Method of diagnosis for recognition of carriers of the Marburg I variant of factor VII activating protease (FSAP) by differential modulation of FSAP activity

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DESCRIPTION

Diagnostic procedure for the recognition of carriers of the Marburg I variant of the protease that activates factor VII (FSAP) with the help of differential modulation of FSAP activity.

The invention is in the field of diagnostics for the Marburg Mutation I (MR I) of the protease that activates blood coagulation factor VII (FSAP), which is found in approximately 5 to 10% of the Western European population. According to a study by Willeit et al., Heterozygous carriers of the FASP MR I mutation have a higher risk of developing a carotid stenosis (Carotis Stenose) compared to the population average [Willeit et al. (s) (2003): Marburg I polymorphism of factor VII activating protease: a prominent risk predictor of carotid stenosis. Circulation 107, 667-670]. Since the presence of a FASP MR I mutation represents a potential marker of a genetic predisposition for the development of atherosclerotic diseases, the reliable detection of homo or heterozygous carriers of the FASP MR I mutation is of interest, especially in view of a treatment individual preventive doctor.

FSAP is a plasma serine protease, which has also been described under the name PHBSP (serine protease that binds plasma hyaluronan). FSAP is present in human plasma at a concentration of approximately 12 jug / ml, and through autocatalysis of the single chain proenzyme (sc-FSAP, single chainFSAP) it can be transformed into the active two-chain protease ( tc-FSAP, two chain-FSAP). Active protease has various functions or activities. It is known that FSAP has, on the one hand, the ability to activate Blood Coagulation Factor VII, thus activating single chain plasminogen activators, such as prourokinases that activate plasminogens, such as scuPA (single chain urokinase plasminogen activator) and sctPA (single chain tissue plasminogen activator). On the other hand, FSAP has the ability to inactivate blood coagulation factors V / Va and VIII / VIIIa).

Various test procedures for the qualitative or quantitative determination of FSAP are described in EP 952 215 A2, which take advantage of these biological activities of FSAP. Another activity of FSAP, which allows a determination of the active protease and which has also been described in the aforementioned patent document, as well as in Romish and Other (s): The FVII activating protease cleaves single-chain plasminogen activators. Haemostasis 29, 292-299 and in Hungfeld and Other (s). (1999): Detection of a novel plasma serine protease during purification of vitamin K-dependent coagulation factors. FEBS Letters 456, 290-294, is the amidolytic activity of FSAP with respect to low molecular weight substrates, especially against the chromogen substrate S-2288^TM (HD-IlePro-Arg-pNA).

In addition to the wild-type sequence of the human FSAP gene, various nucleotide polymorphisms are known, which in two cases also lead to a change in the amino acid sequence (EP 1 182 258 A1). The so-called Marburg I mutation (also called Marburg I polymorphism, allele or variant) leads to an exchange of Gly / Glu amino acids at position 534 of the proenzyme, signal peptide (Gly / Gu 534) inclusive, and results in a decrease 50-80% of the activity that activates prourokinase, while the ability to activate Factor VII remains unchanged. Another mutation, the so-called Marburg II (MR II) mutation (also called MR II polymorphism, allele or variant) leads to an amino acid exchange Glu / Gln at position 370 of the proenzyme, signal peptide (Glu / Gln 370) inclusive . However, the Marburg II Mutation has no influence on the activity of FSAP as an activator of prourokinase.

In the state of the art, the individual detection of persons carrying at least one copy of the FASP MR I variant is possible by two different methodical approaches. Until now, a reliable detection of the Gly / Glu amino acid exchange at position 534 of the proenzyme (Gly / Glu 534) is only possible by sequencing the corresponding coding region in genomic DNA or in mRNA. A G / A base exchange in the genomic sequence, which can be detected at nucleotide position 1601 of the cDNA, constitutes the genetic cause of the FSAP MR I mutant (EP 1 182 258 A1). While DNA sequence analysis provides reliable results, from the point of view of routine laboratory analysis, there is a need for assay procedures that are as inexpensive, rapid and safe as possible, and that can also be implemented automatically in available diagnostic kits. Immunochemical methods for detection or testing are predominantly preferred as they meet the above criteria and have already found wide use in laboratory-based diagnostics.

Another method for the determination of a FASP MR I mutant is based on the determination of the potential of the FSAP present in a sample to activate prourokinase. For this purpose, a specific antibody that is unable to differentiate between wild-type FSAP and known FSAP variants is coupled to a solid phase, and is incubated together with the sample liquid. After the addition of prourokinase as a FSAP substrate and a chromogenic urokinase substrate, the amount of chromogen reacted is determined as a measure of the activity of FSAP as a prourokinase activator. Carriers of the Marburg I mutation show an activity to activate prourokinase, decreased by 50-80%. However, a lower activity to activate prourokinase can also be conditioned (imposed) by a small concentration of FSAP in the sample. For this reason, until now it is necessary to determine not only the activity to activate prourokinase, but additionally also the FSAP antigen concentration in a sample (EP 1 348 761 A1 or US 2002/0142316 A1). Monoclonal antibodies are known in the state of the art, which allow the immunological detection of FSAP. In EP 1 182 258 two monoclonal antibodies are described, which come from hybridoma cells

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DSM ACC2453 or DSM ACC2454, which had been obtained after immunization of mice with FSAP protein. Both antibodies bind not only wild-type FSAP protein, but also the Marburg I and Marburg II variants. Other known FSAP antibodies bind to wild-type FSAP as well as known mutant variants; so that, for example, in a Sandwich ELISA the total FSAP antigen content in a sample is determined (see also DE 100 23 923 A1). Therefore, the state of the art teaches that only if a lower activity to activate prourokinase is found, together with an FSAP antigen concentration in the normal range, is this a concrete indication of the presence of an FSAP MRI variant.

The aim of the present invention was to provide other methods for the reliable diagnosis of an MR I mutation or for the individual detection of heterozygous or homozygous carriers of an MR I mutation, which make it possible to dispense with a determination of the FSAP antigen.

This task is achieved by making available the method according to the invention, described in the claims, which makes it possible to reliably differentiate heterozygous or homozygous carriers of an MR I polymorphism from those that are not carriers. The advantage of the present method, which takes advantage of the differential modulation of the activity of the FASP MR I variant, consists in a reliable discrimination between carriers and non-carriers of the FASP MR I mutation, and in particular that an additional determination of the FASP MR I mutation can be dispensed with. antigen content in a sample.

It has been found that by using suitable modulators of activity, the so-called differential modulators of activity, the activity of FSAP, especially the amidolytic activity of FSAP compared to low molecular weight peptide substrates, as well as the activity of FSAP to activate the plasminogen activator activator, in samples from non-carriers of an MR I polymorphism, is modulated differently than FSAP activity in samples of heterozygous or homozygous carriers of an MR I polymorphism, so that a differentiation between non-carriers and carriers of an MR I polymorphism can be achieved on the basis that the amplitude of modulation of FSAP activity is different.

Within the scope of the present invention, under the term "differential modulator of activity" it is possible to understand a substance or mixture of substances, which:

i) .- inhibits or enhances FSAP activity both in samples of carriers of the MR I variant of FSAP as well as in samples of non-carriers of the MR I variant of FSAP, but does this with a different amplitude; or:

ii) .- inhibits the activity of FSAP in samples of carriers of the MR I variant of FSAP, and that in samples of non-carriers of the variant MR I reinforces said activity, or vice versa; or iii) .- reinforces or inhibits FSAP activity in one of the sample types, but does not cause any significant change in FSAP activity in the other type of sample.

Therefore, a substance or a mixture of substances is suitable for use as a differential modulator of activity, which:

a) .- causes a quantitative difference in the inhibition (reduction) or enhancement (elevation) of FSAP activity in samples of non-carriers or carriers of the MR I variant of FSAP, that is, for example, in both types of sample the FSAP activity rises or falls, but with a different amplitude; or

b) .- causes a qualitative difference in the modulation of FSAP activity in samples of non-carriers or of carriers of the MR I variant of FSAP, that is, in one of the sample types the activity is reinforced, therefore it rises, while in the other type of sample the activity is reduced, that is, it is inhibited.

The diverse (differential) modulation of FSAP activity in carrier and non-carrier samples of the FASP MR I variant allows better differentiation between both types of sample. As depicted in Figure 6, this leads to a reduced overlap of the distributions of FSAP activities in samples taken from the universes of non-carriers of the MR I polymorphism and of carriers of the MR I polymorphism. With this, it is possible to better distinguish between non-carriers and the various carriers, thereby increasing the sensitivity and / or specificity of the diagnosis [Vizthum, F. and Otro (s) (2005) Proteomics: from basic research to diagnostic application. A review of requirements & needs. J. Proteome Res., 4 (4): 1086-97].

By way of example, during the use of aprotinin as differential modulator of activity, a quantitative difference is observed: the activity of the FSAP that activates the plasminogen activator in samples of non-carriers and carriers, is more strongly inhibited than the activity of FSAP that activates plasminogen activator in samples of carriers of the FASP MR I mutation. In this way, it is possible to differentiate between homozygous and heterozygous carriers of the wild type or MR I variant of FSAP, and thereby identify them.

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Another objective of the present invention is an in vitro diagnostic procedure for the individual detection of individuals who have a genetically conditioned heterozygous or homozygous expression of the MR I variant of FSAP, in which the amplitude of the change in activity is determined. of the FSAP that is contained in a sample. For this, the activity of the FSAP that is contained in a sample is determined, in the absence as well as in the presence of a differential modulator of activity, being able to carry out the determination of the activity of the FSAP in the absence and in the presence:

1) .- in parallel, that is, in two reactive preparations, or

2) .- successively, that is, consecutively in a single reactive preparation, first in the absence and then, after the addition of the differential modulator of activity, in the presence of the differential modulator of activity.

A distinction can be made between two preferred test methodologies, which are suitable for determining the extent of change in FSAP activity:

1.- Determination of the change in activity in two reagent preparations

In this methodology, the amplitude of the change in the activity of the FSAP that is contained in a sample is determined, in which methodology, in a first reactive preparation, that is, in a first aliquot of a sample, the activity of the FSAP is measured in the presence of of a differential modulator of activity, and in a second reagent preparation, that is, in a second aliquot of the same sample, the activity of FSAP is measured in the absence of this differential modulator of activity. Comparison of the results of both reactions provides information about the amplitude of the change in FSAP activity in the presence of the activity modulator.

It is preferable to determine the amplitude of the change in the activity of the FSAP that activates the plasminogen activator, of a biological sample, preferably a blood or plasma sample, for which the activity of the FSAP that activates the prourokinase is measured once in absence and once in the presence of a differential modulator of activity, through the kinetics of conversion of a plasminogen activator and its substrate.

In another embodiment, the amplitude of the change in FSAP amidolytic activity of a biological sample, preferably a blood or plasma sample, is determined, the amidolytic activity of FSAP being measured once in the absence and once in the presence of a modulator. differential activity, by means of low molecular weight FSAP substrate conversion kinetics.

2.- Determination of the change in activity in a single reagent preparation

In this assay methodology, the amplitude of the change in the activity of the FSAP that is contained in a sample is determined, for which the sample is subjected to incubation in a single reagent preparation together with one or more reagents that allow the determination of the FSAP activity, a differential modulator of activity being added during the course of the reaction, and the resulting change in the reaction being monitored.

It is preferable that the amplitude of the change in the activity of the FSAP that activates the plasminogen activator is determined, in a biological sample, preferably a blood or plasma sample, for which the activity of the FSAP that activates the prourokinase is measured, first in the absence of a differential modulator of activity through the kinetics of conversion of a plasminogen activator substrate or a urokinase substrate. During the course of the reaction, a differential modulator of activity is then added to the reagent preparation, and the resulting change in reaction is monitored.

In another embodiment, the amplitude of the change in FSAP amidolytic activity of a biological sample, preferably a blood or plasma sample, is determined, the FSAP amidolytic activity being measured first in the absence of a differential modulator of activity, by means of the reaction kinetics of a low molecular weight FSAP substrate. During the course of the reaction, a differential modulator of activity is then added to the reagent preparation, and the resulting change in reaction is monitored.

Within the scope of the invention, the term "sample" refers to the material presumed to contain the FSAP or the FASP MR I variant. The term "sample" encompasses biological fluids or tissues, especially from humans and animals such as blood, plasma, serum, as well as other body fluids, secretions or extracts, presumed to contain the FSAP or the mutant FASP MR I. Eventually it is necessary to apply a preliminary treatment to the samples, so that the analytes are accessible for the detection procedure or to remove undesirable disturbing components from the samples. Such preliminary treatment of samples may include cell separation or lysis, precipitation, hydrolysis or denaturation of sample components such as proteins, centrifugation of samples, treatment of samples with organic solvents such as example alcohols, especially methanol; treatment of samples with detergents. The sample is frequently transferred to another medium, usually water, which should not interfere with the detection procedure if possible.

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In a preferred embodiment for determining FSAP activity, a biological sample, preferably a blood sample or a plasma sample, is incubated on a solid phase to which a binding partner with an affinity for the FSAP. Coligants that bind the wild-type FSAP protein with the same affinity as an FSAP protein with the MR I mutation are to be preferred. Binders that have an affinity for FSAP and are suitable for the enrichment, or isolation, of FSAP protein from complex protein solutions, such as plasma samples, encompass, for example, substances from the heparin group, heparan sulfate, dextran sulfate, and hyalouronic acids. Also suitable are monoclonal and polyclonal antibodies against FSAP or antibody fragments, such as F (ab ') or F (ab') fragments.₂. Monoclonal antibodies that are formed from one of the hybridoma cell strains DSM ACC2453 and DSM2454, deposited with the DSMZ - Deutsche Sammlung von Mikroorganismen und Zellkulturen (German Collection of Microorganisms, GmbH, Mascheroder weg 1b, 38124, Braunschweig are especially preferred. , Germany.

Within the scope of this invention, the term "solid phase" encompasses an object that consists of a porous and / or non-porous material, generally not soluble in water and that can have the most diverse forms, such as the form of containers, small tubes, microtiter plates, spheres, microparticles, rods, strips, filter paper or chromatography paper. In general, the surface of the solid phase is hydrophilic, or it is possible to give it a hydrophilic character. The solid phase can consist of the most diverse possible materials, such as inorganic and / or organic materials, of materials that occur in nature and / or of modified natural origin. Examples of solid phase materials are polymers, such as cellulose, nitrocellulose, cellulose acetate, polyvinyl chloride, polyacrylamide, cross-linked dextran molecules, agarose, polystyrene, polyethylene, polypropylene, polymethacrylate or nylon; ceramics, glass, metals, especially noble metals such as gold and silver; magnetite, mixtures or combinations thereof, etc. The solid phase can present a coating consisting of one or more layers, for example of proteins, carbohydrates, lipophilic substances, biopolymers, organic polymers or mixtures thereof, for the purpose of, for example, repressing or preventing the non-specific binding of components. of samples in the solid phase or for example to achieve improvements regarding the stability of the suspension of particular solid phases, the stability under storage conditions, the stability of the configuration (dimensional stability) or the resistance against UV light, against microbes or against any other agents of harmful action. Microparticles are frequently used as the solid phase. Within the scope of this invention, the notion "microparticles" refers to particles having an approximate diameter of at least 20 nm and not more than 20 pm, usually between 40 nm and 10 pm, preferably between 0.1 and 10 pm, especially preferably between 0.1 and 0.5 pm, more preferably still between 0.15 and 2 pm. The microparticles can have a regular or irregular configuration. They can be balls, spheroids, spheres with cavities or more or less large pores. The microparticles can consist of organic or inorganic material, or of a mixture or combination of both. They may consist of a porous or non-porous material, or a swellable or non-swellable material. In principle, the microparticles can be of any density. Microparticles can consist of several layers such as so-called "core-and-shell" particles with a core and one or more shell layers. The term "microparticle" encompasses, for example, dye crystals, metal sols, silica particles, glass particles, magnetized particles, polymer particles, oil droplets, lipid particles, dextran and protein aggregates, particles consisting of polymeric material, in special substituted polyethylenes, latex particles, for example polystyrene, acrylic acid polymers, methacrylic acid polymers, acrylonitrile polymers, acrylonitrilebutadiene-styrene, polyvinyl acetate, polyvinylpyridine, vinyl chloride-acrylate. Of particular interest are particles that have reactive groups on their surface, such as carboxyl, amino or aldehyde groups, which allow a covalent bond of, for example, the binders, to the latex particle.

For the determination of the activity of the FSAP that activates the plasminogen activator, after washing the solid phase, the FSAP bound to the co-ligand is incubated with an inactive, single-chain plasminogen activator, such as prourokinase or sct- PA and a urokinase substrate or a sct-PA substrate. The FSAP-dependent activation of, for example, prourokinase to obtain urokinase is measured by conversion or cleavage of the urokinase substrate. Preferred substrates are low molecular weight peptide substrates, which have a signal-forming group. The unfolding of the substrate leads to the release of the signal-forming groups. The physical or chemical properties of the released signaling groups differ from the properties of the groups coupled to the peptide and can be determined quantitatively with the aid of suitable procedures. Suitable signal-forming groups are, for example, luminescence, fluorophores or chromophores, known to the skilled person, which can be measured by optical methods such as luminescence, fluorescence or absorption measurements. Since the intensity of the signal is correlated with the amount of substrate cleaved, it is possible in this way to determine the activity of the FSAP that activates the plasminogen activator. A low molecular weight substrate of group S-2444 is preferably used.^TM (Glu-Gly-Arg-para-nitroaniline; Chromogenix Instrumentation laboratory SpA, Milan, Italy), Pefachrom® uPA (Ala-Gly-Arg-para-nitroaniline [Pefa-5221]; Pentapharm Ltd., Basel, Switzerland) and Chromozym ™ U (Roche Applied Science, Indianapolis, USA).

For the determination of the amidolytic activity of FSAP, after washing the solid phase, the FSAP bound to the binder is incubated together with a low molecular weight substrate, and the conversion or unfolding of the substrate is measured. Within the scope of the present invention, low molecular weight substrates are peptide substrates, consisting of a sequence from 2 to 100, preferably 2 to 50, especially preferably 3 to 10, natural or non-natural amino acids, which additionally possess a signal-forming group that unfolds from the FSAP. The cleavage of a low molecular weight substrate leads to the release of the groups

ES 2 332 467 T3 signal formers. The physical or chemical properties of the released signaling groups differ from the properties of the groups formed on the peptide and can be determined with the aid of suitable procedures. Suitable signal-forming groups are, for example, luminescence, phosphor or chromophores, known to the skilled person, which can be measured by optical methods such as luminescence, fluorescence or absorption measurements. Since the intensity of the signal is correlated with the amount of substrate cleaved, it is possible to determine the amidolytic activity of FSAP in this way. A low molecular weight chromogen substrate selected from the group consisting of: Pefa-3297, Pefa-5114, Pefa-5523, Pefa-5773, Pefa-5979, Pefa-3107, Pefa-5329 (all of them from the series Pefachrom®, Pentapharm Ltd, Basel, Switzerland), S-2288^TM, S-2765^TM, S-2338^TM, S-2222^TM, S-2302^TM (all from the S Series, Chromogenix Instrumentation Laboratory SpA, Milan, Italy) (See also Romish and Other (s) (1999) Hemostasis 29, 292-299 and Hunfeld and Other (s) (1999) FEBS Letters 456, 290) -294).

Measurement of the substrate cleavage reaction can take place over the entire time interval of the reaction until the occurrence of equilibrium, or at least in a certain time interval, or at least until a moment in time. Photometric data can be used to determine activity, for example absorption spectra or values ​​under certain wavelengths, per se, or by referring to a time interval. If the activity is determined by photometric data related to a time interval, that is, if it includes the conversion or reaction rate, various procedures can be used for the determination of the conversion or reaction rate. For example, it is possible to determine the conversion rate using time-conversion curves. In the case of the time-conversion curves, the concentration of the cleavage product is plotted as a function of time. For the determination of the conversion rate, a straight line is drawn in the region of the 0-th order reaction of the time-conversion curve, usually at the beginning of the measurement of the reaction of an enzyme. In this case the inclination or slope of the straight line provides the conversion speed, that is, the change in the concentration of the substrate or the product in a given time interval [Bibliography: Bisswanger, H., Enzymkinetik: Theorie und Methoden , 2, vollig neu bearbeite Auflage, VCH Verlaggesellschaft mbH, 1994, Weinheim, Ny, Basel, Cambridge, Tokyo; especially pp. 66-67].

The measured variables or parameters, which are suitable for the evaluation of the conversion kinetics, are for example all the parameters that describe the reaction kinetics, such as the analysis of the curves per se, but in particular certain individual parameters of the reaction kinetics, such as the maximum inclination, that is, the reaction rate (V ^), the parameters related to the sigmoidal kinetics, the area under the curve, etc. The parameters that are suitable for the evaluation of the test are, for example, also the absolute measurement values, such as, for example, the absorption values ​​that are measured at a given instant of time, or an instant in time in which it has been reached. a certain absorption value, for example a maximum value.

For the determination of the change in FSAP activity in two reagent preparations, the difference or the quotient of a test parameter is preferably formed, which was determined once in the absence and once in the presence of the activity modulator, for the purposes of determine the extent of change in FSAP activity. For example, in the case of using an inhibitor that inhibits the activity of FSAP in samples of non-carriers more strongly than in samples of carriers of MR I, the formation of the difference or the quotient obtained from the reaction rate ( V_max) of the conversion index of the substrate in the absence of the inhibitor (V_{max 0}) and the reaction rate of the substrate conversion index in the presence of the inhibitor (V_max inhibitor), makes it possible to differentiate between FSAP MR I carriers and non-carriers of FSAP MR I. Homozygous or heterozygous carriers of the FSAP MR I mutation show a smaller difference, or a smaller value ratio, than non-carriers. Other algorithms such as the sum or the product are also suitable, as long as they allow a differentiation.

In addition to the speed of the reaction, it is also possible to use absolute test signals or measured values, such as, for example, absorption values ​​that are reached at a certain point in time (Cfg. Figures 1 and 2; in this case, a suitable instant in time could preferably be established in a time interval between 10 and 70 minutes, especially preferably between 30 and 45 minutes, especially at 40 minutes, since in this case there is a maximum difference between those prepared with and without aprotinin).

For the determination of the change in FSAP activity in a single reagent preparation, it is necessary to measure the substrate cleavage reaction before and after the differential modulator of activity is added. At least it is necessary to measure the reaction in at least one instant in time or over a discrete time interval, before and at least at one instant in time or over a discrete time interval after adding the differential modulator of activity.

A preferred differential modulator of activity of FSAP for use in the process according to the invention is aprotinin. Aprotinin inhibits FSAP activity in non-carrier samples more intensely than in MR I carrier samples (See Table 3).

Other preferred differential modulators of FSAP activity for use in the method according to the invention are monoclonal or polyclonal anti-FSAP antibodies. Especially preferred monoclonal antibodies are those that are produced from the hybridoma cell strain, deposited with the Registration Number DSM ACC2726 (EP 1 360 175 A1) with the Deutsche Sammlung von Mikrooganismen und Zellkuklturen GmbH, Mascheroder Weg 1b, 38124 Braunschweig , Germany. A

ES 2 332 467 T3 monoclonal anti-FSAP antibody, which binds to an epitope of FSAP, which is bound by a monoclonal anti-FSAP antibody formed from the hybridoma cell strain DSM ACC 2726. These monoclonal antibodies bind to FSAP and modulate FSAP activity in non-carrier samples, at an amplitude that is different from the amplitude corresponding to MR I carrier samples (See Table 3).

To check whether within the scope of the present invention a substance or a combination of substances is suitable for use as a differential modulator of activity, one can proceed as follows:

The influence of the substance under study, or of the combination of substances under study, on the activity of the FSAP that activates the plasminogen activator, is determined using samples known to contain the FASP MR I variant and with samples which are known not to contain the fAsp MR I variant but do contain the FSAP of the wild-type form. In this case, it can be, for example, samples from one or more persons whose FSAP genotype is known. In addition, samples containing a defined amount of wild-type FSAP protein or FSAP MR I protein can be used. The FSAP protein, or the FSAP MRI protein, which can be used for the preparation of such a sample, can be enriched or isolated for example from human biological material, or produced recombinantly or transgenically. Procedures for the enrichment, isolation, preparation or stabilization of FSAP protein are described in EP 1 226 829, EP 1 074 615 A1 and EP 1 074 616 A1.

The activity of the FSAP activating plasminogen activator, which is contained in various samples, is then determined in the absence as well as in the absence of the substance under investigation. In this case, the substance, or the combination of substances, whose aptitude as differential modulator of activity is the object of study, are applied in at least one concentration, but preferably in various concentrations, as represented for example for aprotinin in the Figures 1 and 2.

The amplitude of the change in the activity of the FSAP that activates the plasminogen activator, in samples with the FSAP MR I variant, compared to samples that do not contain the FSAP MR I variant, but do show FSAP activity in the wild-type form , allows the identification of substances or combinations of substances, which can be used as differential modulators of activity. With this, it is possible to identify substances as differential modulators of activity by the fact that they inhibit or enhance both the activity of the FSAP MR I variant as well as the activity of the wild-type form of FSAP, although they do so to a different amplitude. . However, a substance is also a differential modulator of activity if it inhibits the activity of the FSAP MR I variant and enhances the activity of the wild-type form of FSAP.

The first studies to establish whether certain substances are suitable as differential modulators are carried out with a relatively small number of samples, for example with at least one sample of the FSAP MR I variant in each case, but preferably with in each case. two to ten samples. Substances that in this regard provide significant differentiation, preferably statistically significant differentiation, of samples with wild-type FSAP and with FSAP MR I, could be validated with additional studies with larger numbers of samples.

For the subsequent validation of substances or combinations of substances, tests of samples of people whose genotype is known are preferably used, in order to obtain realistic information about the performance of the test, and to determine with sufficient specificity and diagnostic sensitivity the presence or absence of a FSAP MRI variant. The number of samples to be investigated, from such a phase I study, depends on the expected accuracy of the assay and the relationship between the number of samples with or without the FSAP MR I variant [Obuchowsky, NA and Other (s ) (2004) ROC Curves in Clinical Chemistry: Uses, Misuses and Possible Solutions, Clinical Chemistry, 50: 7.1118-1125]. In the case of practically perfect accuracy in the tests, and if the relationship between the samples with and without the FSAP MR I variant is equal to one, in each case ten samples will be sufficient to obtain statistically significant values. The number of samples increases as the ratio between the samples with and without the FSAP MR I variant increases or decreases, as well as as the accuracy of the assays decreases. In this eventuality, it will be necessary to investigate, for example, more than one hundred samples in each case.

The study of substances or combinations of substances to establish their aptitude as differential modulators of activity can be carried out, for example, by determining the activity of the FSAP that activates prourokinase, in the presence and absence of these substances, possibly at various concentrations. By way of example, solid phase associated binders with affinity for FSAP are used as in Example 1, especially an anti-FSAP antibody, which is formed from the hybridoma cell strain DSM ACC2453, and is carried out Carry out an activity assay as described in Example 1. The differentiation between carriers and non-carriers of the FASP MR I variant is determined by forming a ratio between V_max of the reaction kinetics of a sample without additive (V_{max 0}) and with the addition of the substance (V_max substance).

A similar procedure can be followed when substances or combinations of substances are to be studied to establish their suitability as differential modulators of the activity of the amidolytic activity of FSAP. A test procedure for the determination of the amidolytic activity of FSAP, as described in Example 2, can be carried out once in the absence and once in the presence of the substance under study, and the results of the tests can be used. analogously to the assessment of the suitability of a substance, as described above.

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Classes of substances that are particularly suitable for a study in terms of their suitability as differential modulators of activity for FSAP activity include, for example:

a) .- ions (anions such as chloride, carbonate, sulfate, phosphate, etc., or cations such as sodium, lithium, ammonium, magnesium, calcium, manganese, etc.);

b) .- chelating agents, such as ethylenediamine tetraacetate (EDTA), N, N, N ', N'-ethylene glycol-bis (eaminoethyl ether) tetraacetate, citrate, etc .;

c) .- detergents such as sodium dodecyl sulfate (SDS), Triton® X 100, Tween®, etc .;

d) .- redoactive substances such as dithioerythrol, dithiothreitol, β-mercaptoethanol, glutathione, lipoic acid, vitamin C, vitamin E, etc .;

e) .- nucleic acids, especially aptamers;

f) .- antibodies;

g) .- proteins, peptides and oligopeptides such as antithrombin III, C1 esterase inhibitor, tissue factor pathway inhibitor (TFPI), heparin II cofactor, alpha2-macroglobulin, alpha2-antiplasmin, inter-alpha- trypsin, alpha1-antitrypsin, alpha1-antichymotrypsin, plasminogen activator type 2 inhibitor (PAI2), plasminogen activator type 3 inhibitor (PAI-3), quininogen, high molecular weight kinogen (HMWK), etc .;

h) .- synthetic serine protease inhibitors, such as FOY-305 [N, N-dimethylcarbamoylmethyl 4- (4-guanidinobenzoyloxy) -phenylacetate methanesulfonate] and corresponding derivatives;

i) .- protease inhibitors, low molecular weight, such as FOIPAN (mesylate camostat).

Figures (edit)

Figure 1

Figure 1 shows the inhibition of the activity of FSAP activating prourokinase in the presence of aprotinin, which was determined for a plasma sample from a non-carrier (wild type).

Figure 2

Figure 2 shows the inhibition of the activity of FSAP that activates prourokinase in the presence of aprotinin, which was determined for a plasma sample of a heterozygous carrier of the MR I polymorphism.

Figure 3

Figure 3 shows the inhibition of FSAP amidolytic activity in the presence of aprotinin, which was determined for a sample from a collection of plasmas from non-carriers of the FSAP MR I mutation (wild type).

Figure 4

Figure 4 shows the inhibition of FSAP amidolytic activity in the presence of aprotinin, which was determined for a plasma sample of a heterozygous carrier of the MR 1 polymorphism.

Figure 5

Figure 5 shows the change in FSAP amidolytic activity for a plasma sample from a non-carrier (wild type; open circle) and from a heterozygous carrier of the MR I polymorphism (solid squares) in the absence of aprotinin and in the presence of aprotinin. after it has been added (arrow), for a reaction time of approximately one hour, the activity having been measured in a single reagent preparation. Although both samples initially show the same FSAP activity in the absence of aprotinin, so a differentiation is not possible, the activities change in the presence of aprotinin, so that it is possible to distinguish between both samples, since FSAP activity is more inhibited. strongly in the non-carrier sample.

Figure 6

Figure 6 shows by way of example a possible frequency distribution in universe samples of homozygous carriers of the wild-type form (dashed line) and MR I heterozygotes (solid and dotted lines). In the case of the dotted line, a modulator was used that does not influence the activity of FSAP for homozygous carriers of the wild-type form, and that reduces the activity of FSAP to a greater degree.

ES 2 332 467 T3 for MR I heterozygotes. In the presence of the modulator of activity, the overlapping of the distributions of FSAP activities taken from samples of universes of carriers of the wild-type form and of MR I heterozygotes, is less than in the case of the absence of the activity modulator. In this way, in the case of the presence of the activity modulator, the differentiation between the various carriers or forms can be carried out better than in the absence of the activity modulator, thereby increasing the sensitivity and / or specificity of the diagnosis.

The examples described below serve to clarify individual aspects of this invention, and are not to be construed as limiting.

Example 1

Determination of the activity of FSAP as a prourokinase activator in the absence and in the presence of the differential modulator of aprotinin activity

An anti-FSAP antibody formed from the hybridoma cell strain DSM ACC2453 was used as a solid phase associated binder with affinity for FSAP. To carry out the heterogeneous detection procedure, polystyrene microtiter plates (MTPs) were used as solid phase. Anti-FSAP antibody in 50 ml of NaHCO was associated with the solid polystyrene phase overnight at room temperature.₃, pH 8.2, with a coating volume of 120 µg for each cup of MTP and a coating concentration of 20 µg of antibody per ml. Unbound antibodies were removed by triple washing with 50 ml of isotonic NaCl solution buffered with sodium phosphate, 0.2% Tween® 20, pH6.5.

In each of the wells, 100 µl of a plasma sample to be determined was pipetted at a 1:80 dilution in sample buffer (20 mM sodium citrate, pH 6.0 with 150 ml of NaCl, monohydrochloride 100 mM L-arginine, 1% bovine serum albumin, 0.1% Tween® 80, 100 IU heparin / ml). After incubation at + 37 ° C for one hour, the unbound component parts were removed by triple washing with an isotonic NaCl solution buffered with 50 mM sodium phosphate, Tween ^® 20 to 0.02%, pH 6.5.

For the determination of the activity of the FSAP that activates prourokinase, after removing the sample and washing the solid phase, the following were added:

a) .- 30 μl of assay buffer I (50 mM Tris / HCl, pH 7.2 with 150 mM NaCl, 0.2% Tween® 80, CaCl₂ 15 mM and 50 IU of heparin per liter), or:

b) .- 30 µl of the assay buffer I, which also contained aprotinin in a concentration such that a final concentration of 0.055 KIE / ml of aprotinin was achieved in the reagent preparation (1 U = unit that inhibits kallikrein [KIE]; Aprotinin taken from bovine lungs, Sigma-Aldrich Laborchemikalien GmbH, Taufkirchen, Germany), and in each case 50 µl of recombinant prourokinase (Landing Biotech Inc., Brighton, MA, USA; 5 jug / ml in assay buffer I) and in each case 50 μl of the chromogenic substance S-2444^TM (0.6 mM) in Assay Buffer II (100 mM Tris / HCl, 150 mM NaCl, 15 mM Na-azide, 0.1% Tween® 80, pH 8.2) in a beaker of MTP, and was incubated at + 37 ° C. The change in absorption (OD) of the reagent preparation was observed under a wavelength of 405 nm. The results have been summarized in Table 1. A differentiation between carriers and non-carriers of the FASP MR I variant is possible by forming a ratio between V_max of the reaction kinetics of a sample without the addition of aprotinin (V_{max 0}) and with the addition of aprotinin (V_{max arrotinin}). To this end, the following expression applies:

V_max0 FSAP wild-type / V_{max arrotinin} wild-type FSAP> V_max0 by FSAP MR I / ^Vmax aprotinin ^{by FSAP MR} I.

On the other hand, FSAP MR I (MR I) samples show a difference between V_max0 and V_{max arrotinin} which is conspicuously lower than the wild-type (WT) FSAP samples (See Table 1). In the "MW" column the mean value (Mittelwert) of the three individual determinations has been entered.

The maximum steepness (slope) of the time-conversion curves was determined by linear regression in a sufficiently linear region of the time-conversion curve, and provided the maximum reaction rate V_max of a reaction in mOD / minute.

ES 2 332 467 T3

Table 1

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ES 2 332 467 T3

Figures 1 and 2 show the different reaction kinetics of the conversion of S-2444^TM, hence the activity of FSAP that activates prourokinase. Figure 1 shows the reaction kinetics that have been determined for a plasma sample from a non-carrier, with and without the addition of aprotinin (0.036 and 0.0554 KIE / ml, final concentration of aprotinin). In this case, 0.036 KIE / ml of aprotinin corresponds to a dilution of aprotinin of 1: 30,000, and 0.055 KIE / ml corresponds to a dilution of aprotinin of 1: 20,000. Figure 2 shows the reaction kinetics that were determined for a plasma sample of a heterozygous carrier of the FASP MR I variant with and without the addition of aprotinin (0.036 and 0.055 KIE / ml final aprotinin concentration). It can be clearly recognized that the activity of the FSAP activating prourokinase in the non-carrier sample in the presence of aprotinin is manifestly inhibited to a greater degree than the activity of the FSAP activating prourokinase in the sample of the heterozygous carrier.

Example 2

Determination of FSAP amidolytic activity with respect to the chromogenic low molecular weight peptide substrate, S-2288^TM, in the presence and in the absence of the modulator of differential activity aprotinin

The same microtiter plates were used as described in Example 1. In each of the wells, 100 µl of a plasma sample to be determined was pipetted at a 1:50 dilution in sample buffer (See Example 1). After incubation at + 37 ° C for one hour, the unbound component parts were removed by triple washing with an isotonic NaCl solution buffered with 50 mM sodium phosphate, Tween^® 20 to 0.02%, pH 6.5.

To determine the amidolytic activity of FSAP, after removing the sample and washing the solid phase, the following were added:

a) .- 30 µl of the assay buffer I (See Example 1); or

b) .- 30 µl of the assay buffer I, which also contained aprotinin in a concentration such that a final concentration of 0.055 KIE / ml of aprotinin was achieved in the reagent preparation (See Example 1);

and in each case 80 μl of the chromogen substrate S-2288^TM (1.5 mmol / L; Chromogenix Instrumentation Laboratory

SpA, Milan, Italy) in the assay buffer (See Example 1) in the assay well, and incubated at + 37 ° C for one hour. In order to reduce volatilization effects during the relatively long incubation time in the MTP, it was coated with mineral oil.

The change in absorption (OD) of the reagent preparation was monitored with a wavelength of 405 nm, and the maximum velocity of the reaction V was determined._max (See Example 1). The results have been summarized in Table 2.

A differentiation between carriers and non-carriers of the FASP MR I variant is possible by forming a relationship (quotient) between the V_max of the reaction kinetics of a sample without the addition of aprotinin (V_max0) and with the addition of aprotinin (V_{max aprotinin}). To this end, the following expression applies:

V_max0 FSAP wild-type / V_{max aprotinin} wild-type FSAP&gt; V_max0 by FSAP MR I / ^Vmax aprotinin ^{by FSAP MR} I.

On the other hand, the FASP MR I (MR I) samples show a difference between V_max0 and V_{max arrotinin}a manifestly lower than wild-type (WT) FSAP samples (See Table 2). In the column "MW" the mean value of the three individual determinations has been entered.

ES 2 332 467 T3

Table 2

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<td>Without aprotinin: Vmax₀</td><td colspan="3">S (M CM CD O t- 'O CM</td><td colspan="3"> 11,37 9,99 14,06</td>

<td>FSAP-</td><td colspan="3">or. tr 2 s Ξ</td><td colspan="3">WT WT WT</td>

<td>Sample Do not.</td><td> 14942</td><td> 7020538</td><td> 7020551</td><td>WTP2</td><td> 305</td><td> 2176</td>

ES 2 332 467 T3

Figures 3 and 4 show the different reaction kinetics of the conversion of S-2488 ™, hence the amidolytic activity of FSAP. Figure 3 shows the reaction kinetics that have been determined for a plasma sample of a non-carrier of the MR I mutation, with and without the addition of aprotinin (0.036 and 0.055 KIE / ml, final concentration of aprotinin). Figure 4 shows the reaction kinetics that were determined for a plasma sample of a heterozygous carrier of the FASP MR I variant with and without the addition of aprotinin (0.036 and 0.055 KIE / ml final aprotinin concentration). It can be clearly recognized that the amidolytic activity of FSAP in the non-carrier sample in the presence of aprotinin is manifestly inhibited to a greater degree than the amidolytic activity of FSAP in the heterozygous carrier sample.

Example 3

Determination of the change in the amidolytic activity of FSAP with respect to the low molecular weight chromogenic peptide substrate, S-2288 ™, by adding a differential modulator of activity during the course of the reaction.

The same microtiter plates were used as in Example 1. In each of the wells, 100 µl of a plasma sample to be analyzed was pipetted at a 1:15 dilution in sample buffer (See Example 1 ). After incubation at + 37 ° C for one hour, the unbound component parts were removed by triple washing, as in Example 1.

To determine the amidolytic activity of FSAP, at time t₀ 80 μl of the chromogen substrate S-2288 were introduced into the MTP cup in each case^TM (1.5 mmol / L; Chromogenix Instrumentation Laboratory SpA, Milan, Italy) in a 50:50 mixture of Assay Buffer I and Assay Buffer II (Assay Buffer I and II, see Example 1), and subjected incubation at + 37 ° C in the photometer. After approximately 60 minutes, aprotinin was added to a final concentration of 2.95 KIE / ml or the corresponding monoclonal antibody (MAK) to a final concentration of approximately 60 jug / ml for each reagent preparation. The change in the absorption of the reactive preparations was observed under a wavelength of 405 nm, and the maximum velocity of the reaction V was determined._max (see Example 1). In order to reduce vaporization effects during the relatively long incubation period in the MTP, it was coated with mineral oil.

A differentiation between carriers and non-carriers of the FASP MR I variant is possible by forming a relationship, for example the ratio between the maximum rate of the reaction before the addition of the differential modulator of activity (V ^) and the rate Maximum of the reaction after the addition of the differential modulator ^{of the activity (V}_max aprotinin⁾.

According to Figure 5, the following expression applies to the modulator of aprotinin activity:

V_max0 FSAP wild-type / V_llla,. _aprotinin wild-type FSAP&gt; V_llla,.₀ by FSAP MR I / ^Vmax aprotinin ^{by FSAP MR} I.

Table 3 shows, in addition to aprotinin, the influence of monoclonal antibodies (MAKS) on the V ratio._max/ V_{max modulator activ}¡_dad. In the case of the non-carrier sample of the FASP MR I variant (wild-type sample; WT), three independent measurements were carried out. In the case of the sample of the heterozygous carrier of the FASP MR I (MR I) variant, 6 independent measurements were carried out. The mean values ​​(MW) of V were calculated_max/ V_{max modulator activ}i_dad (V_max/ V_maxA) and the standard deviations referred to the statistical universe (Staw). Negative controls, i.e. the addition of a corresponding volume of Assay Buffer I and Assay Buffer II in the ratio 50:50 with no activity modulator, resulted in a MW V_max/ V_{max A}, 0.81 +/- 0.12.

In order to get an indication of the significance of the influence of an activity modulator, a t-test (t-Test) was carried out. The t-test was based on the adoption of two random samples of equal and unequal variance. A one-sided test (one extreme area) was carried out. This t-test shows that aprotinin, anti-FSAP monoclonal antibody MAK 2004-151 / 013 (2) (DSM ACC2726) and anti-FSAP monoclonal antibody MAK 2004-98 / 016 (3), the latter serving here to For illustrative purposes only and not part of the invention, the activity of the WT sample more strongly reduces the activity of the WT sample with respect to the MR I sample, whereby the WT / MR I ratio is&gt; 1. In contrast, the anti-FSAP monoclonal antibody MAK 1102 / 1189-2 (DSM ACC2454) more strongly reduces the activity of the MR I sample relative to the wild-type sample, so the WT / MR I ratio is &lt; 1. This is also the case for the anti-FSAP monoclonal antibody MAK 2004-35 / 90 (1) (DSM ACC2674). Given that in this case the P values ​​do not yet indicate any significance but are comparatively low, it can be assumed that if there are a sufficient number of measurements, that is, in the case of a greater number of cases, the hypothesis to zero. The MAK 1102 / 1189-2 (DSM ACC 2454) and the MAK 2004 200435/05 (1) (DSM ACC 2674), which are for illustration purposes only and are not part of the invention, may also be suitable as modulators of the activity.

ES 2 332 467 T3

In contrast to this, due to their WT / MRI ratios and their t-test values, anti-FSAP monoclonal antibodies, MAK 1102 / 570-09 (DSM ACC2533, see EP 1 334 983 A2), MAK 2004 -9/026 (2) (DSM ACC2676, see EP 1 630 175 A1) and MAK 2004-34 / 08 (2) DSM ACC2725, see EP 1 630 175 A1) are not suitable as modulators of activity .

TABLE 3

<td>Activity modulator</td><td colspan="2">WT</td><td colspan="2">MRI</td><td>WT / MR I</td><td>P-value for equal variance</td><td>P-value for different variance</td>

<td></td><td>MW Vmax / Vmax A</td><td>Staw</td><td>MW Vmax / V max A</td><td>Staw</td><td></td><td></td><td></td>

<td>Aprotinin</td><td> 16,72</td><td> 1,38</td><td> 12,36</td><td> 2,31</td><td> 1,35</td><td> 0,0331</td><td> 0,0224</td>

<td>MAK 1102/57009 (DSM ACC2533)</td><td> 1,32</td><td> 0,20</td><td> 1,36</td><td> 0,15</td><td> 0,97</td><td> 0,7947</td><td> 0,8293</td>

<td>MAK 1102/1189 * (DSM ACC2454)</td><td> 0,72</td><td> 0,03</td><td> 0,78</td><td> 0,04</td><td> 0,92</td><td> 0,0757</td><td> 0,0625</td>

<td>MAK 2004- 9/026 (2) (DSM ACC2676)</td><td> 0,71</td><td> 0,05</td><td> 0,76</td><td> 0,03</td><td> 0,93</td><td> 0,1109</td><td> 0,2555</td>

<td>MAK 2004- 35/05 (1) * (DSM ACC2674)</td><td> 0,71</td><td> 0,07</td><td> 0,87</td><td> 0,19</td><td> 0,82</td><td> 0,2596</td><td> 0,1531</td>

<td>MAK 200434/08 (2) ACC2725)</td><td> 0,74</td><td> 0,05</td><td> 0,77</td><td> 0,06</td><td> 0,95</td><td> 0,4143</td><td> 0,4229</td>

<td>MAK 2004151/013 (2) (DSM ACC2726)</td><td> 0,95</td><td> 0,04</td><td> 0,80</td><td> 0,05</td><td> 1,19</td><td> 0,057</td><td> 0,0119</td>

<td>MAK 2004- 98/016 (3) *</td><td> 0,94</td><td> 0,05</td><td> 0,77</td><td> 0,06</td><td> 1,23</td><td> 0,0063</td><td> 0,0109</td>

<td colspan="8">* Antibodies are for illustration purposes only and are not part of the invention.</td>