in length is preferred, with about 5 to 10 residues on both sides of the junction of the sequence being altered. The technique of site-directed mutagenesis is well known in the art, as exemplified by publications such as Adelman et al, DNA 2: 183 (1983), which is incorporated herein by reference. As will be appreciated, the mutagenesis technique typically employs a phage vector that exists in both a single-stranded and double-stranded form. Typical vectors useful in site-directed mutagenesis the M13 phage (Messing et al, 1981) These phage are commercially available and their use is well known to those skilled in the art. Alternatively, plasmid vectors that contain a single-stranded phage origin of replication (e.g., Veira et al , 1987) may be employed to obtain single-stranded DNA.

In general, site-directed mutagenesis in accordance herewith is performed by first obtaining a single-stranded vector that includes within its sequence a DNA sequence that encodes the PAI-1 protein (or peptide). An oligonucleotide primer bearing the desired mutated sequence is prepared, generally synthetically (e.g., Crea et al, 1978). This primer is annealed with the vector comprising the single-stranded protein-coding sequence and is subjected to DNA-polymerizing enzymes such as E. coli polymerase I Klenow fragment to complete the synthesis of the mutation-bearing strand. Thus, a mutated sequence and the second strand bears the desired mutation. This heteroduplex vector is then used to transform appropriate cells (such as JM1O1 cells) and clones are selected that include recombinant vectors bearing the mutated sequence arrangement.

Afler such a clone is selected, the mutated protein region may be removed and placed in an appropriate vector for protein production, generally an expression vector of the type that may be employed for transformation of an appropriate host.

For producing PAI-1 mutants, the mutagenesis is most preferably performed using the Altered Sites® mutagenesis kit (now designated "Altered Sites II^®") following the manufacturers instructions (Promega). Briefly, PAI-1 cDNA, along with T7 promoter and terminator regulatory sequences, is isolated as an Xbal-ΕcoRV fragment from the PAI-1 expression plasmid pΕT3aPAI-l (Sherman et al , 1992) This fragment is ligated to Pstl/Xbal cut pSELECT-1® (Promega) (now designated "pALTER"), that had been blunt- ended at the Pstl site, creating phagemid pSELPAI-1. This construct is then transformed into E. coli strain JM1O9, and single-stranded DNA is produced by infection with the helper phage R408 (Promega). The following is a list of oligonucleotides used to generate the preferred mutants at the PI and P4 sites of PAI-1.

PI Ala 5'-GTCTCAGCCGCCATGGCCCCC

PI Val 5'-GTCTCAGCCGTCATGGCCCCC

P4 Ala 5'-GCTGTCATAGCCTCAGCCCGC

P4 Ala, PI Val 5'-GCTGTCATAGCCTCAGCCGTCATGGCCCCC

P4 Ala, PI Ala 5'-GCTGTCATAGCCTCAGCCGCCATGGCCCCC

A newer method is available for enhanced site-elimination mutagenesis which can be applied in the preparation of the mutant PAI-1 proteins. The new Chameleon ^~ mutagenesis kit (Stratagene) may be used to produce one or more site-specific mutation in virtually any double-stranded plasmid (containing a unique nonessential restriction site), thus eliminating the need for subcloning into M13-based vectors and single-strand DNA rescue (Pap worth et al, 1994a). The Chameleon™ kit applies a modification of the unique site-elimination mutagenesis procedure of Deng and Nickoloff (Anal, 1992). The improved protocol includes the use of: (I) more target DNA and a new primer template ratio; (2) native T7 DNA polymerase instead of T4 DNA polymerase; (3) a new mutS cell line that does not produce endonuclease A; and (4) highly competent XLmutS and XLI ^~ Blue? cells for transformation of mutated plasmid DNA. These modifications increase the yield and quality of mutated plasmid DNA, resulting in consistently higher colony numbers and mutagenesis efficiencies. The Chameleon™ mutagenesis kit has been used to introduce insertions, point mutations and deletions as large as 48 bp (Pap worth et al, 1994b) and has also been used with three mutagenic oligonucleotides to simultaneously generate triple mutations. The kit includes competent cells of the XLmutS host strain bearing the endA mutation which removes an endonuclease that degrades miiprep DNA, improving the yield and quality of the mutated plasmid DNA and the reproducibility of the mutagenesis procedure.

The mutagenesis procedure involves simultaneously annealing two oligonucleotide primers to the same strand of denatured double-stranded plasmid DNA. One primer (the mutagenic primer) introduces a chosen mutation, and the second primer (the selection primer) alters the sequence of a unique restriction site in the plasmid in order to create a new restriction site. Extension of these primers with T7 polymerase and ligation of the resulting molecules with T4 ligase are followed by restriction enzyme digestion. Any plasmid molecules that renature without inclusion of the selection primer will be linearized, while those that form with the selection primer will not. The resulting mixture is transformed into the highly competent XLmutS E. coli strain, which is unable to perform mismatch repair. The transformed bacteria are grown overnight in liquid culture, and the plasmid DNA is recovered and treated again with the restriction enzyme that digests plasmids containing the original restriction site Plasmids containing the new restriction site and the chosen mutation will resist digestion. Transformation of this DNA into highly competent E. coli such as XLI-Blue results in 70-91 % of the colonies containing mutated plasmids. If a second round of mutagenesis is desired, a switch primer can be used to "switch" from the new unique restriction site back to the original or another restriction site, at the same time incorporating another mutation. This process makes it possible to perform several rounds of mutation.

Selection primers made by Stratagene select against restriction enzyme sites in the antibiotic-resistance genes for ampicillin, chloramphenicol and neomycinl kanamycin. (There are also primers available for the ColEl origin of replication and the polylinker of both SK and KS versions of the pBluescript® II phagemeid.) The switch primers allow a second round of mutagenesis to recreate the original unique restriction site.

Example 2: Expression. Purification and Characterization of PM-1 Mutants

A novel phagemid vector for efficient mutagenesis and protein expression has been designed by one of the present inventors and his colleagues. This construct, pSELPAI-1, eliminates the need to isolate and subclone each new mutant into an expression plasmid. The inclusion of T7 promoter and terminator sequences in the pSELPAI-1 constructs permits efficient PM-1 expression directly from this vector using an E. col, strain producing T7 polymerase (Studier et al, 1990 Using this system, site-directed mutagenesis is generally achieved with greater than 50% efficiency. In addition, sequence analysis of greater than lOkb, from 8 independent clones, has identified no other mutations, indicating a very low rate of secondary mutations ( <0.01 %). Briefly cells of the E. coli strain BL21 (DE3) transformed with the pSELPA-1 mutants are grown to an ODβsoof 0.5, PAI-1 production is induced by the addition of lmM isopropylthio-β-D-galactoside, and growth is continued at 37°C for 2h. Cells are harvested and PAI-1 is purified as described Lawrence et al, 1989, supra; Sherman et al, 1992, supra). Protein yields are approximately 1-5 mg/L of cell culture. Purity is assessed by SDS-PAGE and staining by Coomassie blue. Inhibitory activity against both uPA (American Diagnostica) and tPA (Activase, Genentech) is measured in a single step chromogenic assay as described (Lawrence et al, 1989) and compared to wtPAI- 1 purified from E. coli carrying the expression plasmid pET3aPAI-l (Sherman et al, 1992). Inhibitory activity against elastase is tested as described below. Other activities, enhancement of clearance or inhibition of cell migration are tested using methods described in more detail in the Examples.

All the mutant proteins have specific activities similar to wild type PAI-1, demonstrating approximately 50 % of the calculated maximum theoretical specific activity (Lawrence et al, 1989). The chromatographic profiles of each mutant, from every step of the purification, are similar to those of wtPAI-1. None of the mutations significantly affect heparin binding. Each mutant binds VN with approximately the same affinity as does wtPAI-1.

Example 3: Chemical Modification of the Protein

A "chemical derivative" of PAI-1 contains additional chemical moieties not normally a part of the protein. Covalent modifications of the PAI-1 mutant proteins are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues. Such derivatized moieties may improve the solubility, absorption, biological half life, and the like The moieties may alternatively eliminate or attenuate any undesirable side effect of the protein and the like. Moieties capable of mediating such effects are disclosed, for example, in Remington 's Pharmaceutical Sciences, 1980. Clearly, any chemical modifications included herein will not substantially alter the advantageous properties of the PAI-1 mutants described herein. Histidyl residues are derivatized by reaction with diethylprocarbonate at pH 5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-5 bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succiic or other carboxylic acid anhydrides. Derivatization with these agents has the effect of reversing the charge of the lysinyl residues. Other suitable reagents for denvatizing a- amino-containing residues include imidoesters such as methyl picoliimidate; pyridoxal phosphate; pyridoxal; chioroborohydride; trinitrobenzenesulfonic acid; O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with gly oxy late.

Cysteinyl residues are highly reactive and can be easily modified by many sulfhydryl reactive reagents such as maleimides, iodoacetamides, benzylic halides and DTNB. Cysteinyl residues also react to a limited extent with isothiocyanates and succinimidyl esters. Cysteinyl residues can be easily converted to an amino group through the use of the modifying reagent 2-Bromoethylamine. Use of this reagent results in the conversion to an aminoethyl derivative.

Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3- butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of argiine residues requires that the reaction be performed in alkaline conditions because of the high pK.a of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the argiine epsilon-amino group.

The specific modification of tyrosyl residues per se has been studied extensively, with particular interest in introducing spectral labels into tyrosyl residues by reaction with aromatic diazonium compounds or tetranitromethane. Most commonly, N-acetylimidizol and tetranitromethane are used to form 0-acetyl tyrosyl species and 3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction with carbodiimides (R'-N-C-N-R') such as l-cyclohexyl-3-(2-morpholinyl-(4-ethyl) carbodiimide or 1- ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore, aspartyl and glutamyl residues are converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues may be deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are dearnidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.

Other modifications include hydroxy lation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., 1983), acetylation of the N-terminal amine, and, in some instances, amidation of the C-terminal carboxyl groups. Commonly used cross-linking agents include, e.g., 1,1- bi s(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysucciimide esters, for 10 example, esters with 4-azidosalicylic acid, homobifunctional imidoesters, including d isuccinimi dyl esters such as 3,3'- dithiobis(succiimidylpropionate), and bifunctional maleimides such as bis-N-maleimido- 1,8-octane. Derivatizing agents such as methyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water- 1 5 insoluble matrices such as cyanogen bromide- activated carbohydrates and the reactive substrates described in U.S. Patents No. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.

Example 4: Construction of mutant PAI-1 cDNA

The following method discloses the construction of a specific mutant PAI-1 for use in the claimed method of the present invention for immobilization to the solid substrate. The coding sequence for the six residue peptide tag, His-His-His-His-His-His—His, which contains the ionic binding sequence for the specific binding to nickel or related ions was introduced at the 5 '-end of the mature PAI-1 cDNA by PCR with the following oligonucleotides: forward, 5'-GGC CAT ATG CAT CAC CAC CAT CAC CAC GTG CAC CAT CCC CCA TCC TA-3', and reverse 5'-GCC ATG CAT GTC CCA GAT GAA GGC GTC TTT CC-3'. This creates a protein that begins Met-His-His-His-His-His-His- Val-His-His-Pro-Pro . where the wild-type sequence of PAI-1 is underlined. The Val-His- His-Pro-Pro sequence is the start of the mature PAI-1 sequence. The PCR product was then restricted with Ndel and Nsϊl, and ligated into the pET_3aPAI-l prokaryotic vector (Sherman et al., 1992). The construct was then transformed into E. coli strain BL21 (DE3) and high levels of recombinant PAI-1 containing the His-tag at its amino-terminus were produced. The His-tag protein was then analyzed for anti-PA activity and found to be completely inactive and no inhibitory activity could be demonstrated. Since the first construct did not work then a new one was constructed that deleted the coding sequence for the first three amino acids of the wild-type PAI-1 sequence and the 6-residue His-tag was added to the amino-terminus of this PAI-1 deletion mutant. The following oligonucleotides were used: forward, 5'-GAT CAT ATG CAT CAC CAC CAT CAC CAT CCC CCA TCC TAC GTG GCC-3', and reverse 5'-GCC ATG CAT GTC CCA GAT GAA GGC GTC TTT CC-3'. This creates a protein that begins Met-His-His-His-His-His-His-Pro-Pro-Ser-Tyr- Val, where the wild-type PAI-1 sequence is underlined. This construct retained full activity, and the mutant PAI-ls were purified either similar to wild-type PAI-1 as describe (Kvassman et al. 1995b) or by metal chelate chromatography as described (Linder et al 1992).

The PAI-1 point mutants were constructed using the Transformer Site-Directed Mutagenesis Kit (Clontech) according to the manufacturer's instructions. In order to produce some PAI-1 mutants exclusively in the active conformation they were constructed on a stable PAI-1 background, 14- IB, that has been previously shown to have significantly enhanced functional stability, but to be indistinguishable from wild-type PAI-1 with respect to inhibitory activity, heparin binding, and vitronectin binding (Berkenpas et al., 1995). Its binding to LRP is also indistinguishable from wild-type PAI-1 (Stefansson et al. , 1998). The mutant PAI-ls were purified either similar to wild-type PAI-1 as describe (Kvassman et al. 1995b) or by metal chelate chromatography as described (Linder et al 1992).

Example 5: Methods of Detecting tPA ELISA Using His PAI-1

Reagents:

Wash buffer:

10 mM pH 7.5

.15 M NaCl

0.1 % BSA

.05% Tween 20 .01 % sodium azide Blocking buffer: 10 mM pH 7.5 .15 M NaCl 3.0% BSA .01 % sodium azide

His- 14- IB PAI-1 (immobilized on solid support):

lOμg/ml in wash buffer

(1.2 ml) (lOμ g/ml)/500 μg/ml = 24 μl and 1176 μl blocking buffer

tPA:

1 ml of 05. μg/ml in blocking buffer 16.7μl of 1: 10³ dilution in 1 ml

Primary Antibody (analtyical reagent):

Rabbit Anti-tPA (serum) 1:5 x 10⁵ dilution

5 μl of 1: 10³ dilution in blocking buffer in 2.5 ml blocking buffer

Secondary Antibody:

Biotinylated Goat Anti-rabbit 1:2.4 x 10⁴ dilution

10 μl of 1: 10² dilution in blocking buffer in 2.5 ml blocking buffer

Detection:

Avidin Alkaline Phosphatase 1 : 10 x 10⁴ dilution in blocking buffer

PNPP Reaction Buffer: 0.1 M glycine ImM MgCh ImM ZnCh pH 10.4

Substrate:

20 mg Sigma® PNPP tablet dissolved in 20 ml reaction buffer Method:

1. Added 0.1 ml of 10 μg/ml of His-PAI-1 to row A of a microtiter plate and buffer to row B.

2. Incubated 20 minutes on rotary shaker.

3. Washed wells three times with 0.3 ml wash buffer per well.

4. Blocked 1 hour with blocking buffer.

5. Added 0.2 ml of .05 μg/ml tPA to wells in first column. Transferred 0.1 ml to subsequent columns containing 0.1 ml blocking buffer in 2X serial dilutions. Incubate 30 minutes with shaking. tPA concentrations ng/ml: 50, 25, 12.5, 6.2, 3.1, 1.6, 0.8, 0.4, 0.2, 0.1, and 0.05.

6. Wash 3X then add 0.1 ml of primary antibody to each well and incubate 30 minutes with shaking.

7. Wash 3X then add 0.1 ml of secondary antibody to each well and incubate 30 minutes with shaking.

8. Wash 3X then add 0.1 ml of avidin AP to each well and incubate 30 minutes with shaking.

9. Wash 3X the add 0.2 ml of substrate solution each well and measure ΔA os

The method set forth above is depicted in Figure 6 as a schematic of the method of the present invention. Results from this method are shown in Figure 7. The results of this assay show linear results between 0 and 30ng/ml of tPA. The closest competitor that measures active tPA in plasma and/or biological fluids is the Chromolize™ tPA kit from Biopool Corporation has a very limited useful range.

The Biopool assay measures active tPA in the range of 0 to less than 3 ng/ml and utilizes an indirect measurement scheme. Total, rather than active tPA is captured by an anti-tPA monoclonal antibody on an ELISA plate. The bound tPA is then used to activate added human plasminogen. This activation requires the use of added stimulators to speed up the relatively slow conversion of plasminogen to plasmin. Plasmin, the end product of the reaction is then measured by an added plasmin substrate. The Biopool assay requires more reagents, and therefore, more possilibility of variability from different lots of reagents. In contrast, the present method directly measures active tPA via the use of the immobilized enzyme inhibitor of the present invention. The present method provides greater than 10 times the range of the Biopool Assay as shown in Figure 8, comparing the method of the present invention in panel A that detects the level of tPA as compared with the Biopool Assay the results of which are shown in panel B. The method of the present invention utilizing an immobilized PAI-1 mutant (6x-His 14- IB PAI-1) has a larger dynamic range than the Biopool Assay method. This is important as evidence in Biopool Assay's own product literature which indicates that normal tPA rnages between 0.2 to 2.0 IU/ml and values forllowing venous occlusion vary between 1.4 to 14 IU/ml. The method of the present invention covers this clinical range well whereas the Biopool Assay does not.

Another embodiment of the present invention includes an immobilized enzyme inhibitor that recognizes the enzyme, PSA (Prostate Specific Antigen). PSA is the primary marker used clinically to detect prostate cancer. PSA is a serine proteinase composed of 240 amino acids. PSA circulates in serum as a complex with αi-antichymotrypsin (bound) and a noncomplexed (free) form. The predominate form of PSA in serum exists as the bound form with only a minor fraction present in the free form.

Prostate cancer tissue contributes about 10 times more to serum PSA levels that does an equal amount of normal prostate tissue (Howanitz, 1996). An important clinical distinction must be made between actual prostate cancer and the more common benign prostatic hypertrophy (BPH) which also increases the total PSA levels in serum. The free form of PSA has been shown to be significantly lower in those men having BPH (Christensson et al, 1993). The present method which measures free enzyme and distinguishes free PSA from bound PSA.

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Claims

WE CLAIM:

1. A method for the detection of a functionally active form of an enzyme in a biological sample, comprising: contacting an enzyme inhibitor immobilized on a solid substrate with said biological sample, and measuring the binding of said enzyme inhibitor to said active form of the enzyme by a detectable label, wherein said enzyme inhibitor specifically forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme.

2. The method of claim 1, wherein said enzyme inhibitor is proteinase inhibitor or mutant thereof and said enzyme is a proteinase.

3. The method of claim 2, wherein said proteinase inhibitor or mutant thereof is selected form the group consisting of serine, cysteine, aspartic, thiol, carboxyl, metallo- proteinase inhibitors and mutants thereof.

4. The method of claim 3, wherein said enzyme is selected form the group consisting of serine, cysteine, aspartic, thiol, carboxyl, and metallo-proteinases.

5. The method of claim 3, wherein said proteinase inhibitor or mutant thereof is a serine proteinase inhibitor or a mutant thereof.

6. The method of claim 5, wherein said enzyme is a serine proteinase.

7. The method of claim 6, wherein said serine proteinase is selected from the group consisting of tissue plasminogen activator (tPA), urokinase, thrombin, plasmin, neutrophil, pancreatic elastase, trypsin, chymotrypsin,cathepsin G and prostate specific antigen.

8. The method of claim 5, wherein said serine proteinase inhibitor or mutant thereof is a plasminogen activator inhibitor or a mutant thereof.

9. The method of claim 8, wherein said plasminogen activator inhibitor is plasminogen activator inhibitor-1 (PAI-1) or a mutant thereof.

10. The method of claim 9, wherein said enzyme is a plasminogen activator.

11. The method of claim 1, wherein said solid substrate is selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene.

12 The method of any one of claims 1-11, wherein said enzyme inhibitor is modified to facilitate immobilization to said solid substrate and wherein said immobilized enzyme inhibitor retains the property of specifically forming a covalent bond or binding with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme.

13. The method of claim 12, wherein said enzyme inhibitor is modified by at least one of the addition of, the substitution of or the deletion of one or more amino acid residues.

14. The method of claim 13, wherein said enzyme inhibitor is immobilized to said solid substrate by specific interaction with at least one amino acid residue of said enzyme inhibitor.

15. The method of claim 14, wherein said amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrate; at least one cysteine residue bindable to maleimide derivatized solid substrate and at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid substrate.

16. The method of claim 1, wherein said detectable label is selected from the group consisting of a radioactive label, a chromophore and a fluorophore.

17. The method of claim 16, wherein said detectable label is conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

18. The method of claim 17, wherein said analytical reagent is an antibody.

19. The method of claim 16, wherein said detectable label is generated by a reactive molecule conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

20. The method of claim 19, wherein said analytical reagent is an antibody.

21. The method of claim 1, wherein said enzyme inhibitor is a peptide chloromethyl ketone.

22. The method of claim 21, wherein said enzyme inhibitor is selected from the group consisting of a tripeptide chloromethyl ketone to a dodecapeptide chloromethyl ketone.

23. The method of claim 22, wherein said tripeptide chloromethyl ketone is an X-Y-Z-chloromethyl ketone, wherein X, Y and Z are independently selected amino acids.

24. The method of claim 23, wherein said tripeptide chloromethyl ketone is selected from the group consisting of: D-Phe-Pro-Arg-chloromethyl ketone; D-Glu-Gly- Arg-chloromethyl ketone; D-Val-Gly-Arg-chloromethyl ketone; and D-Ile-Pro-Arg- chloromethyl ketone.

25. The method of claim 21, wherein said solid substrate is selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene.

26. The method of claim of any one of claims 21-25, wherein said enzyme inhibitor is modified to facilitate immobilization to said solid substrate and wherein said immobilized enzyme inhibitor retains the property of specifically forming a covalent bond or binding with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme.

27. The method of claim 26, wherein said enzyme inhibitor is modified by at least one of the addition of, the substitution of or the deletion of one or more amino acid residues.

28. The method of claim 27, wherein said enzyme inhibitor is immobilized to said solid substrate by specific interaction with at least one amino acid residue of said enzyme inhibitor.

29. The method of claim 28, wherein said amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrate; at least one cysteine residue bindable to maleimide derivatized solid substrate and at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid substrate.

30. The method of claim 21, wherein said detectable label is selected from the group consisting of a radioactive label, a chromophore and a fluorophore.

31. The method of claim 30, wherein said detectable label is conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

32. The method of claim 31, wherein said analytical reagent is an antibody.

33. The method of claim 30, wherein said detectable label is generated by a reactive molecule conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

34. The method of claim 33, wherein said analytical reagent is an antibody.

35. An analytical element for use in the detection of a functionally active form of an enzyme in a biological sample comprising: an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein said enzyme inhibitor or mutant thereof forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with said active form the enzyme.

36. The analytical element of claim 35, wherein said solid substrate is selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene.

37. The analytical element of claim 36, wherein said solid substrate is selected from the group consisting of a microtiter plate or a dipstick.

38. The analytical element of any one of claims 35-37, wherein said enzyme inhibitor or mutant thereof is modified to facilitate immobilization to said solid substrate and wherein said immobilized enzyme inhibitor retains the property of specifically forming a covalent bond or binding with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme.

39. The analytical element of claim 38, wherein said enzyme inhibitor is modified by at least one of the addition of, the substitution of or the deletion of one or more amino acid residues.

40. The analytical element of claim 39, wherein said enzyme inhibitor is immobilized to said solid substrate by specific interaction with at least one amino acid residue of said enzyme inhibitor.

41. The analytical element of claim 40, wherein said amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrate; at least one cysteine residue bindable to maleimide derivatized solid substrate and at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid substrate.

42. The analytical element of claim 35, wherein said enzyme inhibitor is a proteinase inhibitor or a mutant thereof and said enzyme is a proteinase.

43. The analytical element of claim 35, wherein said enzyme inhibitor is a peptide chloromethyl ketone or mutant there of and said enzyme is a proteinase.

44. A kit for the detection of a functionally active form of an enzyme in a biological sample comprising: an analytical element comprising an enzyme inhibitor or mutant thereof immobilized on a solid substrate, wherein said enzyme inhibitor or mutant thereof specifically forms a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme; and an analytical reagent that is conjugated to a detectable label or a reactive molecule that generates a detectable label, wherein said analytical reagent specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

45. The kit of claim 45, wherein said solid substrate is selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene.

46. The kit of claim 45, wherein said solid substrate is selected from the group consisting of a microtiter plate or a dipstick.

47. The kit of claim 44, wherein said enzyme inhibitor or mutant thereof is modified to facilitate immobilization to said solid substrate and wherein said immobilized enzyme inhibitor retains the property of specifically forming a covalent bond or binding with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme.

48. The kit of claim 47, wherein said enzyme inhibitor is modified by at least one of the addition of, the substitution of or the deletion of one or more amino acid residues.

49. The kit of claim 48, wherein said enzyme inhibitor is immobilized to said solid substrate by specific interaction with at least one amino acid residue of said enzyme inhibitor.

50. The kit of claim 49, wherein said amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrate; at least one cysteine residue bindable to maleimide derivatized solid substrate and at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid substrate.

51. The kit of claim 47, wherein said enzyme inhibitor is proteinase inhibitor or mutants thereof and said enzyme is a proteinase.

52. The kit of claim 47, wherein said enzyme inhibitor is a peptide chloromethyl ketone or mutant thereof and said enzyme is a proteinase.

53. The kit of claim 44, wherein said detectable label is selected from the group consisting of a radioactive label, a chromophore and a fluorophore.

54. The kit of claim 53, wherein said detectable label is conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

55. The kit of claim 54, wherein said analytical reagent is an antibody.

56. The kit of claim 53, wherein said detectable label is generated by a reactive molecule conjugated to an analytical reagent that specifically binds to said active form of the enzyme that is bound to said enzyme inhibitor.

57. The kit of claim 56, wherein said analytical reagent is an antibody.

58. The kit of claim 44, further comprising a second analytical element comprising a second analytical reagent immobilized on a solid substrate, wherein said second analytical reagent specifically binds to both the active and inactive form of said enzyme.

59 The kit of claim 58, wherein said second analytical reagent is antibody.

60. A method of immobilizing an enzyme inhibitor or mutant thereof to a solid substrate comprising: modifying said enzyme inhibitor or mutant thereof to facilitate immobilization to said solid substrate; and contacting said modified enzyme inhibitor with said solid substrate until said modified enzyme inhibitor is immobilized to said solid substrate, wherein said immobilized enzyme inhibitor or mutant thereof retains the property of specifically forming a covalent bond or binds with a dissociation constant of 1 x 10^"9M or less with said active form of the enzyme

61. The method of claim 60, wherein said solid substrate is selected from the group consisting of glass, plastic, ceramic, polyproplene, polycarbonate and polybutylene.

62. The method of claim 61, wherein said solid substrate is selected from the group consisting of a microtiter plate or a dipstick.

63. The method of claim 60, wherein said enzyme inhibitor is modified by at least one of the addition of, the substitution of or the deletion of one or more amino acid residues.

64. The method of claim 63, wherein said enzyme inhibitor is immobilized to said solid substrate by specific interaction with at least one amino acid residue of said enzyme inhibitor.

65. The method of claim 64, wherein said amino acid residue is selected from the group consisting of a polyhistidine sequence bindable to a metal chelate solid substrate; at least one cysteine residue bindable to maleimide derivatized solid substrate and at least one lysine residue bindable to N-hydroxysuccinimide derivatized solid substrate.