EP1896856A1

EP1896856A1 - Polypeptide marker for detecting the rejection of transplanted kidneys at an early stage

Info

Publication number: EP1896856A1
Application number: EP06763931A
Authority: EP
Inventors: Harald Mischak; Stefan Wittke
Original assignee: Mosaiques Diagnostics and Therapeutics AG
Current assignee: Mosaiques Diagnostics and Therapeutics AG
Priority date: 2005-06-29
Filing date: 2006-06-28
Publication date: 2008-03-12
Also published as: US20150126405A1; US20100200401A1; WO2007000466A1; JP2008545130A; JP5155857B2

Abstract

The invention relates to a method for detecting a rejection after kidney transplantation (NTx). Said method comprises the step of determining the presence or absence of at least one polypeptide marker in a sample (frequency marker) or the step of determining the amplitude of at least one polypeptide marker (amplitude marker), which are characterized by values for the molecular weight and the migration time (CE time).

Description

Polvpeptide marker for early detection of the repression of transplanted kidneys

The present invention relates to the use of the presence or absence of one or more peptide markers in a sample of an individual for the early detection of rejection (rejection) of transplanted kidneys and a method for early detection of rejection of transplanted kidneys, wherein the presence or absence of the or the peptide marker (s) is indicative of the presence of kidney transplant rejection (NTx).

Kidney transplantation is the most commonly performed organ transplantation in Germany. The waiting time for a new kidney is still around 6-8 years. The goal of therapy after successful kidney transplantation (NTx) is to maintain functional capability over the longest possible period and to prevent rejection of the transplant (rejection). On average, 10-15% of organs lose NTx from various complications. A timely detection of the onset of rejection is therefore extremely important, since it must be immediately intervened with medication to obtain the graft.

Surprisingly, it has now been found that certain peptide markers can be used in a sample of an individual to detect the rejection of transplanted kidneys.

Accordingly, an object of the present invention is the use of the presence or absence and amplitude of at least one polypeptide marker in an individual's sample for detecting rejection of transplanted kidney, wherein the polypeptide marker is selected from polypeptide labels Nos. 1 to 767, the characterized by the molecular masses given in Table 1 and their migration times. Table 1: Polypeptide marker for detecting the rejection of transplanted kidneys and their molecular masses (in Da) and migration times (CE time in minutes):

With the present invention it is possible to diagnose the rejection very early. As a result, the onset of rejection can be treated with medication at an early stage. The invention further enables cost-effective, rapid and reliable detection of the rejection by means of interventions that are in part not or only minimally invasive.

The migration time is determined by means of capillary electrophoresis (CE), as described in Example 2, for example. In this example, a 90 cm long glass capillary with an inner diameter (ID) of 50 microns and an outer diameter (OD) of 360 microns at an applied voltage of 25 or 30 kV operated. The eluent used is 30% methanol, 0.5% formic acid or 20% acetonitrile and 0.25 M formic acid in water.

It is known that the CE migration time can vary. Nevertheless, the order in which the polypeptide labels elute is typically the same for each CE system used under the conditions indicated. To even out any differences in migration time, the system can be normalized using standards for which migration times are known. These standards may e.g. be the polypeptides given in the examples (see example point 3).

The characterization of the polypeptides shown in Tables 1 to 2 was determined by capillary electrophoresis mass spectrometry (CE-MS), a method which was described e.g. in detail by Neuhoff et al. (Rapid Communications in mass spectrometry, 2004, Vol. 20, pages 149-156). The variation of molecular masses between individual measurements or between different mass spectrometers is relatively small with exact calibration, typically in the range of ± 0.1%, preferably in the range of ± 0.05%, more preferably ± 0.03%.

The polypeptide markers according to the invention are proteins or peptides or degradation products of proteins or peptides. They may be chemically modified, e.g. by post-translational modifications such as glycation, phosphorylation, alkylation or disulfide bridging, or by other reactions, e.g. in the context of mining, to be changed. In addition, the polypeptide markers may also be chemically altered as part of the purification of the samples, e.g. oxidized, be.

Based on the parameters that determine the polypeptide markers (molecular mass and migration time), it is possible to identify the sequence of the corresponding polypeptides by methods known in the art. The polypeptides of the invention (see Tables 1-4) are used to diagnose incipient rejection of the graft. Diagnosis is the process of gaining knowledge by assigning symptoms or phenomena to a disease or injury. In the present case, the presence or absence or differences in the amplitude of certain polypeptide markers implies rejection. For this purpose, the polypeptide markers according to the invention are determined in a sample of an individual, their presence or absence and their signal intensity / amplitude suggesting the presence of a rejection. The presence or absence and amplitude of a polypeptide marker can be measured by any method known in the art. Methods that can be used are exemplified below.

A polypeptide marker is present when its reading is at least as high as the threshold. If its reading is below that, the polypeptide marker is absent. The threshold value can either be determined by the sensitivity of the measurement method (detection limit) or defined based on experience.

In the context of the present invention, the threshold is preferably exceeded when the sample reading for a given molecular mass is at least twice that of a blank (e.g., only buffer or solvent).

The polypeptide marker (s) is / are used to measure its presence or absence, the presence or absence being indicative of rejection (frequency marker # 1 - # 242, Table 2). Thus, there are polypeptide markers that are typically present in patients after renal transplantation (NTx) and rejection, such as polypeptide markers Nos. 1-5, but are uncommon in NTx patients but without rejection (control). Furthermore, there are polypeptide markers which are frequently present in patients without rejection but which occur less frequently or not at all in patients with rejection, eg 6 to 15 (Table 2). Table 2: Polypeptide markers (frequency markers) for the detection of rejection of kidney transplants, their molecular masses and migration times and their presence and absence in patient groups after NTx with rejection and control groups Patients after NTx without rejection as factor (1 = 100%, 0 = 0%; Sample preparation and measurement as described in the example).

In addition or as an alternative to the frequency markers (determination of the presence or absence), the amplitude markers indicated in Tables 3 and 4 can also be used to diagnose renal transplant rejections (Numbers 243-767). Amplitude markers are used in a manner that does not determine the presence or absence, but decides the magnitude of the signal (amplitude) in the presence of the signal in both groups. Two standardization methods are possible in order to achieve comparability between differently concentrated samples or different measurement methods: In the first approach, all peptide signals of a sample are normalized to a total ampli- tude of 1 million counts. The respective mean amplitudes of the individual markers are therefore given as parts per million (ppm). The amplitude markers resulting from this procedure are shown in Table 3 (number 243-627). In addition, it is possible to define additional amplitude markers via an alternative standardization procedure:

In this case, all peptide signals of a sample are scaled with a common normalization factor. For this purpose, a linear regression is formed between the peptide amplitudes of the individual samples and the reference values of known polypeptides in the sample. The slope of the regression line just corresponds to the relative concentration and is used as a normalization factor for this sample. The markers characterized by this method are shown in Table 4.

All groups used consist of at least 19, preferably at least 20 individual patient or control samples to obtain a reliable mean amplitude. The decision to make a diagnosis (rejection of the kidney transplant or not) will depend on how high the amplitude of the respective polypeptide markers in the patient sample is compared to the mean amplitudes in the control group or rejection group. If the amplitude corresponds more closely to the mean amplitudes of the rejection group, it can be assumed that the kidney transplant is rejected and corresponds more closely to the mean amplitudes of the control group; it can not be assumed to be a rejection. A more precise definition should be given using marker no. 247 (Table 3). The mean amplitude of the marker is markedly increased upon rejection of the kidney transplant (654 ppm versus 75 ppm in the group without rejection). If the value for this marker is 0 to 75 ppm in a patient sample, or a maximum of 20% above this, ie 0 to 90 ppm, this sample belongs to the control group without rejection. If the value is 654 ppm, or 20% lower, or higher, ie between 523 ppm and very high values, rejection after kidney transplantation can be assumed. Table 3: Amplitude markers (ppm normalization)

Table 4: Amplitude marker (with normalization function)

The individual from whom the sample is derived, in which the presence or absence and the amplitude of one or more polypeptide markers is determined, may be any individual who may suffer from a rejection to NTx, e.g. an animal or a human. Preferably, the subject is a mammal, most preferably a human.

In a preferred embodiment of the invention, not only a polypeptide marker but a combination of markers is used to diagnose NTx rejection. Their presence or absence, as well as differences in amplitude, indicate the presence of a rejection after NTx closed. By comparing a plurality of polypeptide markers, the falsification of the overall result can be reduced or avoided by individual individual deviations from the typical probability of presence in the patient or control individual.

The sample measuring the presence or absence as well as the amplitude of the polypeptide marker (s) of the invention may be any sample recovered from the subject's body. The sample is a sample that has a polypeptide composition suitable for making statements about the condition of the individual (rejection to NTx or not). For example, it may be blood, urine, synovial fluid, tissue fluid, body secretions, sweat, cerebrospinal fluid, lymph, intestinal, gastric, pancreatic, bile, tears, tissue, sperm, vaginal fluid, or a stool sample. Preferably, it is a liquid sample.

In a preferred embodiment, the sample is a urine sample or a blood sample, where a blood sample may be a serum (blood) or plasma (blood) sample.

Urine samples may be known as known in the art. Preferably, in the context of the present invention, a mid-jet urine sample is used. The urine sample may e.g. by means of a catheter or also with the aid of a urination apparatus, as described in WO 01/74275.

Blood samples may be taken by methods known in the art, for example from a vein, artery or capillary. Usually, a blood sample is obtained by removing venous blood from an individual, for example, from the arm by means of a syringe. The term blood sample also includes samples obtained from blood by further purification and separation techniques known in the art, such as blood plasma or blood serum. The presence or absence and amplitude of a polypeptide marker in the sample can be determined by any method known in the art suitable for measuring polypeptide markers. Those skilled in such methods are known. In principle, the presence or absence and the amplitude of a polypeptide marker can be determined by direct methods, such as, for example, mass spectrometry, or indirect methods, such as, for example, by means of ligands.

If necessary or desirable, the sample of the individual, e.g. the urine or blood sample, pretreated prior to measurement of the presence or absence and the amplitude of the polypeptide marker (s) by any suitable means, and e.g. be cleaned or separated. The treatment may e.g. a purification, separation, dilution or concentration. The methods may be, for example, centrifugation, filtration, ultrafiltration, dialysis, precipitation or chromatographic methods such as affinity separation or separation by means of ion exchange chromatography, or an electrophoretic separation. Specific examples include gel electrophoresis, two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), capillary electrophoresis, metal affinity chromatography, immobilized metal affinity chromatography (IMAC), lectin affinity chromatography, liquid chromatography, high performance liquid chromatography (HPLC), normal and reverse phase HPLC, cation exchange chromatography, and selective Bonding to surfaces. All of these methods are well known to those skilled in the art and one skilled in the art will be able to select the method depending on the sample used and the method for determining the presence or absence of the polypeptide marker (s).

In one embodiment of the invention, the sample is separated by electrophoresis prior to its measurement, purified by ultracentrifugation and / or separated by ultrafiltration into fractions containing polypeptide labels of a specific molecular size. Preferably, a mass spectrometric method is used to determine the presence or absence and amplitude of a polypeptide marker, which method can be preceded by a purification or separation of the sample. The mass spectrometric analysis has the advantage over current methods that the concentration of many (> 100) polypeptides of a sample can be determined by a single analysis. Any type of mass spectrometer can be used. With mass spectrometry it is possible to routinely measure 10 fmoles of a polypeptide marker, ie 0.1 ng of a 10 kDa protein with a measurement accuracy of approximately ± 0.01% from a complex mixture. In mass spectrometers, an ion-forming unit is coupled to a suitable analyzer. For example, electrospray ionization (ESI) interfaces are most commonly used to measure ions from liquid samples, whereas the matrix assisted laser desorption / ionization (MALDI) technique is used to measure ions from sample crystallized with a matrix. For example, quadrupoles, ion traps or time-of-flight (TOF) analyzers can be used to analyze the resulting ions.

In electrospray ionization (ESI), the molecules present in solution i.a. is sprayed under the influence of high voltage (e.g., 1-8 kV) to form charged droplets, which become smaller due to evaporation of the solvent. Finally, so-called Coulomb explosions lead to the formation of free ions, which can then be analyzed and detected.

In the analysis of the ions by TOF, a certain acceleration voltage is applied, which gives the ions an equal kinetic energy. Then, the time required for the respective ions to travel a drift path through the flight tube is measured very accurately. Since with the same kinetic energy, the speed of the ions depends on your mass, this can thus be determined. TOF analyzers have a very high scanning speed and achieve a very high resolution. Preferred methods for determining the presence or absence and amplitude of polypeptide markers include gas phase ion spectrometry, such as laser desorption / ionization mass spectrometry, MALDI-TOF-MS, SELDI-TOF-MS (surface enhanced laser desorption ionization), LC-MS ( Liquid chromatography mass spectrometry), 2D-PAGE-MS and capillary electrophoresis mass spectrometry (CE-MS). All of the methods mentioned are known to the person skilled in the art.

A particularly preferred method is CE-MS, in which capillary electrophoresis is coupled with mass spectrometry. This process is described in detail, for example, in German patent application DE 10021737, in Kaiser et al. (J. Chromatogr. A ₁ 2003, Vol. 1013: 157-171, and Electrophoresis, 2004, 25: 2044-2055) and in Wittke et al. (J. Chromatogr. A ₁ 2003, 1013: 173-181 and Electrophoresis, 2005, 26 (7-8): 1476-87). The CE-MS technique allows the presence and amplitude of several hundred polypeptide markers of a sample to be determined simultaneously in a short time, with a low volume and high sensitivity. After a sample has been measured, a pattern of the measured polypeptide markers is prepared. This can be compared with reference patterns of ill or healthy individuals. In most cases, it will be sufficient to use a limited number of polypeptide markers to detect rejection of NTx. More preferred is a CE-MS method which includes CE coupled online to an ESI-TOF-MS.

For CE-MS, the use of volatile solvents is preferred, and it is best to work under essentially salt-free conditions. Examples of suitable solvents include acetonitrile, methanol and the like. The solvents may be diluted with water and treated with a weak acid (e.g., 0.1% to 1% formic acid) to protonate the analyte, preferably the polypeptides.

Capillary electrophoresis makes it possible to separate molecules according to their charge and size. Neutral particles migrate at the rate of electro-osmotic flow when creating a current, cations are accelerated and anions delayed. The advantage of capillaries in electrophoresis is the favorable surface-to-volume ratio, which enables a good removal of the Joule heat arising during the current flow. This in turn allows the application of high voltages (usually up to 30 kV) and thus a high separation efficiency and short analysis times.

In capillary electrophoresis, quartz glass capillaries with internal diameters of typically 50 to 75 μm are normally used. The used lengths are 30-100 cm. In addition, the capillaries usually consist of plastic-coated quartz glass. The capillaries may be both untreated, i. on the inside show their hydrophilic groups, as well as be coated on the inside. A hydrophobic coating can be used to improve the resolution. In addition to the voltage, a pressure which is typically in the range of 0-1 psi may also be applied. The pressure can also be created during the separation or changed during the process.

In a preferred method of measuring polypeptide markers, the markers of the sample are separated by capillary electrophoresis, then directly ionized and transferred online to a mass spectrometer coupled thereto for detection.

Advantageously, several polypeptide markers can be used to detect a rejection of NTx in the method according to the invention. In particular, at least three polypeptide markers may be used, for example, markers 1, 2 and 3; 1, 2 and 4; etc.

More preferred is the use of at least 4, 5, or 6 markers.

Even more preferred is the use of at least 10 markers, for example, markers 1 to 10.

Most preferred is the use of all 767 markers listed in Tables 1 and 2, respectively. In order to determine the likelihood of NTx rejection using multiple markers, statistical techniques known to those skilled in the art may be used. For example, the Weissinger et al. (Kidney Int., 2004, 65: 2426-2434) using a computer program such as S-Plus.

Example:

1. Sample preparation:

Urine was used to detect polypeptide markers for the detection of NTx rejection. Urine was collected from patients after NTx with rejection and from patients after NTx without rejection (control group).

The clinical data of the patients of both groups at the time of transplantation are shown in the following Table 5:

Table 5: Clinical data of the patient groups used in the transplantation

For the subsequent CE-MS measurement, the proteins also present in urine of patients in higher concentration such as albumin and immunoglobulins were separated by ultrafiltration. For this purpose, 700 .mu.l urine were removed and treated with 700 ml_ filtration buffer (2M urea, 1OmM ammonia, 0.02% SDS). These 1.4 ml sample volumes were ultrafiltered (20 kDa, Sartorius, Göttingen, Germany). The UF was carried out at 3000 rpm in a centrifuge until 1.1 ml ultrafiltrate was obtained.

The resulting 1.1 ml of filtrate was then desalted on a PD-10 column (Amersham Bioscience, Uppsala, Sweden) and eluted with 2.5 ml of 0.01% NH ₄ OH in water, and the eluate was then lyophilized. For CE-MS measurement, the polypeptides were then resuspended with 20 μl of water (HPLC grade, Merck).

2. CE-MS measurement:

The CE-MS measurements were carried out using a Beckman Coulter capillary electrophoresis system (P / ACE MDQ system, Beckman Coulter Ine, Fullerton, USA) and Bruker ESI-TOF mass spectrometer (micro-TOF MS, Bruker Daltonik, Bremen, D).

The CE capillaries were purchased from Beckman Coulter, having an ID / OD of 50/360 μm and a length of 90 cm. The mobile phase for the CE separation consisted of 20% acetonitrile, 0.25 M formic acid in water. 30% isopropanol with 0.5% formic acid was used for the "sheath flow" on the MS, here with a flow rate of 2 μl / min The coupling of CE and MS was performed by a CE-ESI-MS sprayer kit (Agilent Technologies , Waldbronn, DE).

To inject the sample, 1 to max. 6 psi pressure applied, the duration of the injection was 99 seconds. With these parameters about 150 nl of the sample were injected into the capillary, this corresponds to about 10% of the capillary volume. To concentrate the sample in the capillary, a "stacking" technique was used, injecting an IM NH ₃ solution for 7 sec (at 1 psi) before injecting the sample and injecting a 2M formic acid solution for 5 sec after sample injection the separation voltage (25 kV), the analytes are automatically concentrated between these solutions. The following CE separation was performed with a pressure method: 0 psi for 40 minutes, 0.1 psi for 2 minutes, 0.2 psi for 2 minutes, 0.3 psi for 2 minutes, 0.4 psi for 2 minutes, finally 32 min at 0.5 psi. The total duration of a separation run was thus 80 minutes.

In order to obtain the best possible signal intensity on the MS side, the "Nebulizer gas" was set to the lowest possible value.The voltage applied to the spray needle to generate the electrospray was 3700 - 4100 V. The other settings on the mass spectrometer were made as instructed The spectra were recorded over a mass range from m / z 400 to m / z 3000 and accumulated every 3 seconds.

3. Standards for the CE measurement

To control and calibrate the CE measurement, the following proteins or polypeptides were used, which are characterized under the selected conditions by the CE migration times listed below:

Protein / polypeptide CE migration time

Aprotinin, (SIGMA, Taufkirchen, DE, cat.No. Al 153) 9.2 min

Ribonuclease, Sigma, Taufkirchen, DE; KatNr .; R4875 10.9 min

Lysozyme, Sigma, Taufkirchen, DE; Kat.Nr .; L7651 8.9 min

"REV", sequence: REVQSKIGYGRQIIS 15.6 min

"ELM", sequence: ELMTGELPYSHINNRDQIIFMVGR 23.4 min

"KINCON", sequence: TGSLPYSHIGSRDQIIFMVGR 20.0 min

"GIVLY" sequence: GIVLYELMTGELPYSHIN 36.8 min

The proteins / polypeptides are each used in a concentration of 10 pmol / μl in water. "RE \ T," ELM "," KINCON "and" GIVLY "represent synthetic peptides.

The molecular masses of the peptides and the m / z ratios of the individual charge states that are visible in the MS are given in the following table:

Claims

claims

A method for detecting a rejection after kidney transplantation (NTx) comprising the step of determining an absence or presence of at least one polypeptide marker in a sample, wherein the polypeptide marker is selected from the markers 1-242 (frequency marker), or the determination of the amplitude of at least one polypeptide marker selected from the markers 243-767 (amplitude markers) characterized by the following values for the molecular masses and the migration time (CE time):

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2. The method according to claim 1, characterized in that an evaluation of the specific presence or absence of the marker 1-242 takes place on the basis of the following reference values:

3. The method according to claim 1, characterized in that an evaluation of the amplitude of the markers 243-767 takes place on the basis of the following reference values:

or

4. The method of claim 1 to 3, wherein at least two or at least three or at least five or at least ten or all polypeptide markers are used, as defined in claim 1.

The method of any one of claims 1 to 4, wherein the sample of an individual is a urine sample or a blood sample (serum or plasma sample).

A method according to any one of claims 1 to 5, wherein capillary electrophoresis, HPLC, gas phase ion spectrometry and / or mass spectrometry is used to detect the presence or absence of the polypeptide marker (s).

7. The method according to any one of claims 1 to 6, wherein before the measurement of the molecular mass of the polypeptide marker, a capillary electrophoresis is performed.

The method of any one of claims 1 to 7, wherein mass spectrometry is used to detect the presence or absence of the polypeptide marker (s).

9. The method according to at least one of claims 1 to 8, characterized in that the CE time is based on a 90 cm long glass capillary having an inner diameter (ID) of 50 microns at an applied voltage of 25 kV, being used as eluent 20th % Acetonitrile, 0.25 M formic acid in water.

10. Use of at least one polypeptide marker selected from the markers Nos. 1 to 767, which is characterized by the following values for the molecular masses and the migration time

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to detect a rejection (rejection) to NTx.

11. Marker combination comprising at least three markers selected from

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