WO2003069343A2

WO2003069343A2 - Method for the detection of tumor markers cea, ca19.9, or ca72.4 in the stool, allowing diagnosis of gastrointestinal tumors

Info

Publication number: WO2003069343A2
Application number: PCT/EP2003/001504
Authority: WO
Inventors: Hans Scheefers
Original assignee: Schebo Biotech Aktiengesellschaft
Priority date: 2002-02-14
Filing date: 2003-02-14
Publication date: 2003-08-21
Also published as: WO2003069343A3; AU2003210274A1

Abstract

The invention relates to methods for the qualitative or/and quantitative detection of CEA, CA 19.9, CA72.4 or parts thereof as tumor markers in human or animal stool, by means of which a malignant activity in the gastrointestinal tract and the abdominal cavity (esophagus, stomach, small intestine, bile ducts, pancreas, and colon) can be diagnosed.

Description

Procedure for the detection of tumor markers: CEA, CA19.9 or CA72.4 in the stool for the detection of gastrointestinal tumors

description

The invention relates to methods for the qualitative and / or quantitative detection of CEA, CA19.9, CA72.4 or parts thereof, which are used as tumor markers in human or animal stool for detecting malignancy in the gastrointestinal tract and the abdominal cavity (esophagus, stomach, small intestine, Biliary tract, pancreas and colon).

As listed in the literature, CEA, CA19.9 and CA72.4 are tumor markers that are used in a variety of tumor diseases in body fluids, e.g. in serum or plasma (see e.g. Review: Anticancer Research 19: 2785-2820, 1999) (CEA = Carcinoembryonial Antigen = MG 180kD; CA 19.9 = Cancer Antigen = MG 1,000 kD; CA72.4 = Cancer Antigen = MG 48 kD).

While this is advantageous on the one hand in order to diagnose a tumor disease at all, on the other hand, the detection of a marker for a wide variety of tumor diseases does not permit any statement about the type and location of the organ or tissue concerned.

Definition and classification of tumor markers

Tumor markers are macromolecules or antigens that occur in blood, serum, or other body fluids, and their changes in concentration are somewhat related to the development and growth of an individual's malignant tumors. Criteria for the quality of tumor markers

The "ideal tumor marker" should have the following properties: high specificity, i.e. undetectable in benign diseases and healthy people; high sensitivity, i.e. it can be detected in a high percentage of tumor patients .; Organ specificity; good correlation with tumor stages or tumor mass; - Relation to the forecast: reliable forecast values.

The criteria of 100% specificity and 100% sensitivity as well as the other criteria listed are still not met by any of the known tumor markers.

There are four pyruvate kinase isoenzymes (L, R, M 1 and M2) in higher organisms. Scientific studies have shown that malignant tumors have a special isoenzyme subclass of pyruvate kinase type M2, the tumor M2-PK.

So far, the qualitative or quantitative detection of pyruvate kinase type Tumor M2-PK in blood plasma using a developed ELISA, e.g. according to EP No. 0 444 1 1 8 or PCT / EP89 / 01 384.

Various studies show the usability of the tumor M2-PK in the diagnosis or in the follow-up of various tumors:

From DE 1 99 45 947 or PCT / EPOO / 09303 it is known that the pyruvate kinase isoenzyme of the type tumor M2 (tumor M2-PK) can also be detected in the stool by means of immunochemical methods. The gastrointestinal tract (GIT) is a system on which most tumors occur compared to other organ systems in the body. Regarding mobility and mortality, the main cancers of the GIT are colon carcinoma, gastric carcinoma, pancreatic carcinoma, esophageal carcinoma and liver carcinoma.

Each type of tumor in the GIT has its own special markers or groups of markers according to the prior art. CEA is the best and most widely used marker for colon cancer. CA1 9.9 for pancreatic carcinoma and CA72.4 for gastric carcinoma. It has only recently been shown that the tumor M2-PK is a very good marker for esophageal cancer and other types of tumor.

All of these markers were determined in the prior art by qualitative or quantitative detection of the respective analyte in blood, serum or plasma.

However, since the markers CEA, CA 1 9.9, CA72.4 and tumor M2-PK can be detected in all tumor diseases in the serum or blood plasma of patients, the type and location of the affected organ or tissue in the patient's body is not possible. Furthermore, it would be desirable to have a tumorous event in the gastrointestinal tract as early as possible, particularly in a growth phase in which the tumor has not yet made any contact with the body's vascular system (in the "polyp cancer sequence", before the infiltration of the submucosa) ) to be able to prove.

If you suspect a neoplastic event in the gastrointestinal tract, especially with regard to the so-called adenoma-carcinoma sequence

Polyps (polyposis coli) are tried according to the prior art by means of various determination methods, occult blood in the stool demonstrated. For this, non-immunological tests (e.g. pseudoperoxidase activity, porphyrin detection) and immunological tests are used (Favenne L. et al. (1,992) Ann. Biol. Clin. 50: 31 1 -31 3).

However, both test principles are not very specific, since they can be disturbed by a large number of influencing factors (false positive / false negative, e.g. due to non-compliance with urgently necessary dietary requirements on the part of the patient and from a number of medicinal products, as well as through excessive vitamin C administration ( e.g. in vegetables, fruit juices etc.), Thomas L., Labor und Diagnose, 5th edition, 1 998).

A positive test for occult blood in the stool must be clarified until the source of the bleeding is located or the cause of the bleeding has been found. In any case, the clinical finding justifies further diagnostics to be carried out quickly, e.g. by endoscopy, sonography, x-ray.

It was therefore an object of the invention to provide an easy-to-carry out method with which tumors and in particular the type and location of certain tumors can be detected.

The aim of the invention is, in particular, to meet the continuing need for specific, easy-to-carry out procedures, so that a neoplastic event, particularly with regard to the problem of the so-called adenoma-carcinoma sequence in polyps (polyposis coli) in the gastrointestinal tract, is particularly early and can be proven beyond any doubt.

This aim is now achieved with the method according to the invention for the detection of a malignant event in the gastrointestinal tract, wherein at least one tumor marker, selected from CEA, CA1 9.9 and CA72.4 or / and sections thereof.

It was found that the content of CEA, CA1 9.9 and CA72.4 in the stool of tumor patients can be detected and determined, the content being proportional to the degree of malignancy in the gastrointestinal tract.

This is all the more surprising because extensive investigations into other specific analytes (proteins) in the stool did not reveal any evidence of their use as tumor markers.

Both the structure and the physico-chemical properties of the CEA, CA1 9.9, CA72.4 or parts thereof are evident as a result of the considerable proteolytic activity and the extreme physiological conditions (e.g. pH, acid in the stomach, alkaline in the intestine) of the gastrointestinal tract is not permanently impaired. This also applies to the detection of CEA, CA1 9.9 or CA72.4 using immunological methods.

According to the invention, it has now been found and shown that, despite the protein denaturation and protein digestion in the GIT described above, a specific detection of CEA, CA1 9.9 or CA72.4 can be carried out in the stool of tumor patients. So far, the markers CEA, CA1 9.9 and CA72.4 have only been quantified in native, undigested patient samples (e.g. body fluids, sera, blood plasma) and used in clinical tumor diagnostics for a variety of tumor diseases.

According to the invention, it was surprisingly found that the tumor markers CEA, CA1 9.9 and CA72.4 are detected in the stool can. The determination can be made using commercially available test systems.

In the case of a positive sample, a tumor in the gastrointestinal tract (GIT), i.e. in particular in the esophagus, in the stomach, in the small intestine, in the biliary tract, in the pancreas or / and in the large intestine, is thus indicated, and thus a localization of the tumorous events in the GIT allows. However, the determination of a neoplastic event in the GIT has so far not been possible with the methods of the prior art or has been possible only with great effort. It should be noted that a stool sample cannot be considered a "body fluid" sample because the entire GIT is biologically outside the body.

With the method according to the invention, it is now possible in a non-invasive method to detect malignant tumor events in the stool in a simple manner by determining the tumor marker proteins CEA, CA1 9.9 or / and CA72.4 and, at the same time, to be able to unequivocally determine if the test is positive, that there is a tumor in the GIT and not anywhere else in the body.

Surprisingly, it is possible with the method according to the invention to detect the tumor markers CEA, CA1 9.9 or / and CA72.4 as free proteins in the stool using conventional test systems according to the prior art, for example using commercially available test systems. It is therefore not necessary to isolate cells from the stool and to analyze the proteins contained in the cells. It was surprisingly found that solid tumors release the tumor markers mentioned as soluble proteins into the lumen of the GIT and that these markers are not found in exfoliated gastrointestinal epithelial cells. The tumor markers described are released by tumor cells, pass through the GIT as a protein and can finally be detected as a protein in the stool. This possibility, namely that these proteins remain detectable in the stool after, for example, gastric or intestinal passage, is surprising and is not described in the literature.

The qualitative and / or quantitative determination according to the invention of the tumor markers CEA, CA1 9.9 or / and CA72.4 in stool in combination with a simultaneous qualitative and / or quantitative detection of the same tumor markers in blood or plasma gives the treating physician previously unavailable information as to whether the tumor is "only" in the GIT, especially in the intestinal lumen, or whether it has broken through the submucosa and has already contacted the vascular system or is already in the metastatic stage (the information results from the quotient formation of the blood or stool measurements obtained ).

Surprisingly, it was found that CEA, CA1 9.9 and CA72.4 remain quantitatively detectable even with intensely homogenized, longer-stored stool samples (for example when sending samples). A clear reaction is obtained even with severe thinning of the stool.

Surprisingly, it was also found that the detection takes place selectively in the stool sample even without prior extraction. However, it is preferable to use an extraction method, preferably using a special detergent (CHAPS) (the extraction method is described in more detail in Example 2).

In the method according to the invention, the malignancy in the gastrointestinal tract of a human or an animal is preferably determined. It is further preferred to detect the tumor marker immunochemically, in particular by means of anti-CEA, anti-CA1 9.9 or / and anti CA72.4 antibodies. Monoclonal or polyclonal antibodies, preferably monoclonal antibodies, can be used. Antibodies are advantageously used which do not cross-react with other constituents of the stool, in particular not with other stool proteins.

Antibodies which can be used according to the invention can be produced by methods known in the prior art. Methods for producing specific monoclonal antibodies are well known to those skilled in the art. For example, the antigen, in this case CEA, CA1 9.9 or CA72.4 or parts thereof, is used to generate antibodies. In principle, the method that was first described by Koehler and Milstein can be used for this purpose, modifications and further developments of these methods being known to the person skilled in the art. With this production specific antibodies can be obtained, the specificity can be determined by selection. The selection is made for specific antibodies that bind to CEA, CA1 9.9 or CA72.4, but not to other stool proteins.

In a particularly preferred embodiment, the detection is based on the principle of the immunoassay. An immunoassay which is preferred according to the invention is carried out by a) bringing the sample into contact with at least two different receptors, of which the first receptor R1 is in the solid phase and is bindable with CEA, CA1 9.9 or CA72.4 and the second

Receptor R2 is in the liquid phase and is also capable of binding with the same tumor marker, the receptor R2 bearing a label or mediating the binding to a detectable molecule, b) separating the solid from the liquid phase, and c) the label or the detectable molecule determined in one of the phases, preferably in the solid phase and accordingly the The amount of tumor markers present in the sample was quantified, using an antibody as at least one of the receptors R1 or R2 which is specifically bindable to CEA, CA1 9.9 or CA72.4 and in particular does not bind to any other stool protein.

In a particularly preferred embodiment, an antibody against the respective tumor marker to be determined is used both as receptor R1 and as receptor R2.

In addition to carrying out the method according to the invention in the form of an immunoassay and in particular an ELISA, other detection technologies can also be used, e.g. the quartz crystal technology, microbalance technology, lateral flow technology, K a n d e l a b e r - T e c h n o l o g i e, T R A C E - T e c h n o I o g i e o d e r electrochemiluminescence technology.

In addition to the method according to the invention, another object of the present invention is a test kit for the detection and / or diagnosis of malignant tumor occurrence in the gastrointestinal tract in humans or animals, which is intended in particular for carrying out the method described above, by determining a tumor marker CEA, CA1 9.9 or / and CA72.4 or parts thereof, the test kit containing at least one monoclonal or polyclonal antibody against CEA, CA1 9.9 or CA72.4. The test kit preferably contains an antibody for the corresponding tumor marker, which does not cross-react with any other stool protein. The test kit can optionally contain further reagents necessary for carrying out an immunoassay or for carrying out the test method provided in each case.

The test kit preferably contains an antibody bound to a solid phase and specific for at least one of the tumor markers mentioned. The invention further relates to the use of antibodies against CEA, CA1 9.9 or CA72.4 for the qualitative or quantitative determination of tumor markers in human or animal stool and a reagent for the selective quantitative determination of CEA, CA1 9.9 or / and CA72.4.

The invention furthermore relates to antibodies, in particular monoclonal antibodies, which specifically bind CEA, CA1 9.9 or CA72.4 and do not cross-react with any other stool protein. The antibodies can be generated, for example, by the method of Koehler and Milstein (Nature 256, 495-497 (1 995)).

Furthermore, the present invention relates to aptamers which bind specifically to CEA, CA1 9.9 or CA72.4 and do not cross-react with any other stool protein.

Aptamers are oligonucleotide sequences that are specific

Have binding properties. Such aptamers can, for example, according to those in US 5,270,163 or in Sumedha, Clin. Chem. 45 (1 999), 1 628-1 650 described methods can be produced or identified.

The invention is further illustrated by the following examples.

Execution - Example 1: CEA determination was carried out using ECLIA technology

The immunochemical determination of the CEA was carried out according to the manufacturer's instructions with the Elecsys from Röche AG. The Elecsys-CEA is a 1-step sandwich test based on the ECLIA principle. (ECLIA = ElektroChemiLuminescenceImmunoAssay). Execution - Example 2

CA 1 9.9 was determined using ECLIA technology

The immunochemical determination of CA1 9.9 was carried out in accordance with the manufacturer's instructions using the Elecsys from Röche AG. The Elecsys-CEA is a 1-step sandwich test based on the ECLIA principle. (ECLIA = ElektroChemiLuminescenceImmunoAssay).

Execution - Example 3: CA72.4 determination was carried out using ECLIA technology

The immunochemical determination of the CA72.4 was carried out according to the manufacturer's instructions with the Elecsys from Röche AG. The Elecsys-CEA is a 1-step sandwich test based on the ECLIA principle. (ECLIA = ElektroChemiLuminescenceImmunoAssay).

Basically, the tumor markers CEA, CA1 9.9 and CA72.4 can also be determined using other commercially available immunochemical test systems.

According to the invention, it is also possible to determine the analyte concentration by means of biosensors, such as, for example, amperometric sensors, potentiometric, ion-selective potentiometric or photometric sensors or else those using semiconductor electrodes such as field effect transistors (FET), chemosensitive field effect transistors (CHEMFET), suspended gate field effect transistors (SGFET) or ion sensitive field effect transistors. Such biosensors are summarized in EAH Hall and G. Hummel in "Biosensors", Springer Verlag Heidelberg, Germany, 1 995 described. Further developments of ion-sensitive field effect transistors (ISFET) or optical detectors are described, among others, by F. Aberl and H. Wolf in "Current Trends in Immune Sensor Technology", Labor 2000, pp. 70-96 (1 993) described. The method according to the invention is also suitable for implementation by means of piezoelectric quartz crystals and surface wave elements which can be used as microbalances. The primary antibody (the so-called catcher) is immobilized on a piezoelectric substrate and measured after binding with the CEA, CA1 9.9 or CA72.4 to be analyzed. Such sensors are described, for example, by A. Leidl et al. in "Proceedings of the Second International Symposium on Minaturized Total Analyzes Systems μTAS", Basel 1 996. Quartz crystal microbalances as described by C. Köslinger et al., Fresenius J. Anal. Chem. (1 994), 349: 349-354, have been found to be particularly suitable or, for example, candelabrum technology from IBM, Inc.

According to the invention, it is also possible to determine the analyte concentration by means of immunochromatographic methods, i.e. to be determined by means of so-called visual rapid tests (lateral flow tests).

A significant improvement in sensitivity, dynamic measuring range, analysis kinetics and format flexibility can be achieved by using electrochemilluminescence technology. Electrochemilluminescence is a process in which light is released. The light release is induced by applying an electrical potential to an electrode that mediates a cyclic oxidation / reaction of a ruthenium metal ion (Bruno, G. (1 997) Rec. Rp. S. 1 75-1 79; Williams R . (1 996), Amer. Biotech., P. 26). A similar technology is the TRACE technology from CIS. example 1

Weigh in the stool samples

An approximately 1 2 ml disposable tube and a microbiological inoculation loop (e.g. Sarstedt) are balanced to 0 using a sensitive digital laboratory balance. The stool is then weighed in with the inoculation loop by pricking the stool sample with the eyelet and introducing the amount of stool remaining at the tip (approx. 100 mg) into the disposable tube. A toothpick can also be used instead of the inoculation loop.

The volumes of the extraction buffer to be added are varied according to the weighed stool sample mass (e.g. 100 mg stool + 10 ml buffer or 75 m g stool + 7.5 ml buffer). Final concentration: 1 0 mg stool / ml extraction buffer.

Example 2

Homogenization and extraction of stool samples

The stool sample suspension is mixed vigorously at room temperature with a test tube shaker (e.g. VORTEX). The chairs must be well homogenized. In order to ensure complete extraction, it is important to add the detergent CHAPS (3 - [(3-cholamidopropyl) dimethylammonio] -1-propanesulfonate, 10 mM (Sigma) to the phosphate-buffered extraction buffer.

Example 3

The stool test can be advantageously carried out using the ScheBo ^{® «} Biotech AG stool dosing system (Quick-Prep).

Claims

Expectations

Procedure for the detection of malignant events in the

Gastrointestinal tract, that means that at least one in human or animal stool

Tumor markers selected from CEA, CA19.9 and CA72.4 or / and

Parts of the same determined.

A method according to claim 1, which means that the tumor marker CEA, CA19.9 or / and CA72.4 or / and parts of the same are detected immunochemically.

3. The method according to any one of claims 1 to 2, so that the content of CEA, CA19.9 or / and CA72.4 or / and of portions thereof are immunochemically mixed by means of anti-CEA, anti - CA19.9 or / and anti-CA72.4 antibodies detected.

4. The method according to any one of the preceding claims, so that the detection is carried out by means of antibodies, which are monoclonal or polyclonal antibodies and which are not with others

Components of the stool, especially not cross-react with other proteins.

5. The method according to any one of the preceding claims, characterized in that one carries out the detection according to the principle of the immunoassay.

6. The method according to any one of the preceding claims, d a d u rc h g e k e n n z e i c h n et that one determines the tumor marker CEA, CA19.9 or / and CA72.4 and / or parts thereof qualitatively and / or quantitatively.

7. The method according to any one of the preceding claims, dadu rc hge ke nnzeichn et that one uses CEA, CA19.9 or / and CA72.4-specific antibodies for the selective determination of the tumor marker CEA, CA19.9 or / and CA72. 4 or / and of portions thereof according to the principle of immunoassay by a) incubation with at least two different receptors, of which the first receptor R1 is in the solid phase and is bindable with CEA, CA19.9 or / and CA72.4 and of which at least one further receptor R2, which is also capable of binding with CEA, CA19.9 or / and CA72.4, is present in the liquid phase, in particular in solution, and wherein the receptor R2 bears a label or mediates binding to a detectable molecule, b) separation the solid from the liquid phase and c) determining or measuring the marking in one of the phases, an antibody being used as at least one of the receptors which is specifically bindable with CEA, CA19.9 or / and CA72.4 ig is.

8. The method of claim 7, d a d u rc h g e ke n n zei c h n et that one as an antibody bound to the solid phase

Antibody is used that is specifically bindable with CEA, CA19.9 or CA72.4 and as another soluble receptor with CEA, CA19.9 or CA72.4 bindable antibody is used, which carries a label or mediates the binding to a detectable molecule.

9. The method according to any one of claims 1 to 8, d a d u rc h g e k n nze i c h n et that this uses ELISA, quartz crystal, microbalance, lateral flow technology, candelabra technology, TRACE technology or electrochemiluminescence technology.

10. Test kit for the detection of a malignant tumor in the

Gastrointestinal tract, especially when performing a

Method according to one of claims 1 to 9, by determining the tumor marker CEA, CA19.9 or / and CA72.4 or parts thereof, so that it has at least one monoclonal or polyclonal antibody against CEA, CA19.9 or CA72 .4 contains.

11. Test kit according to claim 10 for the diagnosis of malignant tumor growth in the gastrointestinal tract (GIT), so that it contains at least one receptor for CEA, CA19.9 or CA72.4, which does not cross-react with any other stool protein, and optionally further reagents necessary for carrying out an immunoassay.

12. Test kit according to one of claims 10 or 11, d a d u rc h g e ke n n ze ic h n et that it has an anti-CEA, anti

Contains CA19.9 or anti-CA72.4 antibodies.

13. Use of antibodies against CEA, CA19.9 or CA72.4 for the qualitative or quantitative determination of a tumor marker, selected from CEA, CA19.9 and CA72.4 or parts thereof in human or animal stool.

14. Reagent for the selective, quantitative determination of CEA, CA19.9 or / and CA72.4, containing a receptor R1 bound to a solid phase, which is capable of binding to CEA, CA19.9 or CA72.4 and at least one in a soluble phase receptor R2 present, which is capable of binding to the CEA, CA19.9 or CA72.4, and wherein at least one soluble receptor R2 bears a label or mediates the binding to a detectable molecule, so that at least one of the receptors is an antibody that is specifically bindable to CEA, CA19.9 or CA72.4.