EP1404874A2

EP1404874A2 - Screening method

Info

Publication number: EP1404874A2
Application number: EP20020745418
Authority: EP
Inventors: Michael Neumaier
Original assignee: Individual
Current assignee: Ivonex gesellschaft fur Biomedizinische Forschung
Priority date: 2001-06-28
Filing date: 2002-06-28
Publication date: 2004-04-07
Also published as: WO2003002764A3; WO2003002764A2; CA2452094A1; US20050100903A1; DE10131382A1; AU2002317009A1

Abstract

The invention relates to a method which allows to identify therapeutically useful compounds via the detection of the expression of glycoprotein antigens of the CEACAM family. The therapeutically useful compounds have properties that prevent the development of hyperplastic modifications that have been identified as precursors of a neoplastic transformation and that may lead to the formation of a carcinoma, or that reestablish a normalization of the tissue. The invention specifically relates to a method for identifying a compound/compounds that are useful in tumor prevention or for treating tumor precursors. The selected compounds are capable, by controlling gene expression, of increasing the apoptosis sensitivity of cells of the colon mucosa, especially of the precursor cells (or of reducing an apoptosis resistance). The invention further relates to a diagnostic method and to the use of the cells identified according to the invention in pharmaceutical compounds for preventing tumor formation and for treating the precursors thereof.

Description

Screening method

DESCRIPTION

Field of the invention

The invention describes a method for identifying therapeutically useful compound (s). These compounds have properties to prevent the earliest morphological tissue changes that can lead to cancer, or to drive them into programmed cell death (apoptosis). In this way, the tissue situation is normalized.

State of the art

The development of tumors using the example of colon carcinoma

Carcinomas develop in successive steps from normal tissue via intermediate forms (nor al-to-tumor transition or multi-step carcinogenesis). For this, genetic changes (mutations) that are associated with this change are described. The multi-step carcinogenesis was formulated by the group around Bert Vogelstein (e.g. Fearon E.R. and Vogelstein B., Cell, 1990, 61 p.759ff) and has since been widely confirmed in the literature (Fig. 1). It says that

1. several genetic events are necessary to complete the transition from normal to carcinoma tissue;

2. Critical genes, called "gatekeepers", whose mutation / loss represent a point-of-no-return from which progression to carcinoma results. An example of a gatekeeper is the gene for adenomatous polyposis coli (APC), the Mutation / loss leads to the development of polypous colon cancer;

3. The gene mutations must occur in a specific order in order to lead to a tumor. For example, isolated, i.e. Without a prior "gatekeeper defect", k-ras mutations arise and the affected cells undergo apoptosis (programmed cell death). Conversely, k-ras mutations become permissive after a previous gatekeeper defect and constantly contribute to the tumor phenotype. The time of occurrence and persistence are also important for the importance of a change in the context of tumor development.

The terminology of the different tumor levels and qualities in the context of carcinogenesis is important for the classification of the present invention. A basic distinction is made between hyperplasia and dysplasia. Hyperplasia is characterized by a pure increase in morphologically normal differentiated cells. According to the prior art, the development of hyperplastic polyps does not lead to carcinogenesis. Dysplasia is characterized by the appearance of qualitatively changed cells, ie cells that have been differentiated in different ways. Dysplasia is based on an uncontrolled unregulated cell formation (neoplasia), from which an adenomatous polyp results, depending on the degree of differentiation, as an optional or obligatory precancerosis. In contrast to hyperplasia, neoplasia is associated with a gatekeeper defect, as explained above. It has not yet been clarified whether there is a systematic pathobiochemical connection between hyperplastic and dysplastic (adenomatous) polyps, since no change was known to date, which can be demonstrated in all forms of morphologically modified tissue. The gatekeeper APC

The initiation of carcinogenesis in most colorectal cancers presupposes the functional loss of the multifunctional APC protein. An important function is the regulation of the β-catenin concentration in the cell. ß-Catenin is associated with membrane-bound adhesion molecules and migrates between the cell membrane and the cell nucleus, where it is responsible for the activity of various genes via the activation of transcription factors. APC controls the breakdown of ß-catenin. Defective APC proteins lead to increased intracellular ß-catenin concentration both in the cytoplasm and in the nucleus. This can be demonstrated using standard immunological and molecular biological methods. ß-catenin is therefore a surrogate marker for the APC function (Fearnhead et al., Hum Mol Gen 2001, Vol.10, No.7, p. 721ff).

Well over 700 APC mutations have been described. APC mutations are observed in dysplasias, but not in hyperplastic changes (pure cell increase without qualitative changes). For this reason too, hyperplastic polyps or hyperplastic "aberrant crypt foci" (ACF, micro-growths) are considered harmless.

CEACAM Fami1ie

The CEACAM family consists of 29 genes on chromosome 19q. The glycoproteins are expressed in a variety of tissues. In the large intestine there are essentially CEACAM-1, CEA (CEACAM-5), CEACAM-6 and CEACAM-7, are on the cell surface facing the intestinal lumen (apical-luminal localization) and represent a large part of the glycocalix there , The CEACAM molecules are adhesion molecules that bind to each other and form a highly organized network in the Glykocalix. Among them, the CEACAM-1 maintains contact with the inside of the cytoplasmic domain Cell membrane and participates in signal transduction phenomena. Their physiological importance has not been proven in the colon.

In colorectal carcinomas, the expression of the CEACAMs is changed significantly, i.e. it is dysregulated or altered. CEACAM-1 and CEACAM-7 expression is greatly reduced or is completely lost, while CEA and CEACAM-6 are partially strongly overexpressed (dysregulation of CEACAM expression is also observed in carcinomas of the esophagus, stomach, mammary gland and lungs) , The changes in expression are not limited to carcinomas. Thus, the loss of CEACAM-1 is found in colorectal adenomas and thus early forms of dysplasia / neoplasia at the same frequency (> 90% of cases) (Nollau P. et al., (1997), Cancer Res. 57, 2354- 57).

The cause of the changes in CEACAM expression is unknown. No mutations in the coding or non-coding regions of the DNA sequence have been reported so far. Specific transcription factors that regulate the tissue-specific expression of CEACAM-1 are not yet known. Epigenetic phenomena (methylation of regulatory sequences) were also not found.

The physiological significance of the CEACAM molecules has not been clearly clarified. The example of the CEACAM-1 shows the multitude of hypotheses given in the literature. These have been proven by in-vitro experiments or derived from clinical observations:

CEACAM-1 has an adhesive function. Cells transfected with the CEACAM-1 gene show greater adherence to one another than non-transfected starting cells (Teixeira, AM, et al. (1994), Blood 84, 21.1-219.) The adherence is in one for Cadherine described ways Temperature and calcium dependent (Rojas et al., 1990, Cell Growth Differ 1, 527-533).

CEACAM-1 also has a receptor function. The murine homolog bgp serves as a receptor for the mouse hepatitis virus (MHV) (Blau et al., 2001; J Virol 75, 8173-8186). Human CEACAM-1 expressed on human granulocytes serves as a receptor for the OPA proteins of microbial pathogens such as Sal onella (Chen et al., 1997, J Exp Med 185, 1557-1564).

In tissues that regularly express CEACAM-1, there is a loss of expression at the RNA and protein levels in the corresponding carcinomas, e.g. in colorectal cancer (Neumaier et al., 1993; Proc Natl Acad Sei 90, 10744-10748), an exception is gastric cancer, in which an increased expression compared to normal tissue is regularly found in the tumor (Kinugasa et al., 1998, Cancer 76, 148-153).

A comparative gene expression analysis (SAGE) on tumor cell lines shows that CEACAM-1 is one of the genes that are most frequently and most clearly lost in their expression (Zhang et al., 1997, Science 276, 1268-1272). The molecular causes of the CEACAM-1 loss have not yet been clarified.

Adenomas, which can be regarded as precursors of colorectal cancer, already show a loss of CEACAM-1 expression that is identical in terms of degree and frequency as malignant tumors (Nollau et al., 1997, Cancer Res 57, 2354-2357). This shows that CEACAM-1 loss is an early event in carcinogenesis. Animal models show that tumor genetics in the tumor transplant model decrease if the cells have been previously transfected with CEACAM-1 (Fournes et al., 2001; Oncogene 20, 219-230).

Expression of CEACAM-1 within the colon crypts begins in the lower half of the crypts and increases towards the lumen steadily increasing (Frangsmyr et al., 1995, Cancer Res 55, 2963-2967). In the area of proliferating crypt cells, CEACAM-1 production is undetectable, and CEACAM-1 is also involved in the morphogenesis of epithelia.

The cytoplasmic domain of CEACAM-1 is involved in signal transmission (Afar et al., 1992, Biochem Biophys Acta 1134, 46-52.). Tyrosine residues as well as serine and threonine have been identified as phosphorylation substrates. The motifs of the domain which control the phosphorylation have been identified as activating and inactivating sequence motifs for kinases and phosphorylases (ITAM or ITIM motifs) (Beauchemin et al., 1997, Oncogene 14, 783-790).

All examinations of CEACAM-1 functions so far use established tumor cell lines or transfectomes as model systems. To date, nothing is known in the prior art about the expression behavior of CEACAM-1 and the functional role of CEACAM-1 in tumor stages before gatekeeping mutations and in hyperplastic lesions (hyperplastic ACF and polyps). Furthermore, a connection between the hyperplastic lesions and dysplasia is not described. There are no known changes in genes or gene products or changes in the expression patterns of these genes and gene products in hyperplasia, which also continues in the dysplastic tissue.

The inventor has now succeeded in proving that the expression of CEACAM-1, as a member of the CEACAM family, is downregulated in hyperplastic lesions at the frequency known from dysplasia (adenoma and carcinoma). get lost . This is the first time that a direct pathobiochemical connection between hyperplasia and dysplasia has been demonstrated. This allows the conclusion that both individual stages of the same tissue development represent. On the other hand, the invention is based on the observation that the loss of CEACAM-1 expression in hyperplasia is associated with a loss of apoptosis of the affected tissue. The causality between CEACAM-1 and apoptosis loss is shown here, in that the apoptosis ability is shown as a function of the level of expression and possible degree of cross-linking of the cell surface-bound CEACAM-1.

The object of the present invention is now to provide a method for identifying therapeutically useful compounds which regulate the expression of CEACAM molecules. These compounds can be used for the prevention of tumors; in particular, these compounds can prevent gastrointestinal tumors or bring about regression of existing hyperplasias by increasing apoptosis.

Another object of this invention is to provide a test system for screening the compounds.

A further object of the present invention is the use of these identified compounds in pharmaceutical compositions for the prevention of tumor formation in individuals, in particular those with predispositions to tumor formation, for example those who have a gatekeeper defect on a chromosome.

Summary of the invention

The present invention relates to a method for identifying compound (s), comprising the steps:

incubating a sample which can express a gene (s) or a gene product (s) of the CEACAM family with one or more compounds; incubate a second sample that can express a gene (s) or a gene product (s) of the CEACAM family in the absence of the compound (s);

Comparison of the expression of the gene (s) or gene product (s) of the CEACAM family in the samples.

If necessary, the connection (s) can also be identified by determining the apoptosis rate in the samples.

Compounds that can directly or indirectly regulate an alteration in the expression of gene (s) or gene product (s) or that contribute to an increase in the apoptosis rate of the cells can be used in a therapy concept that prevents the formation of early precursor lesions Can lead to carcinomas, prevented or treated such precursor lesions.

According to the invention, the expression of members of the CEACAM family is used as a parameter in order to identify substances which influence the expression of the antigens. In a functional sense, the determination of the apoptosis rate can be used to identify compounds that can treat precursor lesions.

In addition, the present invention provides a test system and kit that can be used to test compounds for their ability to regulate the dysregulation of expression in members of the CEACAM family, for example due to the level of expression of an unmodified cell. Furthermore, the test system allows the investigation of substances that influence the signal cascade via a member of the CEACAM family, so that the apoptosis rate is increased. This means that compounds can also be identified that contribute to signal transmission, for example by crosslinking CEACAM molecules, which as a result leads to an increase in the apoptosis rate. These substances can be used in pharmaceutical compositions which are used in the preventive treatment of tumors prior to carcinogenesis.

Furthermore, the present invention relates to a diagnostic method comprising the determination of the CEACAM expression in samples to be examined. This procedure allows the detection of precursor lesions at an early stage of tumor formation.

The invention is based on the new observations that certain molecules of the CEACAM family are important for the regulation of apoptosis (programmed cell death) in a hitherto unknown manner, but these molecules cannot fulfill their expression due to a disturbance in their expression that occurs in the earliest tissue changes.

Indeed, it has surprisingly been found that there is a direct correlation between the expression of members of the CEACAM family, such as CEACAM-1 and CEACAM-7, and the apoptosis sensitivity of the colon cells.

pictures

Fig.l: shows the development of hyperplastic and dysplastic (neoplastic) colorectal tumors according to the prior art positions (according to Kinzler KW and Vogelstein B. 1996, Cell, 87, 159-170; Jen J. et al. 1994, Cancer Res ., 54.5523-5526). While hyperplastic lesions do not lead to tumor progression due to the lack of gatekeeping mutations, gatekeeping mutations initiate this dysplastic adenoma-carcinoma sequence. 2 shows the development of hyperplastic and dysplastic (neoplastic) colorectal tumors in accordance with the findings on which the invention is based: in the colon crypt there is a reduction / loss of CEACAM-1 expression with the result of reduced apoptosis. This leads to hyperplasia of the affected crypt (s) in the form of a so-called aberrant crypt focus (ACF) or hyperplastic polyp (HP). Mutations within ACF or HP can affect different genes. In the case of the mutation of non-permissive genes (eg k-ras), the destruction of the hyperplastic tissue results. In the case of gene mutations that are critical for tumor progression (eg gatekeepers such as APC), a dsyplastic change results in the form of a new formation, which represents a precancerous condition.

Fig. 3: shows the expression of CEACAMl and the

Accumulation of ß-catenin (as a surrogate marker of the APC defect) in hyperplastic (ACF, HP) and dysplastic (adenoma = AD, carcinoma = CA) lesions. The loss of CEACAM1 expression is detectable in all tissue changes in a high and constant frequency, while APC defects are only found from the stage of dysplasia (neoplasia). The result of the overarching joint CEACAM1 change suggests a pathobiochemical connection between dysplasia and hyperplasia.

Fig.4: shows that controlled by ribozyme

Overexpression of CEA leads to apoptosis resistance. Cells that show increased CEA expression are less sensitive to apoptosis stimuli such as interferon ga ma. The effect of CEA on the programmed cell death is therefore opposite to that of CEACAM1.

Fig. 5: shows the importance of the height of the CEACAM-1-

Expression and CEACAM-1 crosslinking on the cell surface for the triggerability of apoptosis in the colon cell line HT29. By adding ga ma interferon (gIFN) at short notice, HT29 cells can be stimulated to produce different levels of CEACAM-1. By cross-linking the CEACAM-1 molecules on the cell surface of activated cells, apoptosis can be triggered specifically. Irrelevant antibodies have no effect.

6 shows the stimulability of CEACAM-1 expression in HT29 cells after treatment with tumor necrosis factor alpha (TNF-α) in a Western blot.

HT29 colon carcinoma cells were incubated with TNF-α (10ng / ml) for 6 hours. The cells were microscopically checked for vitality, the total protein isolated and this separated electrophoretically. Lane 1 shows the treated cells, lane 2 the untreated control. 8μg protein were separated per lane. The detection was carried out with a CEA antibody and a peroxidase-labeled anti-mouse secondary antibody according to the standard protocol. To check the protein loading, the blot was additionally stained for β-actin.

Detailed description of the invention

The term "CEACAM molecules" or "CEACAM family" as used herein includes all molecules that are considered to be members of the CEACAM family, such as CEACAM-1, CEACAM-6, CEACAM-7 and CEA. The term "identify" here means that a connection can be named which has the designated properties. This includes the screening of compounds and the subsequent evaluation of the results obtained from the screening in order to then clearly name the compound.

The term "screening" here means that a large number of compounds are examined in one process. This investigation then provides information regarding certain properties of the compounds.

The term "alteration" or "dysregulation" here means that the level of the molecule in or on a cell has changed compared to the normal situation. For CEACAM-1 this means e.g. that expression at CEACAM-1 is decreased in the cells compared to cells in the normal mucosa. This alteration or dysregulation can lie both at the level of the genes and at the level of the gene products.

The method according to the invention is a method for identifying connections, comprising the steps

incubating a sample which can express a gene (s) or a gene product (s) of the CEACAM family with one or more compounds; incubate a second sample which can express a gene (s) or gene product (s) of the CEACAM family in the absence of the compound (s); Comparison of the expression of the gene (s) or gene product (s) of the CEACAM family in the samples.

The invention further relates to a method for identifying connections, comprising the steps incubate a sample that contains a gene (s) or a

CEACAM family gene product (s) can express with one or more compounds; incubate a second sample that contains a gene (s) or

Can express gene product (s) of the CEACAM family in

Absence of connection (s);

Comparison of the apoptosis rate of the cells in the samples.

In particular, the expression of the members CEACAM-1, CEA, CEACAM-6 and CEACAM-7 is determined using the method according to the invention. The level of expression is related to the increase in the apoptosis rate of the cells. Furthermore, the method according to the invention allows the determination of compounds which show the ability to influence the signal cascade via CEACAM-1, CEA, CEACAM-6 and CEACAM-7 and thus to increase the apoptosis rate in the cells. Ie, with the inventive method, compounds are identified which of these result in expression of a member of the CEACAM family through networking ^'an increase in the rate of apoptosis.

The determination of the compounds to be examined as compounds for regulating the alteration of the expression of members of the CEACAM family, like for the compounds which cause crosslinking of the members, can be carried out indirectly by determining the susceptibility to apoptosis, i.e. the apoptosis rate.

The determination of the apoptosis rate thus provides information about the effectiveness of the compound to influence the expression of the members of the CEACAM family and also about the effectiveness of the compounds to influence the signal transduction by networking a member of the CEACAM family.

The determination of the apoptosis susceptibility (apoptosis resistance), ie the apoptosis rate, can be done by generally known methods, such as staining with Annexin V or Propidiu iodide. In addition, commercially available methods are suitable with which the nucleosomes released in the course of apoptosis can be measured with a sandwich ELISA. Another method immunochemically measures the caspase-induced proteolysis of cytokeratins and the resulting neoepitopes of this filament protein (mab M30, Röche Molecular Systems).

A useful use of the identified compounds, when present, results in an increased apoptosis readiness (the apoptosis resistance is decreased) compared to the sample incubated in the absence of the compound.

The following can be mentioned as examples of methods for detecting the expression of gene (s) or gene product (s). These methods are generally established and known to the person skilled in the art.

The CEACAM expression can be detected immunohistochemically in situ on tissue sections or in cell cultures by using specific antibodies. These can be both monoclonal and polyclonal. Antibodies against CEA (clone T84.66, C1P83), CEACAM-1 (clone 4D1C2 and clone 29H2), CEACAM-6 (clone Bu33) and CEACAM-7 (clone Bac-2) are mentioned here as exemplary monoclonal antibodies.

The expression can also be detected immunochemically quantitatively in tissue homogenates and cell culture lysates after electrophoretic separation and Western blot, it being possible to use the above-mentioned antibodies. For this purpose, densitometric evaluations of Western blots can be carried out, which also enables the relative expression of the individual antigens to be compared with one another. Finally, the expression can be quantitatively detected immunochemically in tissue homogenates and cell culture lysates. Specific antibodies against the corresponding gene products of the members of the CEACAM family are used. Standard enzyme immunoassays such as ELISA, RIA, etc. are suitable methods.

Expression can also be examined in situ at the mRNA level. The person skilled in the art can easily obtain corresponding gene probes from the published sequences of the members of the CEACAM family. The in-situ hybridization takes place according to standard procedures.

Expression can also be determined semiquantitatively or quantitatively by densitometry by analyzing Northern blots. Northern blots are standard procedures and can be carried out with appropriate gene probes obtained from the corresponding sequences of the CEACAM members. The person skilled in the art can easily obtain and use the gene probes that can be used.

Expression can also by

Amplification methods can be determined using CEACAM-specific primer oligonucleotides. The primers can easily be derived from the known sequences and can be examined for their specificity. For this purpose, quantitative methods are available which deliver quantitative results of gene expression (e.g. real-time RT PCR with LightCycler (RÖCHE Molecular Systems) or TaqMan ^® (Applied Biosystems)

The detection method for determining the expression of gene (s) or gene product (s) according to the invention is not limited to the abovementioned. Other methods can also be used which allow a comparison of the expression of the members of the CEACAM family in samples in the presence and absence of the compound (s) to be investigated. Array systems are particularly suitable for this purpose, which allow the simultaneous assessment of various factors influencing the CEACAM system. Suitable arrays basically contain those genes which are involved in the regulation and function of the CEACAM system.

The arrays can be designed both for measuring mRNA expression (transcription array) and for measuring gene products (protein array).

The sample in which the expression of the gene (s) or. Gene product (s) determined in the presence and absence of the compounds to be investigated according to the invention can be exemplary:

Cells, e.g. B. Primary cells or genetically modified cells. These genetically modified cells can be cells that contain a gene (s) or. Can express gene product (s) of a member (s) of the CEACAM family by transfecting them with appropriate vectors.

A cell culture line, e.g. B. HT29, A818 etc. These cell lines, which are commercially available from the corresponding centers, can be used in an appropriate amount, for. B. be sown in microtiter plates and are then incubated for a predetermined time with and without the compound to be examined.

Furthermore, cell homogenates or extracts from CEACAM-1 expressing tissues can be used by examining with the aid of quantitative assays (as stated above) for factors which regulate CEACAM-1 expression.

Compounds as can be used in the process according to the invention are not particularly restricted. For example, you can his : Peptides, such as oligo- or polypeptides, in particular also recombinant CEACA domains,

Proteins such as cytokines, antibodies and lectins,

Carbohydrates, simple or complex forms

Small molecules, with a molecular weight between 50 and 1000 Da.

Polymers that link the individual CEACAM

Enable molecules. The material is the

Polymers are not particularly limited. Essential functional properties of the polymers can be the repetitive arrangement of binding domains (e.g. CEACAM-

Domains, binding peptides, cross-linking

Carbohydrate structures)

This to be examined compounds may exist as libraries, such as those used in gen Hochdurchsatzuntersuchun ^'example. These libraries are commercially available, among others.

The compounds can exist as such or as a salt.

Compounds which are identified and selected by the method according to the invention are distinguished by the fact that they can selectively upregulate the expression of CEACAM-1 and / or CEACAM-7 compared to CEACAM-6 and CEA. On the other hand, they can e.g. downregulate CEACAM-6 and CEA expression. The regulation causes an increase in the apoptosis tendency of the sample (the apoptosis resistance is reduced).

The connections identified with the method according to the invention can alternatively influence the signal transmission by a member of the CEACAM family, e.g. CEACAM-1 so that the apoptosis rate of the cells is increased.

These compounds according to the invention, identifiable with the method according to the invention, include compounds such as Factors that have an influence on the regulation of the CEACAM molecules. Ie it can be connections that regulate factors like

Affect transcription factors and thus lead to an increased transcription of the CEACAM-DNA, such as CEACAM-1 and CEACAM-7, or in the case of CEA and CEACAM-6 to a reduced transcription.

It was found according to the invention:

that e.g. the expression of CEACAM-1 is already lost or at least greatly reduced in most of the precursors of hyperplastic tumors of the colon. The percentage of loss is comparable to that found in dysplastic tumor forms known as cancer precursors and in carcinomas. It can be concluded from this that hyperplastic (inherently harmless) tumors can develop into dysplastic cancer precursors in the sense of continuous development.

that the networking of e.g. CEACAM-1 transmits a specific signal to trigger apoptosis on the cell surface. Depending on expression and cross-linking, this signal can be demonstrated in the in vitro model in apoptosis-sensitive reporter cells as well as in human colon cells of the HT29 line.

In vivo, a loss of CEACAM-1 correlates in the examined hyperplastic tissue lesions with a decreased apoptosis. Reduced apoptosis is considered to be the main cause of the formation of hyperplastic colon crypts (Roncucci L. et al. 2000, Cell Prolif., 33, 1-18). The biological function of CEACAM-1 described here as a regulation of the tendency to apoptosis is so far unknown. In this respect, the loss described here as an example for CEACAM1 the earliest molecular change that results in a pathobiochemical change in the phenotype of the colon crypts.

Thus, according to the invention, it has been possible for the first time to establish a long-sought pathophysiologically relevant connection between (usually regarded as harmless) hyperplasia and neoplasia (which leads to carcinomas in the course of progression). The CEACAMs causally intervene in the regulation of apoptosis, whereby the loss of the signaling CEACAM-1 is probably the causative trigger for the earliest morphologically recognizable changes in the colon. This is of particular importance since the CEACAMl loss is not a genetically fixed, but a functional-regulatory defect. Accordingly, pharmacalogic intervention strategies that restore the CEACAMl function are ideally suited to achieve a rationally based prevention of hyperplasia (derived from pathophysiological findings).

The results shown above are also applicable to CEACAM-7. As already explained above, the situation is different with CEA and CEACAM6. There the expression of the molecule is up-regulated in the changed state of the cell and the apoptosis rate is thereby reduced. Compounds identified according to the invention are selective for a CEACAM-1 and / or a CEACAM-7 increase and do not lead to an increase in the expression of CEA and / or CEACAM-6. The compound even advantageously leads to a reduction in the expression of CEA and CEACAM-6.

However, compounds identified according to the invention can also selectively lead to a CEA and / or a CEACAM-6 reduction and do not lead to a reduction in the expression of CEACAM-1 and / or CEACAM-7. Advantageously leads the compound even increases expression of CEACAM-1 and CEACAM-7.

The compounds identified according to the invention enable the following:

The restoration or enhancement of the CEACAM-1 and / or CEACAM-7 expression or the reduction of the CEA and the CEACAM-6 expression and thus the increase in the apoptosis ability of the affected crypt cells. By reducing hyperplasia, there is a reduction in the risk of critical mutations, a use in the prevention of cancer.

Candidate compounds can be tested in vivo in animal models. Test methods are known in rodents which test the carcinogenicity of e.g. Check dietary substances by examining the resulting ACF frequency in the gastrointestinal tract (Sorensen I.K., 1997, Carcinogenesis 18, 777-781).

A clinical examination is also conceivable in patients who have a predisposition to cancer in the sense of an obligatory precancerosis, such as is given by the hereditary defect of an APC allele. The effect of compounds that can be used for prevention can be e.g. during diagnostic interventions e.g. check against the development of numbers of colon tumors in the course. For this purpose, the gene expression of members of the CEACAM family, in particular of CEACAM-1, can also be examined using the described methods after taking a biopsy.

The test system according to the invention can be an automated test system which carries out the above-mentioned steps for examining influencing factors on the CEACAM system carries out. In particular, it can be a test system that is suitable for high-throughput analysis.

The kit according to the invention can contain the necessary components for carrying out the method according to the invention and includes the sample and all necessary reagents for incubation.

The identified compounds can be used in pharmaceutical compositions in customary amounts and dosages. The pharmaceutical compositions can be in the formulations customary for the route of administration with the selected compound as an active agent.

The predisposed groups, in which a preventive therapeutic application of the identified compounds is particularly advantageous, are individuals who develop FAP (familial adenomatous polyposis) and HNPPC (hereditary nonpolyposis colorectal cancer) or risk groups for somatic mutations of gatekeepers genes.

But also in carcinomas of other tissues e.g. Changes in CEACAM expression have been observed in the esophagus, stomach, mammary gland and lungs. The compounds identified according to the invention can also be used here.

The diagnostic test method according to the invention can serve for the early diagnosis of precursor lesions. The method comprises determining the expression of a member of the CEACAM family of a sample which comprises cells of the tissue to be examined and comparing the expression with a normal tissue. In particular, this diagnostic method can be a method in which the expression of CEACAM-1 or CEACAM-7 in a sample is determined. The The sample is usually a biopsy of the tissue to be examined.

Furthermore, a diagnostic kit is provided according to the invention, comprising components for determining the CEACAM expression in a sample. These components can be primary antibodies to a member of the CEACAM family (either monoclonal or polyclonal in origin) that are either directly labeled or unlabeled. Furthermore, the kit can contain a secondary antibody directed against the primary antibody, which is marked with a marker.

The proof can be directly or indirectly with known e.g. immunohistological and immunofluorescence methods are carried out. For this purpose, the antibodies against a member of the CEACAM family contained in the kit according to the invention can be directly labeled with generally known molecules, including enzymes such as alkaline phosphatase and peroxidase, and

Fluorescent dyes, such as FITC, TRITC, rhodamine, Texas Red, ALEXA® dyes, Cy® dyes. However, the labeling can also be carried out indirectly by using secondary antibodies or the antibodies against CEACAM which are labeled with molecules such as biotin, digoxigenin or the like and then detected using a secondary reagent.

Furthermore, the kit can contain enzyme substrates for a peroxidase or alkaline phosphatase, which allow the enzymatic detection of the bound secondary molecule.

Examples

example 1

CEACAM-1 expression in colon adenomas Fresh samples of patient adenomas were examined for the expression of various CEACAM genes (Nollau P. et al., (1997), Cancer Res. 57, 2354-2357). For this purpose, the total RNA was isolated from the samples by a conventional method and subjected to analysis by means of a Northern blot (Neumaier M. et al., PNAS, (1993), 90 (22), 10744-10748).

The Northern blots were evaluated with the aid of quantitative densitometry and image analysis and the changes in the expression of CEACAMs compared to the corresponding normal tissue of the same patient were determined (matched-pair analysis). The results for the adenomas are shown in Table 1 below.

Table 1

0 no more specific CEACAM1 RNA detectable (+) 60% loss of expression ((+))> 80% loss of expression Example 2

Immunohistochemical detection of the change in CEACAMl expression in various precursor lesions

Normal colonic mucosal tissue was freshly obtained from surgery. The material was fixed in 4% formaldehyde / PBS at 4 ° C for one to four hours according to the standard protocol. Samples were stained with 0.2% methylene blue (Sigma, Taufkirchen, Germany) for 3 min and examined for ACF with magnifying glasses. Samples with more than seven foci were used for further investigations. Samples of hyperplastic polyps, adenomas and carcinomas were obtained from the pathological institute of the UKE-Hamburg for comparison purposes.

5 μm sections were made from the tissue samples embedded in paraffin using standard technology. These sections were deparaffinized on the slides and washed with PBS. The sections were then pretreated in the microwave for 10 min at 650W in 10MM citrate buffer (pH 6.0) or for 3 min at 650W in ImM EDTA buffer (pH 8.0), followed by 7 min at 160W.

The sections were then stained with the following antibodies as follows:

IMMUNHISTOCHEMICAL DETECTION OF CEACAMl. Monoclonal CEACAMl-specific antibodies clone 29H2 (Novocastra, Newcastle, UK, diluted 1/50) or clone 4D1.C2 (Prof. C. Wagener, Dept. of Clinical Chemistry, University Clinic Hamburg, Germany, 4μg / ml).

IMMUNHISTOCHEMICAL DETECTION OF CEA. Monoclonal antibody T84.66 (Prof. JE Shively, City of Hope National Cancer Center, Duarte, CA, USA) against CEA high specificity; in particular no cross-reaction with CEACAMl or CEACAM6 (5μg / ml).

IMMUNHISTOCHEMICAL DETECTION OF THE APC FUNCTION. Monoclonal antibody 7D11 (Alexis, Grünberg, Germany) against human ß-catenin (lμg / ml).

IMMUNHISTOCHEMICAL DETECTION OF APOPTOSIS. Monoclonal antibody M30 (RÖCHE, Molecular Systems, Mannheim, Germany) against a neoepitope of human cytokeratin-18 caused by apoptosis-induced caspases. IMMUNHISTOCHEMICAL DETECTION OF APOPTOSIS. Monoclonal antibody CH11 (Coulter Immunotech, Heidelberg, Germany) against human CD95 (lμg / ml).

The primary antibodies were incubated overnight at 4 ° C. in moist chambers on the sections. Specific peroxidase-based staining was carried out with the VECTOR Elite KIT (Vector Laboratories, Burlington, CA, USA) using diaminobenzidines according to the manufacturer's instructions. All washing steps were carried out with PBS (pH 7.4) strictly according to the manufacturer's instructions. The immunohistochemistry was evaluated visually semi-quantitative.

The expression of CEACAM1 and β-catenin (as a measure of the β-catenin accumulation caused by APC defect) of the examined tissues is shown in FIG. 3.

The data shown make it clear that the loss of CEACAM1 expression can already be found in the ACF and HP, while the surrogate marker ß-catenin used for APC shows no change, but can only be found in the neoplastic tumors.

This proves that the change in the expression of CEACAM1 represents a significantly earlier event in tumor development than the defect which, according to the state of the art, is generally regarded as the first event for colon cancer of the APC gene (accumulation of β-catenin expression). Furthermore, the loss of CEACAM1 expression persists throughout the development of multi-step carcinogenesis. The change in CEACAM1 expression already occurs in hyperplasia and is similar to that in advanced neoplasia. On the other hand, the accumulation of ß-catenin as a surrogate marker for changes in the APC is only detectable in neoplasia; hyperplastic changes are not recognized.

Example 3

Significance of the expression of CEA (CEACAM-5) for apoptosis sensitivity in HT29 cells

The influence of CEA protein expression on the susceptibility of cells to apoptosis is investigated with the help of ribozyme-regulated mRNA expression. HT29 colon cancer cells (available from ATCC, Rockville, USA) were transfected with CEA-targeted hammerhead ribozyme expression vector.

The vector was produced analogously to Schulte et al. , PNAS 1996, 93, pp. 14759ff and Juhl, H., et al, 1997, JBC 272, pp. 29482ff.

The following oligonucleotides were used as ribozyme sense and antisense nucleotides.

5 '-agcttTGCTCTTCTGATGAGTCCGTTAGGACGAAACTATGGAgggcc-3' (sense) and

5 '-cTCCATAGTTTCGTCCTAACGGACTCATCAGAAGAGCAa-3' (antisense)

These were hybridized and inserted into the pTET vector as described in Schulte et al. , PNAS 1996, 93, p. 14759ff. The plasmid pTET / Rz2113 obtained now contains CEA-specific flanking regions at the 5 "end (7 nucleotides long) and at the 3 "end (8 nucleotides long). These include the catalytic core sequence of the hammerhead structure and target it at the B3 domain of CEA.

Transfection of the HT29 cells

First, the cells were transfected using the LipofectAmine system (Life Technologies) with the vector pUHG15-1 (Gossen and Bujard, PNAS, 1992, 89, p. 5547ff) in order to obtain the cell clone HT29 / tTA-5. This clone showed the best transactivated tetracycline activity.

This clone was then co-transfected with the plasmid pTET / Rz2113 (10 μg) prepared above and mixed with 1 μg pZeo (Invitrogen, San Diego, USA). Zeocin-resistant clones with the ribozyme could thus be obtained. The clones were selected in culture with 0.4 mg / ml zeocin and 1 μg / l tetracycline and examined for regulated CEA expression using FACS analysis (FACStar plus, Becton-Dickinson). The HT29 cells were also cultured in IMEM medium (Life Technologies Inc., Gaithersburg, USA) mixed with 10% heat-inactivated fetal bovine serum and glutamine at 37 ° C and 5% CO 2.

A clone HT29 / Rz4 was obtained in which the CEA expression is regulated depending on the tetracycline. This regulation was about 50% here and is generally at least 30%, preferably at least 50%.

Determination of apoptosis

Ixl0E6 cells were harvested, washed twice with 300 μl cold PBS, pH 7.4 and then stained in 100 μl propidium iodide / Annexin V-FITC double staining solution (TACSTM Annexin V-FITC protocol, Trevigen, Gaithersburg, USA) according to the manufacturer's protocol. They were incubated for 15 min at room temperature in the dark. 400 ul 1-fold concentrated binding buffer was then added to the cell suspension and the cells were analyzed with the flow cytometer within one hour.

The results of the analysis are shown in Fig.4. It can clearly be seen that the HT29 cells with an increased CEA expression (without the addition of tetracycline for ribozyme activation) show a lower apoptosis rate when apoptosis is induced by the addition of gamma-interferon. In contrast, the cells with reduced CEA expression (after induction of the CEA-specific ribozyme by administration of tetracycline) have a significantly higher sensitivity to apoptosis. This result showed that CEA (CEACAM5) decreases the ability to apoptosis, depending on the concentration and indirectly.

According to the results of the data, CEACAM-1 and CEA have an antagonistic effect in HT29 (see below).

Example 4

Importance of CEACAM-1 expression for apoptosis sensitivity in Jurkat cells

The Jurkat cell line is a standard reporter cell line for apoptosis assays. Jurkat cells are themselves CEACA negative. To investigate the importance of CEACAM-1 for apoptosis, Jurkat cells were transfected with the cDNA coding for transmembrane CEACAM-1 using standard methods (BGP-Jurkat). BGP-Jurkat cells express CEACAM-1 on their surface, as can be demonstrated by FACS analyzes with CEACAM-1-binding antibodies. Total protein extracts from BGP-Jurkat cells have a regular CEACAM-1 band in the Western blot with a relative molecular weight of around 160 kDa.

BGP-Jurkat cells were in logarithmic growth phase under constant cell culture conditions held and incubated for either 4 or 16 hours with different antibodies. The following were used:

- clone 7D11 (10 μg / ml, against intracellular ß-catenin) as a negative control,

Clone CH11 (100 ng / ml, against human CD95) as a positive control,

- clone 4D1.C2 (4μg / ml, specific for human CEACAM-1)

The primary antibodies were added to the medium for 24 hours each. After changing the medium, polyclonal goat anti-mouse immunoglobulin (15 μg / ml) was added to the cell cultures as a secondary antibody for cross-linking. No secondary antibody was added to the approach with anti-CD95 antibody CH11 (IgM). After a total experiment time of 80-96 hours, the cells were stained with trypan blue and propidium iodide according to standard protocols and the number of dead or apoptotic cells was counted microscopically as a blinded evaluation. All approaches were carried out in triplicate determinations and the experiments were repeated three times independently. The results of the individual tests were statistically evaluated according to the Wilcoxon rank test.

Human apoptosis reporter cells of the Jurkat type, which had previously been transfected stably with the cDNA encoding the transmembrane CEACAM-1, show a significantly increased apoptosis if the CEACA-1 molecules on the surface are specifically affected by the murine monoclonal anti-CEACAM-1 Antibodies are bound to 4D1C2 and crosslinked by an anti-murine secondary antibody. The secondary antibody and an irrelevant monoclonal antibody (mab 7D11) do not trigger apoptosis itself. The antibodies have no effect in untransfected Jurkat cells. As a positive control, the anti-CD95 antibody mab CH11 in the CEACAM-1-expressing and non-expressing cells shows that Jurkat cells are apoptosis-sensitive. This result shows that CEACAM1 can send an apoptosis signal. Since no other members of the CEACAM family are expressed in addition to CEACAM-1, the result is specifically attributable to CEACAM-1 and can be triggered by binding and cross-linking using CEABAM-1 antibody mab 4D1C2 or secondary antibodies.

Example 5

Importance of the modulated expression of CEACAM-1 for apoptosis sensitivity in HT29 cells

HT29 cells were treated with 500 U / ml interferon gamma for 6 hours. In a second approach, no interferon stimulation was used. After changing the medium, the cells are treated with primary antibodies as in Example 4. These are: mab 4D1C2 (anti-CEACAM-1; 4μg / ml) or irrelevant mab 7D11 (anti-ß-catenin lμg / ml). As a second negative control, an approach without antibodies was carried out. After changing the medium, secondary antibodies (anti-mouse; 15 μg / ml) were added to all cell batches. After a For a total experiment duration of 80-96 hours, the apoptosis was determined using a standard propidium iodide stain or by the apoptosis ELISA from RÖCHE Molecular Systems, strictly according to the manufacturer's protocol. All experiments were performed in triplicate and repeated triplicately independently. The significance of the data was evaluated in the Wilcoxon rank test.

The result (FIG. 5) confirms the importance of CEACAM-1 for apoptosis and the data from the Jurkat reporter cell system. In addition, the experiment shows that the sensitivity to apoptosis trigger depends on the one hand on the level of CEACAM1 expression. However, this requires their cross-linking and not an increase in the number of CEACAM-1 molecules on the cell surface alone, as apoptosis in ^' stimulated HT29 (black column in "untreated control") shows. Even unspecific immunoglobulin-mediated adsorption effects do not lead to an increase in apoptosis in stimulated HT29 (black columns in "irrelevant antibody" and "anti-mouse IgG"). These apoptosis rates correspond to those in the unstimulated HT29 cells. With the basally low CEACAM-1 expression in HT29, no increase in apoptosis is observed despite crosslinking, while induction of apoptosis can be observed in the cells of higher expression.

This experiment shows that in addition to the level of CEACAM-1 expression, cross-linking is also of further importance for the sensitivity of the HT29 cells to apoptosis. The cross-linking (which is made possible in vitro by the antibody cross-linking) is ensured in vivo by the presence of the adhesion of the molecules of the other members of the CEA family in the Glykocalix. Example 6

Specific upregulation of CEACAM-1 by TNF-alpha

HT29 colon carcinoma cells were incubated with TNF-alpha (10ng / ml) for 6 hours. The cells were examined microscopically for vitality, lysed, the total protein isolated and this separated electrophoretically. 8μg protein were separated per lane. The detection was carried out with an anti-CEA antibody and an anti-mouse HRP coupled secondary antibody (Dianova, Hamburg, Germany). The use of a cross-reacting anti-CEA antibody was chosen to test the selectivity of the candidate substances for the expression of the individual antigens. The selectivity of the substance effects is important because CEACAM-1 and -7 or CEA and CEACAM-6 can have antagonistic effects on apoptosis.

The proportionality of the expression of individual CEACAMs can be quantified in the Western blot after densitometric evaluation (e.g. public domain NIH software IMAGE 1.6.1). The antigens are identified in the blot by assignment to the relative molar mass. The molecular sizes of the individual CEACAMs are described in the literature. The blot was normalized with β-actin (Sigma-Aldrich, Taufkirchen, Germany). This blot (FIG. 6) was then evaluated densitometrically using the NIH image software 1.6.1.

Treatment with TNF-alpha showed an approximately 2-fold upregulation of CEACAM-1 after 6 hours compared to the untreated sample (lanes 1 and 2). This investigation shows the possibility of the specific up-regulation of the CEACAM-1 expression by exogenously added substances without influencing other proteins of the CEACAM family. In contrast to TNFa, other substances had no effect on CEACAM-1 expression:

200nM Wortmannin (Calbiochem-Novabiochem, Nottingham, UK) was incubated with lxlOE6 cells / well (6-well plate) for 3, 8 or 24 hours. After removing the reagent, the cells were washed with PBS, then the

Total protein fraction isolated and examined for changes in CEACAM1 expression as described by Western blot analysis. No changes in the expression of members of the CEA family could be found.

5x10E5 cells were cultured in a T75 bottle for 2 days. 3 μg / ml 5-azacytidine (Sigma-Aldrich, Taufkirchen, Germany) was used for the experiment. A medium change with 5-azacytidine (3μg / ml) took place on the 3rd day. A medium change without 5-azacytidine took place on the 4th day with subsequent cultivation overnight. A medium change with 5-azacytidine took place on day 5 with cultivation overnight. A medium change without 5-azacytidine took place on day 6 with cultivation overnight.

Protein isolation was performed on day 7. The proteins were examined in a Western blot. For this purpose, an antibody clone mAb C1N3 (Prof. Dr. Kalthoff, Molecular Oncology of the Surgical University Clinic, Kiel, Germany) was used in a concentration of 5μg / ml. The detection was carried out using peroxidase-labeled anti-mouse immunoglobulin according to the standard protocol.

The antibody against human β-actin, which was used to normalize the signals on the Western blot, was used in a dilution of 1: 10000 (v / v) and the signal was then developed according to the standard protocol.

Claims

1. A method of identifying connections comprising the steps

incubating a sample which can express a gene (s) or a gene product (s) of the CEACAM family with one or more compounds; incubate a second sample that can express a gene (s) or gene product (s) of the CEACAM family in the absence of the compound (s); Comparison of the expression of the gene (s) or gene product (s) of the CEACAM family in the samples.

2. The method according to claim 1, characterized in that the sample comprises cells.

3. The method according to claim 1 or 2, characterized in that the sample comprises cell lines.

4. The method according to claim 1 or 2, wherein tissue cells are used.

5. The method according to any one of claims 1 to 4, wherein genetically modified cells are used as a sample.

6. The method according to any one of claims 1 to 5, wherein the gene (s) or gene product (s) is / are CEACAM-1, CEA, CEACAM-6 and CEACAM-7.

7. The method according to any one of claims 1 to 6, wherein the gene or gene product is CEACAM-1 and / or CEACAM-7.

8. The method according to any one of claims 1 to 7, wherein the compound increases the expression of CEACAM-1 and / or CEACAM-7 in the presence compared to the absence.

9. The method according to any one of claims 1 to 6, wherein the compound lowers the expression of CEA and / or CEACAM-6 in the presence compared to the absence.

10. The method according to any one of claims 1 to 6, wherein the compound increases the expression of CEACAM-1 and / or CEACAM-7 in the presence compared to the absence and at the same time the compound the expression of CEA and / or CEACAM-6 in the presence of Decreased compared to absence.

11. The method according to any one of claims 1 to 10, wherein the determination is carried out at the mRNA level.

12. The method according to any one of claims 1 to 10, wherein the expression is determined at the protein level.

13. The method according to any one of claims 1 to 10, characterized in that the determination is carried out by measuring the apoptosis rate.

14. The method according to any one of the preceding claims, characterized in that compounds which cause crosslinking of the CECAM molecules are identified.

15. The method according to claim 14, characterized in that the compounds are antibodies or polymers.

16. Compounds that alter the expression of a gene (s) or. Gene product (s) of a member (s) of the CEACAM family in cells regulate, to a level which is present in cells which do not show any dysregulation and these dysregulated cells after regulation by the compound show a higher apoptosis susceptibility compared to the cells which have not been exposed to the compound.

17. A compound according to claim 16 selected from the group of peptides such as oligo- or polypeptides, proteins, cytokines, carbohydrates, antibodies, polymers and small molecules, with a molecular weight between 50 and 1000 Da.

18. A compound according to any one of claims 16 or 17, characterized in that the compound increases the expression of CEACAM-1 and / or CEACAM-7.

19. The method according to any one of claims 16 or 17, characterized in that the compound lowers the expression of CEACAM-6 and / or CEA.

20. A compound according to any one of claims 16 to 19, characterized in that the compound increases the expression of CEACAM-1 and / or CEACAM-7 and that the compound lowers the expression of CEACAM-6 and / or CEA.

21. Pharmaceutical compositions containing as active compound one or more compounds according to claim 16 or 20.

22. Use of a compound according to any one of claims 16 to 20 for the manufacture of a pharmaceutical composition for the preventive treatment of tumors.

23. Use according to claim 22, wherein the tumor is a tumor of the gastrointestinal.

24. Use according to claim 22 or 23, wherein the tumor is a colon carcinoma.

25. Use according to any one of claims 22 to 23, wherein the individuals have a predisposition to the development of tumors.

26. Kit for performing the method according to one of claims 1 to 15.

27. Test system for performing the method according to one of claims 1 to 15.

28. A diagnostic method for the detection of tumor precursor cells comprising the step of determining the expression of a member (s) of the CEACAM family in a sample.

29. The diagnostic method according to claim 28, wherein the sample is a cell homogenate or a tissue section.

30. Diagnostic method according to claim 28 or 29, characterized in that the expression of CEACAM-1, CEACAM-6, CEACA -7 and / or CEA is determined.

31. The method according to claim 28 or 30, characterized in that the expression of CEACAM-1 or CEACAM-7 is determined.

32. The method according to any one of claims 28 to 31, characterized in that the lowering of the CEACAM-1 and / or the CEACAM-7 expression is determined and / or that the increase in CEACAM-6 and / or CEA expression is determined.

33. Diagnostic kit comprising components for determining the expression of a member (s) of the CEACAM family.