EP1317275A1 - Medicament comprising a dna sequence which codes for the rna-binding koc protein, and comprising a koc protein or a dna sequence of the koc promoter - Google Patents

Abstract

The invention relates to a medicament containing an RNA-binding protein KOC or a DNA sequence that codes this protein. The invention also relates to a diagnostic method with regard to tumors associated with expression of KOC, and to different applications of the inventive medicament, preferably for artificially inducing the pluripotentiality of body cells. The invention additionally relates to a DNA sequence of the KOC promoter, whereby the KOC promoter is characterized in that it is only activated in embryonic tissues or malignant tumors, and, finally, relates to different applications of the KOC promoter.

Description

 Medicament with a DNA sequence coding for the RNA-binding KOC protein, a KOC protein or a DNA sequence of the KOC promoter

The present invention relates to a medicament which contains an RNA-binding protein KOC or a DNA sequence coding for this protein, preferably inserted on a vector. The present invention further relates to a diagnostic method with regard to malignant tumors associated with the expression of KOC and various uses of the medicament according to the invention, preferably for artificially inducing the pluripotency of body cells. Finally, the present invention also relates to a DNA sequence of the KOC promoter, the KOC promoter being characterized in that it is activated only in embryonic tissues or malignant tumors, and various uses of the KOC promoter.

The failure of individual organs or differentiated cells within an organ is the cause of many diseases that are still difficult to treat today. Despite advances in medical technology in transplantation medicine, the lack of donor organs is the reason for the annual deaths of thousands of patients. In addition, the functionality of the transplanted organs is only possible through continuous postoperative immune suppression, some of which associated with serious side effects.

For these reasons, the development of new technologies for the regeneration of diseased organs or for the reconstitution of organs has become a focus in medical research. The use of the body's own cellular development and differentiation potential for therapeutic approaches is the goal of these new treatment strategies. This will look for suitable Wanted methods that lead to the achievement of cell differentiation and proliferation while maintaining the function of the individual cell type without having a transforming potential. This could make it possible in future to generate stem cell populations from, for example, healthy cell material obtained from biopsies by targeted manipulation and cultivation, from which specific and differentiated cells can develop on the basis of their proliferation potential. This could finally resolve bottlenecks in solid replacement organs in transplant medicine.

The coordinated process of a complex cellular differentiation program, which ultimately leads to the development of a functioning organ, is, however, a largely unsolved problem (Lanza et al., Nature Medicine 5, No. 9 (1999), 975-977; Vogel, G. , Science 283 (1999), 1432-1434; Solter et al., Science 283 (1999), 1468-1470). An implementation of this concept only appears tangible in cases in which a differentiated individual cell ultimately "determines organ function", such as the insulin-producing beta cell of the pancreas or in the case of antibody-producing B cells, as well as in precursor cells of the bone marrow that are caused by specific growth factors can be differentiated.

The subject of intensive research is therefore the development of new methods for the development of functional organs, tissues and stem cells for transplantation purposes. For this purpose, individual genes have already been identified that are essential for the control of development processes. Furthermore, it was possible to isolate divisible, undifferentiated cells (embryonic stem cells, ES cells) that can be differentiated in a targeted manner by transferring certain genes. For example, ES cells can differentiate into mesenchymal cells by treatment with the "bone morphogenetic protein 4" (bmp4). In addition, these cells can also be manipulated by defined culture conditions such that three-dimensional, tissue-like structures with organ-specific expression patterns be developed. Another example of the pluripotency of stem cells can be seen in the fact that it has been possible to implant neuronal stem cells in mice with destroyed bone marrow, which differentiate there into blood cells.

So far, however, a controlled reprogramming of adult, differentiated cells to ES cells has not been so successful that this is medically justifiable because, for example, mouse ES cells are per se tumorigenic and, if injected into adult mice, can form teratocarcinomas. Applied to the human field, this procedure is not only technically very complex, but also touches on ethical aspects, since this process requires human blastocysts or mammalian oocytes ("therapeutic cloning").

Thus, the invention is essentially based on the technical problem of providing means with which a differentiation of a mature cell type into an embryonic phenotype that is divisible but still has characteristics of the respective differentiated cell is made possible.

This technical problem has been solved by providing the embodiments characterized in the patent claims.

In the course of an investigation of differential gene expression in pancreatic carcinoma, a gene was originally cloned that codes for an RNA-binding protein of the KH family, hence the name KOC (KH domain containing protein overexpressed in cancer) (Müller-Pillasch et al., Oncogene 14 (1997) 2729-2733). KOC is only expressed in pancreatic carcinoma, not in normal pancreatic tissue or in chronic pancreatitis. The gene codes for a 69 kDa protein with four KH domains ("hnRNP K homologous"); see Figure 1. The sequence is in the NCBI database under Accession No. U97188 deposited. KH domains are highly conserved, functionally important structures of RNA-binding proteins that are responsible for direct mRNA binding. Studies have shown that RNA-binding proteins play a central role in the post-transcriptional regulation of gene expression. In this function, RNA-binding proteins are essential for the regulation of growth and development. Examples include patterning and cellular differentiation during embryonic development.

Two mechanisms of post-transcriptional gene regulation by proteins containing KH domains are the control of the mRNA stability and the specific subcellular localization of the mRNA. Although a number of proteins containing KH domains have already been identified, no KH protein has so far been shown to be able to reverse the phenotype of differentiated cells to a phenotype of immature, undifferentiated, divisible cells and thus to be possible therapeutic agent is suitable for the production of syngeneic stem cell populations which provide large numbers of divisible, specifically differentiable cells.

It could be shown in the present invention that the problems described above can be solved with the aid of the gene which codes for the RNA-binding protein KOC. With the RNA-binding protein KOC, a mature cell type can be dedifferentiated into an embryonic phenotype, but which still bears the characteristics of the differentiated cell, so that stem cells of any cell type can be generated in the same organism. The KOC gene reverts the phenotype of differentiated cells into a phenotype of immature, undifferentiated, divisible cells. Thus, the RNA-binding protein KOC is suitable as a possible therapeutic agent for the production of syngeneic stem cell populations that use large numbers of divisible, specifically differentiable cells Make available, which can then be used, for example, in the field of transplantation medicine and oncology up to the development of biologically functional replacement organs. Furthermore, the RNA-binding protein KOC can be used to influence physiological or physical aging-induced aging processes, for example human skin.

The investigations were carried out on pre-B cells and by genetic manipulation of these differentiated cells with KOC, they could be converted into an undifferentiated state, which corresponds to an earlier, embryonic precursor form or stem cells of these cells. The pre B-cell model showed that, for example, the DNA synthesis of the KOC / p210 cells is significantly stronger compared to that of the p210 cells and that the KOC / p210 cells compared to the p210 cells have a large number of embryonic cells Up-regulate markers, or down-regulate markers that distinguish adult pre-B cells from fetal pre-B cells. Cells "re-differentiated" with the KOC gene are significantly less sensitive to cell death induced by radiation and dexamethasone than untreated cells. In addition, these cells re-enter the cell cycle and proliferate. KOC thus differs fundamentally from conventional growth factors used to date, which always only stimulate the proliferation of a differentiated cell type or transform a cell type into a malignant phenotype. The properties mentioned result in numerous potential applications for KOC in transplantation medicine and cancer treatment. The use of KOC is important for the development of new techniques for the restoration and regeneration of damaged organs (organ replacement), especially at the level of the generation of type-specific syngeneic stem cells and artificial tissue, such as in diseases such as Alzheimer's (neuronal stem cells), diabetes (pre- Beta cells), heart failure (precursor to cardiac muscle cells), liver (precursor to hepatocytes). Furthermore, the use of KOC in the case of skin injuries, such as burns, can in the future by inducing rapid extracorporeal multiplication of Skin cells may be important for transplant purposes.

In summary, the results for KOC (either as part of gene therapy or as protein administration) include following areas of application:

(1) Achieving resistance of differentiated cells to cell death (e.g. radiation) induced by different noxae, and

(2) Transfer of cells from a differentiated, non-proliferation-capable to an undifferentiated, proliferation-capable precursor state, which still bears clear features of the differentiated phenotype and thus enables the differentiation into the original cell type after expansion of the cell population by switching off the KOC expression. This is syngeneic, i.e. in the same organism, the unrestricted extraction of stem cells of any cell type and thus also the unrestricted extraction of differentiated, in part. cells that are not capable of dividing, or only very poorly, are possible at any age.

Thus, the administration of KOC itself or the gene encoding this protein is useful for the purposes described above. On the other hand, the possibility of inactivating KOC in diseases which are associated with an excessively high activity of KOC or an excessively high expression of the gene can also be therapeutically useful. Such inactivation can take place at various levels, for example at the genetic level ("knock out", inhibition of translation by means of antisense RNAs, ribozymes or aptamers) or protein level (via inhibiting ligands, for example antibodies or antagonists, etc.).

In addition, the DNA sequence of the KOC promoter was also cloned and sequenced in the course of the investigations leading to the present invention, it being shown that this is only activated in embryonic tissue or malignant tumors. The present invention thus relates to a DNA sequence of the KOC promoter which comprises the nucleic acid sequence shown in FIG. 9 or a nucleic acid sequence which deviates therefrom and whose biological function essentially corresponds to that of the KOC promoter.

The term "a different nucleic acid sequence" used in connection with the KOC promoter in the present invention refers to any nucleic acid sequence which differs from the nucleic acid sequence shown in FIG. 9 by the deletion, addition or substitution of bases, however the biological function of the Promotors is retained, ie to be able to control the transcription of a gene, the promoter being activated essentially only in embryonic tissue or malignant tumors. The deviating nucleic acid sequences are preferably nucleic acid sequences which hybridize with the nucleic acid sequence shown in FIG. 9, reference being made to the definition below with regard to the term “hybridize”, or fragments of the nucleic acid sequence shown in FIG. 9. Preferably, the "different nucleic acid sequences" are 70%, more preferably 80% and most preferably 90% identical to the DNA sequence of Figure 9.

The present invention also relates to a medicament which contains a DNA sequence which codes for an RNA-binding protein KOC with the amino acid sequence shown in FIG. 1, or a DNA sequence which contains the nucleic acid sequence shown in FIG. The DNA sequence is preferably cDNA.

The present invention further relates to a pharmaceutical composition containing a DNA sequence of the promoter KOC according to the invention or a DNA Se ^acid sequence, the binding for a protein having the biological properties of an RNA encoding the protein KOC, wherein said DNA sequence of the DNA sequence of Figure 1 in the codon sequence due to the degeneration of genetic codes, hybridizes with the above DNA sequences or is a fragment, an allelic variant or another variant of the above DNA sequences.

The term “hybridized” used in the present invention refers to conventional hybridization conditions, preferably to hybridization conditions in which 6xSSC, 1% SDS, IxDenhardts solution is used and the hybridization temperatures between 35 ° C. and 70 ° C., preferably be at 45 ° C. After hybridization, washing is preferably carried out with 2xSSC, 1% SDS or with 0.2xSSC (stringent conditions) at temperatures between 40 ° C. and 75 ° C., preferably at 50 ° C. (for the definition of SSC and Denhardts solution see Sambrook et al ., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Stringent hybridization conditions are particularly preferred, as described, for example, in Sambrook et al., Supra, or Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.

The terms "variant" or "fragment" used in the present invention encompass DNA sequences which differ from the sequences indicated in FIG. 1 by deletion (s), insertion (s), exchange (s) and / or others in the prior art Distinguish known modifications or comprise a fragment of the original nucleic acid molecule, the protein encoded by these DNA sequences still having the biological properties of the RNA-binding protein and being biologically active in mammals. This also includes allele variants. Methods for generating the above changes in the DNA sequence are known to the person skilled in the art and are described in standard works in molecular biology, for example in Sambrook et al., Supra. The person skilled in the art is also able to determine whether a protein encoded by a DNA sequence modified in this way still has the biological properties of the RNA-binding protein KOC. Below are all these DNA sequences are generally referred to as "KOC DNA sequences".

By lowering or inhibiting the expression of the DNA sequences described above, the synthesis of the proteins encoded by them can be reduced or eliminated, which is desirable, for example, in certain disease states. Therefore, a further preferred embodiment of the present invention relates to antisense RNA which is characterized in that it is complementary to the above DNA sequences and can reduce or inhibit the synthesis of the protein encoded by these DNA sequences and a ribozyme which is characterized in that that it can specifically bind to and cleave part of the above DNA sequences and to the RNA transcribed from these DNA sequences, thereby reducing or inhibiting the synthesis of the protein encoded by these DNA sequences. These antisense RNAs and ribozymes are preferably complementary to a coding region of the mRNA. The person skilled in the art is able to prepare and use suitable antisense RNAs based on the disclosed DNA sequences. Suitable procedures are described for example in EB-B1 0223399 or EP-A1 0458. Ribozymes are RNA enzymes and consist of a single strand of RNA. These can intermolecularly cleave other RNAs, for example the mRNAs transcribed by the DNA sequences according to the invention. In principle, these ribozymes must have two domains, (1) a catalytic domain and, (2) a domain that is complementary to the target RNA and can bind to it, which is a prerequisite for cleaving the target RNA. Based on procedures described in the literature, it is now possible to construct specific ribozymes that cut a desired RNA at a specific, pre-selected location (see, for example, Tanner et al., In: Antisense Research and Applications, CRC Press, Inc. (1993 ), 415-426). The DNA sequences coding the RNA-binding protein KOC or the related protein or the DNAs coding for the antisense RNAs or ribozymes described above can also be inserted into a vector, preferably an expression vector. The present invention thus also includes medicaments containing these vectors or expression vectors. The term "vector" refers to a plasmid (pUC18, pBR322, pBlueScript, etc.), a virus or another suitable vehicle. In a preferred embodiment, the DNA molecule according to the invention is furcally linked in the vector to regulatory elements which allow its expression in prokaryotic or eukaryotic host cells. In addition to the regulatory elements, for example a promoter, such vectors typically contain an origin of replication and specific genes which allow the phenotypic selection of a transformed host cell. The regulatory elements for expression in prokaryotes, for example E. coli, include the lac, trp promoter or T7 promoter, and for expression in eukaryotes the AOX1 or GAL1 promoter in yeast and the CMV, SV40 , RSV, metallothione and polyhedrin promoter, CMV or SV40 enhancer for expression in animal cells. Suitable regulatory sequences are also described in Goeddel: Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter. Suitable expression vectors for E. coli include, for example, pGEMEX, pUC derivatives, pGEX-2T, pET3b and pQE-8. Vectors suitable for expression in yeast include pY100 and Ycpadl, for expression in mammalian cells pMSXND, pKCR, pEFBOS, cDM8 and pCEV4, and of pcDNAI / amp, pcDNAI / neo, pRc / CMV, pSV2gpt, pSV2neo, pSV2-dhfr Vectors derived from pTk2, pRSVneo, pMSG, pSVT7, pko-neo and pHyg. The expression vectors according to the invention also include vectors derived from bacuiovirus for expression in insect cells, for example pAcSGHisNT-A. The KOC-DNA sequences described above, which may also comprise a DNA sequence of the KOC promoter, are preferably inserted into a vector suitable for gene therapy, for example under the control of a tissue-specific promoter, and introduced into the cells. In a preferred embodiment, the vector containing the DNA sequences described above is a virus, for example an adenovirus, vaccinia virus or retrovirus. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. Vectors suitable for gene therapy are also disclosed in WO 93/04701, WO 92/22635, WO 92/20316, WO 92/19749 and WO 92/06180. For the purposes of gene therapy, the KOC-DNA sequences described above can also be transported to the target cells in the form of colloidal dispersions. These include, for example, liposomes or lipoplexes (Mannino et al., Biotechniques 6 (1988), 682). Finally, the KOC-DNA sequences or the KOC protein can be administered locally, for example in the form of a cream, ointment, etc.

General methods known in the art can be used to construct expression vectors which contain DNA sequences coding for the RNA-binding protein KOC and / or a DNA sequence of the KOC promoter and suitable control sequences. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as are described, for example, in Sambrook et al., Supra. The DNA sequences according to the invention can also be inserted in connection with a DNA coding for another protein or peptide, so that KOC can be expressed, for example, in the form of a fusion protein. The person skilled in the art is also familiar with processes for the recombinant production of KOC, ie the cultivation of suitable host cells under conditions which allow the expression of the protein (or fusion protein) (preferably stable expression), and the extraction of the protein from the culture or from the host cells. suitable Purification methods (for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are also generally known.

An essential goal in the diagnosis of malignant diseases is the specific and sensitive detection of early malignant changes. For example, more and more gene mutations that occur in the context of the adenoma-carcinoma sequence are used in epithelial tumors. The disadvantage of these methods is that gene mutations sometimes occur in normal tissues, such as, for example, ki-ras in normal pancreatic tissues or in highly inflammatory tissues, which makes it impossible to distinguish between chronic, inflammatory or tumorous tissues, which are of great therapeutic importance , The aim of the research is therefore to identify genes that are only expressed / upregulated in malignant degenerate cells and are therefore suitable for unambiguous tumor diagnosis.

As discussed above, KOC is expressed in pancreatic cancer, but not in normal pancreatic tissue or in chronic pancreatitis. This finding that KOC is only expressed in malignant tissues (organs or cells) but not in benign tissues has been confirmed in many different tissues and cancers. Please refer to Tab. 1. KOC is therefore suitable for the differential diagnosis between malignancy and benignity, for example in the case of inflammation, of a tissue, preferably of the pancreas or colon. KOC is particularly characterized by the fact that its expression can be detected very early in the development of cancer, long before any auto-antibodies against KOC can be formed. Furthermore, KOC enables the differentiation of different stages of malignancy, especially in pancreatic and colon carcinoma. It has been shown here that KOC expression increases with the severity of the dysplasia. KOC thus represents a universally applicable Tumor indicator. Therefore, the present invention also relates to diagnostic methods for the detection of a malignant tumor associated with the expression of KOC or its precursors, in which a sample with a probe suitable for specific hybridization with the mRNA transcribed from a KOC-DNA sequence or a primer or an antibody against the KOC protein or a fragment thereof and then directly or indirectly determines whether the concentration of KOC and / or the KOC mRNA in the sample differs from a control sample. The person skilled in the art knows methods for suitable sampling and also suitable control samples. The control sample is preferably a tissue which corresponds to the same tissue as the tissue affected by the tumor, but comes from a healthy source. An increased KOC expression compared to the control sample is a diagnostic sign of a tumor or a predisposition to a tumor. The probes that can be used for this diagnostic method include primers based on the KOC-DNA sequences, for example for PCR, RT-PCR or aptamer diagnostics (SELEX method). Preferably, the probes (or primers) are oligonucleotides that comprise a nucleic acid region that hybridizes under stringent conditions with at least 10, preferably at least 15 consecutive nucleotides of the sequence of FIG. 1 or naturally occurring variants thereof. The probe (or the primer) can also carry a label, for example a radioisotope, a fluorescent compound, an enzyme or an enzyme cofactor. The person skilled in the art is also familiar with conditions which allow that only the KOC mRNA is not amplified in the PCR etc. but not the KOC DNA or that a distinction can be made between DNA amplification products and mRNA amplification products, for example by treating the sample with DNAse or by using primers which also lead to the amplification of intron regions, so that a DNA amplificate is longer compared to the mRNA amplificate. Alternatively, an oligo (dT) anchored primer for PCR, RT-PCR etc. can also be used. The antibodies for the diagnostic method according to the invention can be monoclonal, polyclonal or synthetic antibodies or fragments thereof. In this context, the term “fragment” means all parts of the monoclonal antibody (for example Fab, Fv or “single chain Fv” fragments) which have the same epitope specificity as the complete antibody. The production of such fragments is known to the person skilled in the art. The antibodies according to the invention are preferably monoclonal antibodies. These antibodies can be produced according to standard procedures, the KOC protein or a (synthetic) fragment thereof preferably serving as an immunogen. Methods for obtaining monoclonal antibodies are known to the person skilled in the art.

A preferred embodiment of the method according to the invention relates to the differential diagnosis between chronic pancreatitis and pancreatic carcinoma.

Furthermore, the present invention relates to a diagnostic kit for performing the diagnostic method according to the invention, which contains a probe suitable for specific hybridization with a mRNA transcribed from a KOC-DNA sequence or a primer and / or an anti-KOC antibody or a fragment thereof. Depending on the configuration of the diagnostic kit, the sample or the probe or the primer or the antibody or the fragment thereof can be immobilized. The antibodies can also be present in the liquid phase, for example in immunoassays. The antibodies can be labeled in different ways. Suitable markers and labeling methods are known in the art. Examples of immunoassays are ELISA and RIA.

The medicament according to the invention allows a number of (therapeutic) measures to be carried out. The medicament is preferably combined with a suitable carrier. Suitable carriers and the formulation such medicinal products are known to the person skilled in the art. Suitable carriers include, for example, phosphate-buffered saline solutions, water, emulsions, for example oil / water emulsions, wetting agents, sterile solutions, etc. The medicaments can be administered orally or parenterally. Methods for parenteral administration include topical, intraarterial, intramuscular, subcutaneous, intramedullary, intrathecal, intravenous, intravenous, intraperitoneal, or intranasal administration.

The present invention thus relates to the use of the compounds described above (KOC-DNA sequences / KOC-protein) for artificially inducing the pluripotency of body cells. This is e.g. important for the development of replacement organs, especially for the production of artificial pancreatic, liver or nerve tissue. Thus, the present invention also relates to the use of the above compounds for the production of tissues or organs via the differentiation of stem cell populations.

The present invention also relates to the use of the compounds described above (KOC DNA sequences / KOC protein) for improving the ex vivo expansion of hematopoietic stem cells, for example for autologous bone marrow transplants. In particular, syngeneic progenitor cells can be generated without "purging". This makes autologous transplants possible on a much larger scale than before. The cells to be expanded can be clearly characterized beforehand in FACS (Fluorescence activated Cell Sorting), so that an expansion of malignant cells is excluded. Immunological problems, which often occur with allogeneic transplantation, are excluded. Problems and complications of a delayed regeneration of the marrow due to an insufficient number of stem cells reinfused, such as in sepsis or life-threatening fungal infections, are eliminated, since the amount of stem cells that are available can be determined as desired. But this also becomes one further dose increases possible with chemotherapy, since the endogenous stem cell population is no longer required exclusively for the regeneration of the marrow. Use of the method in high-dose chemotherapy with repeated administration of chemotherapy and reinfusion of autologous bone marrow stem cells is therefore of use.

Thus, the present invention also relates to the use of the above compounds for high dose chemotherapy.

Another therapeutic effect is that as long as the protein is expressed, KOC stem cells are less sensitive to chemotherapy or radiation therapy. That under therapy with expression of KOC, the number of re-infusions of expanding stem cells required is reduced. The subsequent shutdown of KOC can then restore the normal sensitivity of the cells to noxae / apoptotic stimuli after the completion of chemotherapy or radiation therapy.

The present invention thus also relates to the use of the above compounds for producing a prophylactic effect in chemotherapy or radiation therapy.

Since it was shown in expression analyzes that not only the mRNA of KOC at the nucleic acid level but also the protein can be detected accordingly by means of a specific antibody, it can be assumed that the protein as such has its specific effect on the respective cells. Therefore, the use of KOC on skin cells, preferably by means of protein dissolved in creams, is also advantageous. Since only superficial layers of the skin are reached, especially when KOC is applied topically in creams or lotions, there is no lasting (side) proliferation-promoting effect on the entire dermis go out. Rather, a lasting effect is only achieved in the upper layers of the skin, which are particularly damaged by exposure to environmental influences (UV) and can malignant (spinalioma, basalioma). This provides a therapeutic-prophylactic effect of KOC on skin exposed to radiation - also in the context of therapeutic irradiation of body regions with gamma rays.

Thus, the present invention also relates to the use of the above compounds to produce a prophylactic effect on skin exposed to radiation.

Furthermore, the donor marrow can be transfected with KOC in an allogeneic bone marrow transplant. A faster and less complicated engraftment is then to be expected after the transplant. This resulted in bone marrow transplants performed on mice in the context of this invention. Mark, which was transfected with KOC, showed significantly faster engraftment.

Thus, the present invention also relates to the use of the above compounds for the improvement of the engraftment in allogeneic bone marrow transplants.

In addition, the use of KOC can also contribute to the slowing down or reversal of aging processes, the aging processes preferably being aging processes in the area of the vascular endothelium, neurons, keratinocytes and myocytes.

Thus, the present invention also relates to the use of the above compounds for slowing and / or reversing through physiological or physical pollutants induced aging processes, preferably aging processes, which are induced for example by pollutants, such as UV radiation and other environmental influences. Here, KOC (gene or protein) is preferably applied topically to a suitable carrier (for example, as liposomes, lotions, plasters, plasters, bandages, injections, etc.) dissolved in the skin or administered therein. This use also includes a cosmetic application of KOC (gene or protein) on the skin to influence acid skin / wrinkles.

The present invention further relates to the use of the above compounds for the regeneration of skin defects or for accelerated wound healing. This also includes the formation of granulation tissue, e.g. after trauma, burns or in the context of UIcera cruris of different origins.

Furthermore, it has been shown that an individual can be immunized using KOC against malignant tumors associated with the expression of KOC and their precursors.

The present invention thus relates to the use of the compounds described above (KOC-DNA sequences / KOC-protein) for immunizing an individual, for example an animal or a human, against malignant tumors associated with the expression of KOC and their precursors. In particular, the present invention relates to a medicament suitable therefor which contains these compounds in an amount suitable for the immunization of an individual. The KOC protein can be in wild-type or modified form. The latter form includes changes in the amino acid sequence, such as additions, deletions, substitutions and / or inversions of one or more amino acids. Fragments of the KOC proteins can also be present as such or in conjunction with carriers, the fragments having a wild-type or an altered amino acid sequence can. It is favorable if the carriers do not act as immunogenic in the individual. Such carriers can be individual's own proteins or foreign proteins or fragments thereof. Carriers such as serum albumin, fibrinogen or transferrin or a fragment thereof are preferred. It is particularly favorable if the fragments of the KOC proteins contain epitopes which are recognized by cytotoxic T cells, for example CD8 ⁺ T cells, and which can induce a cytotoxic immune response. Such epitopes of the KOC proteins can be determined by methods known to the person skilled in the art, in particular by using a software of the NIH (NIH bioinform at ionse rv ice http: /bimas.dcrt.nih.gov.cgi-bin/molbio/ken_parker_comboform) become. It can also be advantageous if different of the KOC proteins or fragments thereof, to which the above statements apply accordingly, are present at the same time. For the production of the KOC proteins or their fragments, in addition to the above statements, reference is made to Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989).

If a KOC-DNA sequence is used to immunize an individual, it must be in an expressible form, i.e. as such it can be present together with elements suitable for its expression or in connection with a vector. Reference is made to the above explanations.

The term "malignant tumors and their precursors" encompasses malignant tumors of any kind and origin, which are associated with the expression of KOC, and their precursors. For example, they can be carcinomas of epithelial origin, in particular pancreas, colon, breast, bronchial and anogenital carcinomas.

The term "amount suitable for immunization of an individual" includes any amount of the KOC protein to which the above statements apply, or an expressible KOC DNA sequence for which the above explanations apply accordingly, with which an individual can be immunized. The amount depends on whether the KOC protein or the expressible KOC DNA sequence is used. The amount also depends on whether the immunization of the individual aims more at induction of antibodies directed against the KOC protein or at stimulation of cytotoxic T cells directed against the KOC protein, eg CD8 ⁺ T cells. Both possibilities of immunization can be achieved by the present invention. Furthermore, the amount depends on whether the immunization is intended as a prophylactic or therapeutic treatment. In addition, the age, gender, and weight of the individual speak a role in determining the amount. It is favorable if the individual is injected with 100μg - 1 g of the KOC protein or 10 ⁶ - 10 ¹² MOI of a recombinant virus containing an expressible KOC-DNA sequence. The injection can take place intramuscularly, subcutaneously, intradermally or in any other form of application in several places of the individual. Furthermore, it may be advantageous to carry out one or more “booster injections” with approximately the same amount, it being particularly advantageous to use different fragments of the KOC protein in the individual injections. Furthermore, it is convenient if the drug contains immunization adjuvants such as GM-CSF or Freund's adjuvant to immunize the individual.

Furthermore, a non-human mammal ("knock-out") can be provided by conventional methods, the KOC gene of which is changed. A method is favorable which comprises the following steps:

(a) Production of a DNA fragment, in particular a vector, containing an altered KOC gene, the gene being altered by inserting a heterologous sequence, in particular a selectable marker;

(b) preparation of embryonic stem cells from a non-human mammal (preferably mouse); (c) transforming the embryonic stem cells from step (b) with the DNA fragment from step (a), the KOC gene in the embryonic stem cells being changed by homologous recombination with the DNA fragment from (a),

(d) culturing the cells of step (c),

(e) selection of the cultured cells from step (d) for the presence of the heterologous sequence, in particular the selectable marker,

(f) Generating chimeric non-human mammals from the cells of step (e) by injecting these cells into mammalian blastocysts (preferably mouse blastocysts), transferring the blastocysts into pseudo-pregnant female mammals (preferably mouse) and analyzing the progeny obtained for a change in the KOC gene.

In step (c) the mechanism of homologous recombination (cf. R.M. Torres, R. Kühn, Laboratory Protocols for Conditional Gene Targeting, Oxford University Press, 1997) is used to transfect embryonic stem cells. The homologous recombination between the DNA sequences present in a chromosome and new, added cloned DNA sequences enables the insertion of a cloned gene into the genome of a living cell instead of the original gene. With this method, embryonic germ cells can be used to obtain via chimeras animals that are homozygous for the desired gene or the desired gene part or the desired mutation.

The term "embryonic stem cells" refers to any embryonic stem cells from a non-human mammal that are suitable for mutating the KOC gene. The embryonic stem cells are preferably from the mouse, in particular the cells E14 / 1 or 129 / SV.

The term "vector" encompasses any vector which, by recombination with the DNA of embryonic stem cells, alters the KOC gene. made possible. The vector preferably has a marker which can be used to select for existing stem cells in which the desired recombination has taken place. Such a marker is, for example, the loxP / tk neo-cassette, which can be removed from the genome again using the Cre / loxP system.

Furthermore, the person skilled in the art knows conditions and materials in order to carry out steps (a) - (f).

The present invention provides a non-human mammal whose KOC gene is altered. This change can be a function deactivation. With such a mammal or cells from it, the function of the KOC protein can be examined selectively. It is also possible to find substances, drugs and therapeutic approaches that can be used to selectively influence the function. The present invention therefore provides a basis for acting on a wide variety of diseases. Such diseases are e.g. induction of cancer due to errors in the control of cell proliferation. In addition, the mammal with altered KOC function enables studies to be carried out as to whether weakened, if not no, tumor development is observed in these animals through the application of various cancerogens. The animal represents a new disease model for the development of new therapeutic approaches.

Finally, the present invention also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence which deviates therefrom, the biological function of which essentially corresponds to that of the KOC promoter for the isolation and / or enrichment and / or selective multiplication of stem cells , preferably non-human embryonic stem cells.

Stem cells are the primary cells from which the individual tissues of an organism develop. Because all cell types in an organism consist of just one stem cell, the zygote (fertilized egg cell), all stem cells have precursors that have different abilities (potencies) to form new tissues depending on the development stage of the individual. One speaks of toti-, pluri- and multipotent stem cells. The earliest stem cells, the embryonic stem cells, are totipotent. Any tissue can arise from them. Later stem cells lose this ability and are then only able to generate individual tissues. If stem cells are required for scientific or medical purposes, careful separation of other differentiated cells must be ensured during stem cell extraction, since these can overgrow the embryonic stem cells under culture conditions. Furthermore, the differentiation or programmed cell death of stem cells is induced by a continuous presence of certain differentiated cells. For this reason, it is desirable to be able to select stem cells, for example embryonic stem cells from the "mixed culture". Genetic manipulation makes it possible to introduce genetic selection markers into the cells whose gene products ensure selective survival of the stem cells under certain culture conditions. For example, a gene product can cause resistance to certain antibiotics. All cells that do not express this resistance gene can be eliminated by adding the respective antibiotic in the culture medium. A crucial prerequisite for a selection of stem cells is that the respective selection marker is under the control of a promoter that is activated exclusively in the stem cells. Since the KOC promoter is only activated during this time, it can be used as a control unit for the selection of stem cells. For this purpose, infection / transfection of a vector in which a selection marker is under the control of the KOC promoter gives the stem cells a selection advantage under conditioned culture conditions (eg addition of antibiotics) compared to cells that are no longer in the embryonic state or already differentiated a selection advantage. Procedure for infection / transfection of Stem cells, suitable vectors and suitable selection markers, for example the neo-gene / G418 system, are known to the person skilled in the art; see also the above explanations regarding the description of the non-human mammal. The person skilled in the art is also familiar with suitable cultivation methods and media in order to be able to cultivate stem cells, for example embryonic stem cells; see, for example, DE 196 08 813 C2, EP-B1 0 695 351. The "basic medium", which contains, for example, the antibiotic in a suitable concentration for selection, can be any medium which is usually used for the cultivation of mammalian cells. The cells are grown in the above medium under suitable conditions, optionally with (partial) renewal of the medium at suitable time intervals. Suitable conditions, for example with regard to suitable containers, temperature, relative atmospheric humidity, 0 ₂ content and C0 ₂ content of the gas phase, are known to the person skilled in the art. Preferably, the cells are cultured in the medium under the following conditions: (a) 37 ° C, (b) 100% rel. Humidity, (c) 10% 0 ₂ and (d) 5% C0 ₂ . The person skilled in the art can also monitor the desired inhibition of cell differentiation on the basis of customary criteria (morphological criteria, presence or absence of specific surface proteins, etc.).

The present invention also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence which deviates therefrom, the biological function of which essentially corresponds to that of the KOC promoter, for determining the degree of differentiation or dedifferentiation of a cell or a tissue. For this purpose, it is favorable to combine the D NA sequence from FIG. 9 or the nucleic acid sequence deviating therefrom with a reporter gene and to introduce the construct into the cell or the tissue and to demonstrate the expression of the reporter gene, which provides information about gives the degree of differentiation or degree of differentiation. The present invention also relates to a diagnostic kit for carrying out the diagnostic method described above, which contains a probe or a primer suitable for specific hybridization with a DNA sequence of the KOC promoter. With regard to the design of the kit, the statements made above apply mutatis mutandis to the diagnostic kit relating to the KOC protein or KOC gene. The inventors have characterized pairs of primers which are ideally suited for the purposes mentioned above, for example as a tumor indicator. These can be provided as part of the kit. The preferred primers are:

Forward primer:

ATG 14 5'-ATATCGGAAACCTCAGCGAG-3 '

ATG 651 δ'-CATTCGGAACATCACCAAAC-S '

ATG 778 δ'-GGGGTATTAAACGTGCATTT-S ¹

ATG 1428 5'-CACTGTCATGAGCCTCACTA-3 '

Reverse primer:

ATG 98 5'-GTAGCCAGTCTTCACCAGGA-3 '

ATG 341 δ'-TGCAGTTTCCGAGTCAGTGT-S '

ATG 757 5'-GACTTACAAGCCGCAGAGGT-3 'ATG -217 5'-TGACAG TTTAAACCAGCA-3'

The following primer pair combinations are very particularly preferred:

ATG 14 - ATG 98 (1) ATG 651 - ATG 757 (2) ATG 14 - ATG 341 (6) 4. Fettfuar ~ UUJ.

ATG ATG ATG ATG ATG ATG

AT ^ G (D ATG 14 757

The conventional tumor markers used so far in the clinic are usually acute phase proteins, which increase in their measurable value in the course of a tumor disease, but also in numerous pathophysiological conditions, e.g. renal insufficiency, inflammation, etc., above the normal value in serum are to be found elevated. These tumor markers only have their clinical significance if, firstly, they are significantly increased and secondly, their value changes in the course of a disease or therapy. This means that most of the common tumor markers are only suitable for course assessment or therapy evaluation. However, they are primarily of no diagnostic value. The use of KOC as a tumor indicator makes it possible for the first time to predict with accidental findings, e.g. in imaging procedures, with excellent specificity and sensitivity whether a lesion is a malignant or benign mass. This is of particular interest since KOC provides this information with regard to any tissue and is therefore a universally applicable tumor indicator for confirming the diagnosis of Is mass. It is also noteworthy that KOC is not affected in its expression behavior by other pathophysiological conditions, such as inflammation, and thus guarantees 100% sensitivity and specificity. The importance of KOC as a tumor indicator also results from the fact that investigator-dependent influences can be ruled out absolutely. As a surrogate parameter, KOC can therefore significantly improve cytological diagnostic reliability. In addition, reference is made to the above explanations regarding the use of KOC as a tumor indicator.

It has also long been known that certain chemotherapeutic agents are initially good in humans, but gradually become less effective over time. The cause of this "resistance" is a mechanism that tumor cells get themselves going, since chemotherapy drugs can activate the so-called multi-drug resistance (md) gene. The gene product itself is located on the cell membrane and is responsible for transporting foreign substances out of the cell. In this way, the medication becomes ineffective. This resistance can be prevented, for example, using the tumor necrosis factor (TNF-alpha). TNF-alpha inhibits the expression of the MDR1 gene and thus the development of resistance if it is supplied from the outside or transferred as a gene. For example, the TNF-alpha gene is packaged with the promoter sequence of the mdr-1 gene in a vector and treated Animals with this vector construct and with chemotherapeutic agents that act on the promoter sequences of the mdr-1 gene turn on the production of TNF alpha, prevent the formation of resistance and promote the destruction of the tumor cell by apoptosis. A major disadvantage of this method, however, is that chemotherapy, which is stressful for the patient, must also be carried out. Another example is therapy with cisplatin (cis-diaminedichloroplatinum), which is used as an active agent in a variety of malignant diseases as a chemotherapeutic agent. Because the KOC promoter is activated during the malignant transformation of a cell (see below Example 6), this is excellently suitable as a control unit for a so-called "gene-directed enzyme pro drug (suicide gene) therapy" (GDEPT) for the treatment of a large number of malignant diseases. The HSV-TK gene (in combination with the application of ganciclovir, which becomes toxic through phosphorylation via the HSV-TK), a tumor suppressor gene, for example p53, p16, p21, DPC4, APC, etc., is preferred for this gene therapy cancer treatment ., or an apoptosis-inducing gene, for example bad, caspases, etc., functionally linked to the KOC promoter.

Thus, the present invention also relates to the use of the DNA sequence of the KOC promoter shown in FIG. 9 or the nucleic acid sequence which deviates therefrom, whose biological function essentially corresponds to that of the KOC promoter, for gene therapy cancer treatment. With regard to the implementation of a gene therapy or the vectors suitable therefor, the above explanations apply analogously with regard to the KOC-DNA sequences.

Furthermore, the present invention relates to a method for identifying compounds which bind to the DNA sequence of the KOC promoter according to the invention and modulate its activity, the method comprising the following steps: a) bringing a DNA sequence of the KOC promoter according to the invention into contact with a Test compound in a cellular assay; and b) determining the modulation of the activity of the KOC promoter, where modulation indicates that the test compound is active.

The test compounds can be very different compounds, both naturally occurring and synthetic, organic and inorganic compounds, and also polymers, for example oligopeptides, polypeptides, oligonucleotides and polynucleotides, as well as small molecules, antibodies, sugars, fatty acids, nucleotides and nucleotide and Analogs, analogs of course occurring structures, for example peptide "imitators", nucleic acid analogs etc., and numerous other compounds. Transcription factors are particularly suitable as test compounds.

When performing the cellular assay, it must be ensured that the test compound is brought into contact with the DNA sequence of the KOC promoter which controls the transcription of a reporter gene under such conditions that specific binding is made possible. Then it is preferably determined in the same test system whether the transcription of the promoter functionally linked to the reporter gene has been modulated by the presence of the test compound. Examples of suitable reporter genes are GFP, Lacz, luciferase or CAT. The values found in terms of the transcription rate are preferably compared with the values in a second test system that differs from the first only in the absence of the test compound. Basically suitable assay formats for the identification of compounds that affect the modulation of the activity of the KOC promoter are well known in the biotechnological and pharmaceutical industries and additional assays and variations of the assay provided above for illustration are obvious to the person skilled in the art.

Changes in promoter activity can be measured by any suitable method. Changes in expression level can be examined using methods well known to those skilled in the art. This includes monitoring the mRNA concentration, eg using suitable probes or primers, immunoassays for protein concentration, RNAse protection assays, amplification assays or any other means suitable for detection that is known in the art. Further detection methods regarding the activation of the promoter depend on the specific properties of the respective reporter gene. The search for compounds which are effective for modulating the activity of the KOC promoter can also be carried out on a large scale, for example by screening a very large number of candidate compounds in substance libraries, the substance libraries being synthetic or natural molecules, eg cDNAs from expression libraries. Thus, in a preferred embodiment of the method according to the invention, the test compound is part of a substance library.

The above method according to the invention can be modified on the basis of protocols which are described in the scientific literature and the patent literature and are known in the art. For example, a large number of potentially useful molecules can be screened in a single test. For example, if a field of 1000 compounds is to be screened, in principle all 1000 compounds can be placed in a microtiter plate well and tested simultaneously. If a promoter modulator is discovered, the pool of 1000 can then be divided into 10 pools of 100 and the process repeated until an individual modulator is identified. In any event, the production and simultaneous screening of large banks of synthetic molecules can be carried out using well known combinatorial chemistry techniques, see for example van Breemen, Anal. Chem. 69 (1997), 2159-2164 and Lam, Anticancer Drug Des. j2 (1997), 145-167.

The method according to the invention can also be greatly accelerated as high throughput screening. The assays regarding promoter modulation described here can be modified accordingly for use in such a method. It will be apparent to those skilled in the art that numerous methods are available for this purpose. In addition, a large number of potentially useful promoter activity-modifying compounds can be screened in extracts from natural products as a starting material. Such extracts can come from a large number of sources, for example the species fungi, actinomycetes, algae, insects, protozoa, plants and bacteria. The extracts that show modulation can then be analyzed to isolate the active molecule. See, for example, Turner, J. Ethnopharmacol. 51 (1-3) (1996), 39-43 and Suh, Anticancer Res. 15 (1995) 233-239. The compounds found which modulate the activity of the KOC promoter can in many cases be used therapeutically, for example in the context of tumor therapy.

Brief description of the figures:

FIG. 1: DNA sequence of the gene coding for the RNA-binding protein KOC and amino acid sequence derived therefrom

Figure 2: Proliferation of cells that express KOC and p210 and cells that only express p210

Cells expressing KOC and p210 proliferate 10-fold more than cells expressing only p210.

Figure 3: Growth of cells expressing KOC and p210 and cells expressing only p210

Cells expressing KOC and p210 grow independent of the stroma compared to cells expressing only p210

Figure 4: Resistance of cells that express KOC and p210 and cells that only express p210. towards dexamethasone

Cells that express KOC and p210 are compared to cells that only express p210, resistant to dexamethasone

Fig. 5: Overexpression of the embrvonal marker IGF-II in transgenic mice

Fig. 6: Use of KOC as a molecular biological tumor indicator

7: Detection of the embryonic-specific activation of the KOC promoter via Northem blot analyzes

Northern blot analysis of KOC expression in the development of murine embryos. Each lane contains 2 μg poly (A) ⁺ RNA from mouse embryos from different stages of development (days 7-17 postcoitum) (Fig. 7a).

KOC expression during human embryonic pancreatic development. Each lane contains 2μg poly (A) ⁺ RNA from human embryonic pancreas at different stages of development. The hybridization signals appear as smear due to the slight degradation of the mRNA. (Fig. 7b)

8: Results of the RT-PCR of different tissues. Result of an RT-PCR analysis with the primer pair 6. The total RNA from six different human healthy pancreatic tissues, six different chronic pancreatitis tissues, two healthy colonic tissues, one colon carcinoma tissue (1 ), a soft tissue sarcoma tissue (2), a gastric carcinoma tissue (3) and eleven pancreatic carcinoma tissues.

Fig. 9: DNA sequence of the KOC promoter

The following examples illustrate the invention. Example 1: Overexpression of KOC leads to apoptosis resistance in adult pre-B lines

A well-studied model system to study development processes are murine pre B cells. Murine pre B cells were isolated from either adult bone marrow or fetal liver. The tibiae and femura of mice were isolated to isolate the adult bone marrow. Then they were sprayed with medium (DMEM, Life Technologies, Karlsruhe, Germany). To isolate the fetal progenitor cells, the liver was isolated from day 15 mouse embryos. This was then ground through a sterile cell culture sieve, releasing the individual blood precursor cells. 1 X 10 ⁷ cells per 6 cm were cultivated from adult bone marrow and fetal liver. Both the adult cells and the fetal cells could be cultivated comparatively easily in vitro and thus analyzed for differences in gene expression and for functional differences. Different in vitro culture systems were available for both cell populations.

One system was to cultivate the cells on a stromal cell line in the presence of the growth factor Interleukin 7 (R&D Systems, Wiesbaden, Germany). The current cell line ST2 was used in the present case. The cells were expanded until they were approximately 80% confluent. Then they were irradiated with 3000 rad. Cells treated in this way were growth-inactive, but still had a metabolism for a few days. Bone marrow or fetal liver cells were plated on this cell line with a cell density of 2 x 10 ⁶ cells with a concentration of 10ng IL7 per ml. Under these conditions, only pre-B cells grew. The cells were plated onto fresh stromal cells twice a week.

In a second system, the bone marrow or the fetal liver cells with the comparatively low tumorigenic oncogene p210 BCR-ABL transformed. This is a fusion protein that is produced by a chromosomal translocation, with parts of the BCR (break point dark region) gene from chromosome 22 fused to c-ABL (tyrosine kinase) on chromosome 9 (Philadelphia chromosome), resulting in the kinase c-ABL is activated. The translocation mentioned is associated with chronic myeloid leukemia. Retroviral infection was performed. For this purpose, p210 BCR-ABL was cloned into the retroviral vector pBMNZ (available from Garry Nolan, Stanford; vector map: http://www.stanford.edu/group/nolan/pmaps.htmp). 10 ⁷ cells were incubated with 3 ml of retroviral supernatant in the presence of 8 mg / ml polybrene in 50 ml conical tubes for at least 3 hours. The cells were then plated with IL7 on stroma for 48 hours. After 48 hours, IL7 was washed out and the cells were plated without IL7. Since BCR-ABL made the pre-B cells IL7 independent, only cells that had been infected with the retrovirus coding for BCR-ABL grew out (FIG. 3). A selection for eg G418 could therefore not be made. As mentioned, the BCR-ABL transformed cells were initially IL7-independent and later also stroma-independent. However, the cells transformed in this way were still able to differentiate B cells that mature in vivo.

It was previously known that adult pre B cells differ from fetal ones by the expression of two genes: the terminal deoxynucleotide transferase (TdT) and the "myosin light chain 2" (PLRLC) are expressed in adult pre B cells, but not in fetal ones , Known functional differences are that fetal pre B cells are resistant to steroids, while adult cells become apoptotic after steroid administration. Furthermore, fetal pre B cells can be kept in culture indefinitely in vitro, while the adult cells die after a few months of passage. We were able to show that KOC is expressed in fetal pre B cells but not in adult ones. With KOC there is a marker available to discriminate fetal pre B cells from adults. To study the functional consequences of KOC expression, adult pre B cells transformed with p210 BCR-ABL were infected with a retrovirus (pBMZ) encoding KOC. The infection was carried out analogously as described above for the retrovirus encoding the BCR-ABL. In this way, KOC overexpression was successful in the adult pre B cells. Compared to non-KOC expressing cells, the expressing cells were significantly more resistant to apoptosis, which was triggered by different stimuli. Resistance to steroids was the most impressive. While the cells expressing KOC proliferated uninhibited at a concentration of 10-6 M dexamethasone, all control cells died. It could also be shown that the DNA synthesis of the KOC / ρ210 cells in the tymidine incorporation assay is significantly stronger than that of the p210 cells (FIG. 4).

Example 2: Overexpression of KOC induces fetal gene expression

After the clear phenotypic differences between the two cell populations described in Example 1 could be observed, it seemed sensible to create a gene expression profile of these cells using the "Gene Chip" ® system from Affymetrix. This is an oligonucleotide array that is hybridized with different fluorescence-labeled RNA of the respective cell population. The specificity of the hybridization is ensured by the presence of 20 nucleotide probes per gene with perfect sequence agreement and 20 nucleotide probes with a base mismatch. Using this method, numerous genes could be identified that are differentially expressed. For example, Cyclin DI and IGF-II were only detectable in the cells that also expressed KOC in addition to p210. In the context described here, it is particularly worth mentioning that in the cells expressing KOC the marker genes for the adult preB cell phenotype TdT and PLRLC were completely downregulated. Since, as described above, these two markers are the only ones described which distinguish adult preB cells from fetal ones, it can be assumed that overexpression of KOC induces fetal gene expression. It should be emphasized that firstly the degree of differential change in the pancreas of transgenic mice clearly depends on the expression level of KOC and secondly that the embryonic markers of infected preB cells are upregulated analogously to KOC expression. This fact makes it possible to achieve corresponding dose effects in the respective organs by administering different amounts of KOC.

As a result of the above experiments, a selection of genes is given below which are differentially expressed by KOC in the cells expressing BCR-ABL:

36a

37

Example 3: Transgenic mouse model

In order to confirm the redifferentiation of hematopoietic cells shown in vitro on other cell types (e.g. on epithelial cells), the effect of overexpression of KOC in a transgenic mouse model was investigated.

For a controllable and ubiquitous overexpression, a mouse line was constructed in which the expression of KOC is under the control of the metallothio no promoter (Palmiter et al., Mol. Cell. Biol. 13 (9) (1993), 5266-5275 ). The induction of KOC transcription could be activated and controlled by applying zinc in drinking water.

In order to produce the transgenic mice, the coding cDNA of the human KOC gene was cloned in sense orientation directly behind the metallothionein promoter of the expression vector MT-LCR 2999B4 (Palmiter et al., 1993, Mol Cell Biol, 13, 5266-5275 ). The DNA construct (Dral fragment containing the complete open reading frame of KOC was cloned into the Nrul site of the MT-LCR2999B4) was cut with the restriction endonuclease Sall (eg from Röche, Penzberg). The cut construct was injected into the female pronucleus according to the standard protocol using a microinjection system (standard protocol described in: "The Molecular Biology of the Gene", James D. Watson et al., 4 ^th Ed., Transgenic Mice: Mice with new germ line genes, p. 814 ff.). The cells were then transferred to a pseudopregnant female. The animals which integrated the transgenic construct were identified by means of PCR and Southern blot analysis of the genomic DNA from tail tips of the 1-2 month old mice. These founders were crossed with B6 mice. The lines that inherit the transgene were determined by renewed PCR analysis of the genomic DNA of the offspring of this F1 generation. 02/20036

38

The genomic DNA was isolated from mouse tails using the "Qiagen tissue Kit" and was carried out according to the manufacturer's protocol (Qiagen GmbH, Hilden).

The PCR reactions took place under the following conditions:

PCR reaction: starting material 500ng genomic DNA or 100ng cDNA

Primer each 100ng (20pmol)

5x Mg ²⁺ free buffer, pH 9.5 10μl

10mM dNTPs 5μl

HotWax ™ beads 2.5mM gCI 1 piece

Taq polymerase (5U / μl) O.δμl ddH ₂ O ad 50μl

PCR conditions: time temperature number of cycles

Hot start 2 min 95 ° C 1x

Denaturation at 95 ° C for 30 seconds

Annealing 30 sec see below 35x

Extension 2 min 72 ° C

Final extension 10 min 72 ° C 1x

Primer: Koc2117for: 5'-GAGGACCAGGCAACTTTTG-3- (KOC-specific) hGHIrev: ₅ '-ACTGGAGTGGCAACTTCC-3' (from sequence section of the human growth hormone, serves to stabilize the mRNA)

HotWaxTM, Invitrogen, Groningen, Netherlands 39

Histological examinations of the pancreas of the mice showed significant changes in the organ structure. Tumors were also not observed in animals that express KOC very strongly in a one-year observation period (with a total age of the animals of about two years). The ectopic KOC overexpression also had no influence on the general condition / behavior of the mice. Finally, the embryonic markers upregulated in the pancreas of transgenic mice were checked immunohistologically. The RT-PCR data showed that, in addition to other genes, IGF-II, which is normally only expressed in the embryonic stage in mice, is also overexpressed in adult transgenic animals (FIG. 5).

Transgenic mice were characterized by Western blot analysis of various organs to identify the transgenic mice that express KOC most strongly. Two of these lines were used for breeding.

Production of Western blots: The proteins were separated in an SDS polyacrylamide gel and, according to the manufacturer, on a nitrocellulose membrane (OPTITRAN BA-S83, Schleicher & Schüll, Dassel) in an electroblot process (Sammy dry Schleicher & Schüll, Dassel). transferred. In order to prevent non-specific binding of the antibodies, the blots were incubated with 5% skimmed milk powder in TBS 30min at 37 ° C. and then washed three times with TBS. The incubation with the anti-KOC rabbit serum was carried out for 2 hours at RT in TBS with 0.2% BSA and 0.01% Tween 20. After washing three times with TBS, the blots were incubated with the second anti-rabbit antibody coupled to peroxidase (from Amersham ) incubated for 1h at RT. The detection was carried out after washing three times with TBS using the ECL Western blotting detection reagents (from Amersham).

Example 4: Differential diagnosis of malignant tumors compared to normal or inflammatory tissue 40

Tissue samples were used that were obtained from tissues / tumors of unclear dignity in the liver and lymph nodes using fine needle puncture. Control samples were taken from inflammatory areas of the colon. The biopsies were taken up in lysis buffer (either standard guanidinium buffer or RLT buffer from Qiagen, Hilden, Germany) and mechanically comminuted.

The total RNA was extracted using the "RNAeasy Kit" from Qiagen, Hilden. This was followed by a DNase digestion. An RT-PCR was carried out directly with the "One-Step RT-PCR Kit" (from Qiagen, Hilden): Primers used:

KOC primer: 5'-TGCAGTTTCCGAGTCAGTGT-3 '

5'-ATATCGGAAACCTCAGCGAG-3 'Annealing temperature: 55 ° C, 35 cycles

5 ml of the respective PCR mixture were applied to a 1% agarose gel. The result is shown in Fig. 6. It should be noted that no band can be seen in lane 1 which would indicate a tumorous event, but rather represents a "smear" due to overloading of the lane. Corresponding KOC bands are only visible in lanes 3 and 4, so that the presence of a tumor can be assumed here.

Example 5: Identification of the DNA sequence for the KOC promoter.

The KOC gene is located on chromosome 7 in the 7p11.5 region. In this respect, a chromosome 7 specific genomic library (vector Lawrist 4) was screened with a KOC probe. The DNA of positive clones was digested with different restriction enzymes and separated by gel electrophoresis. The gel was blotted using standard procedures. The DNA fixed on the nylon membrane was hybridized with a radioactively labeled 5'-KOC probe. 41

A positive 7 kb fragment was then cloned into the vector Bluescript KS (Pharmacia). The clone was deposited on November 1 with the DSMZ (German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig) under the entry number DSM 13810. The clone was also sequenced. The first 1946 bp upstream from the ATG are shown in FIG. 9. The binding sites of transcription factors such as SP1, SRF, TFIID / TBF, AP1, AP2, BPV-E2, c-Myb, c / EBP, E1A-F, GATA-1, HNF-5, IBP- are located on this DNA sequence. 1, NF-IL6, NF-kB, SP1, T-antigen, TCF-1, v-Myb etc.

A sequence comparison of the DNA sequence of FIG. 9 with an international genomic sequence database (NCBI, National Center for Biotechnology Information) showed that sequence homologies of approximately 98% are located on chromosome 4 and 7. It can be assumed that the KOC promoter is located on chromosome 7 as a duplicate on chromosome 4, with numerous chromosomal in situ hybridizations and the presence of classic exon-intron structures proving that the functional KOC gene is located on chromosome 7. Experiments with restriction endonucleases or targeted deletions (e.g. with the Erase-a-base kit from Promega) and subsequent subcloning or connection of the subfragments obtained with reporter genes, cf. the above explanations help to clarify the structure of the entire deposited clone. DNA sequences that had the smallest possible KOC promoter region were also determined.

Example 6: The KOC promoter is only expressed in malignant tissues and tumor cell lines.

It could be shown that the KOC promoter is only expressed in malignant tissues and tumor cell lines (eg colon and pancreas). No KOC transcripts could be detected in human and also murine adult tissues. 42

Due to the fact that a large number of KH proteins play an essential role during embryogenesis, it was assumed that KOC could also play a role during embryogenesis. Northern blot analysis with poly A ⁺ RNA of mouse embryos from different developmental stages showed that KOC is most strongly expressed on day 11 of the murine development (FIG. 7a). It was shown that KOC expression decreased rapidly during advanced stages of development. In order to obtain the first indications for the function of KOC in organogenesis, hybridizations were carried out in situ on mouse embryos of different stages. It was shown that KOC is most strongly expressed at the time of organogenesis, namely in the CNS, intestine, pancreas, thymus, kidney and hematopoietic organs. During later stages of mouse development, KOC expression is limited to the thymus and intestinal epithelium, with KOC mainly found in the intestinal crypts. KOC expression is completely switched off two days after birth. Due to the differential gene expression of KOC in pancreatic carcinoma, the expression of the protein was studied in human pancreatic development. For this purpose, poly (A) ⁺ RNA from human fetal pancreatic tissues from "The Genetics Institute", Boston, USA was used. This was separated by gel electrophoresis and blotted using standard methods. The Northern blot was then hybridized with a radiolabelled human KOC probe. It was shown that KOC is clearly expressed during the 12th week of pregnancy. The highest KOC expression was observed in the 18th week of pregnancy (FIG. 7b).

Finally, the transcript detection was carried out by RT-PCR, which showed that the KOC promoter is activated only in pancreatic carcinoma tissue, but not in the pancreas in chronic pancreatitis or in healthy pancreas. For this purpose, samples were taken from various tissues and in liquid nitrogen immediately after the surgical removal 43

snap frozen and stored at -70 ° C. The tissue was pulverized in a sterile mortar in liquid nitrogen for the extraction of the total RNA. The RNA extraction from this human pancreatic tissue was carried out with the RNAeasy Midi Kit according to the manufacturer's protocol (Qiagen). After a DNase I digestion, the mRNA was rewritten directly into cDNA. For this purpose commercially available reverse transcriptases (e.g. Superscript II RT, Gibco BRL) were used according to the manufacturer's protocol. To adjust the amount of cDNA to be used, the primers of the acidic ribosomal phosphoprotein (GenBank (NCB) xs13, Acc.Nr .: Z36785) were used for an internal control PCR. This protein is known to be present in normal, inflammatory and malignant pancreatic Tissue is expressed equally strongly (forward primer: 5'CTGGCTAAGTTGGT TGCTTT-3 '; reverse primer: 5'-GCAGCT-GATCAAGACTGGA-3'; attachment temperature 58 ° C; 18 cycles).

The following KOC primers were used for the actual KOC RT-PCR: Forward primer: 5'-TGCAGTTTCCGAGTCAGTGT-3 '; Reverse primer: 5'- ATATCGGAAACCTCAGCGAG-3 '; Annealing temperature 55 ° C; 35 cycles). The amplified DNA fragments were visualized by gel electrophoretic separation in an ethidium bromide-stained agarose gel under UV light. The results are shown in Figure 8. The following findings were obtained for the individual tissue samples:

Normal pancreatic tissue: 0/6 positive; Chronic pancreatitis: 0/6 positive; Healthy colon tissue: 0/2 positive; Various tumors: 2/2 positive; Pancreatic carcinoma tissue: 7/7 positive. 44

Example 7: Regression of a tumor by administration of an adenovirus with a cDNA encoding TNF-alpha which is under the control of the KOC promoter

An adenovirus was used as a vector to investigate the possible suitability of the KOC promoter for gene therapy treatment of a tumor. For this purpose, the KOC promoter sequence was functionally cloned by homologous recombination into the adenoviral vector adenovirus type 5 (ATCC, VR-5), so that the E1A gene (which is essential for the replication of the adenovirus) was selectively only in KOC-positive cells is expressed (Ad.KOC-E1) In addition, the TNF-alpha cDNA was cloned into this construct under the control of the KOC promoter (Ad.KOC-E1 / KOC-TNF); this procedure is described in Kurihara et al. (J. Clin. Invest. 106: 763-771, 2000). The virus was produced by infection in the human embryonic kidney cell line (HEK293, ATCC). For the first investigations, tumor cells (panic carcinoma cells Panel or MiaPaCa2; available from the Amercican Type Culture Collection (ATCC)) were injected subcutaneously into the flanks of athymic nude mice (strain NMR nu / nu I). The tumors were grown to a size of 1 cm. A clear regression of the tumor was observed after intratumoral injection of the Ad.KOC-E1 / KOC-TNF construct. The antitumor effect of this adenoviral construct was confirmed in the histopathological analysis.

These results were also confirmed in vitro. Cells in which the KOC promoter has been shown to be active (human pancreatic carcinoma cell line Panel and MiaPaCa2 (American Type Culture Collection, ATTC)) were infected with the vector construct described above. These tumor cells underwent apoptosis compared to cell lines that do not express KOC (NIH 3T3 fibroblasts, ATCC).

Furthermore, an adenovirus in which the herpes simplex thymidine kinase 45

(HSV-tk) is under the control of the KOC promoter (Ad.KOC-tk, adenovirus type 5) is used to form the HSV-tk after infection with this virus in KOC-expressing tumor cells (see above) and with simultaneous administration of ganciclovir, this non-toxic substance was converted into a toxic substance by phosphorylation, as a result of which the tumor cells were destroyed.

Example 8: Use of KOC as a diagnostic tool to discriminate benign and malignant lesions from biopsy material

Various biopsy materials (tissue / cells) were taken up directly in RNA lysis buffer (50 g guanidinium thiocyanate, 2.5 ml 1M sodium citrate pH 7.0, 0.5 g N-lauryl sarcosine, 0.7 ml 2-mercaptoethanol in 100 ml H ₂ O). The total RNA was isolated from the biopsies using the "RNAeasy Mini Kit" (Qiagen). The following RT-PCR reaction was carried out using the "One-Step RT PCR Kit" (from Qiagen). The PCR conditions were as follows:

KOC primers used: reverse: 5'-TGCAGTTTCCGAGTCAGTGT-3 'forward: δ'-ATATCGGAAACCTCAGCGAG-S' annealing temperature: 55 ° C, 35 cycles

The amplified DNA fragments are visualized by gel electrophoretic separation in an ethidium bromide-stained agarose gel under UV light.

The results are summarized in Table 1. 46

Example 9: Stimulation of CD8 ⁺ T cells against the KOC protein and lysis of carcinoma cells expressing the KOC protein.

(A) Stimulation of CD8 ⁺ T cells against the KOC protein

Peripheral mononuclear cells are obtained from a healthy donor and subjected to a so-called ELISPOT analysis. The principle of this experiment is that lymphocytes in culture vessels are stimulated with specific antigens. If the lymphocytes are activated because they recognize the antigen, the activated lymphocytes release cytokines, which in turn bind to specific antibodies which are immobilized on the bottom surface of the culture vessels. After the lymphocytes have been washed out, the bound cytokines can then be detected in the culture vessels with the aid of a second antibody, which is made visible in a subsequent color reaction.

Peripheral blood lymphocytes (PBL) were purified from an HLA-A0201 positive healthy subject by density centrifugation using a Ficoll Paque ^® gradient. T-lymphocytes were obtained by separating the B-lymphocytes or the monocytes using antibody-coupled magneto beads (CD11, CD16, CD19, CD36 and CD56) (Pant T cell isolation kit ^* , Milteny, Bergisch Gladbach, Germany). About 2 x 10 ⁷ T cells were obtained from 30 ml of blood.

HLA-A0201 restricted peptides of the KOC protein were identified using a software system from the NIH (NIH bioinformation Service [httpJbimas.dcrt.nih.gov.cgi- bin / molbio / ken_pa rker_comboform). The peptides were as follows: 47

9mer peptides: 10mer peptides:

199 RLLVPTQFV 33 FLVKtGYAFV

280 KILAHNNFV 142 FQLEnFTLKV

552 KIQEILTQV 26 KIPVsGPFLV

The isolated T cells were incubated with T2 cells which were (a) loaded with a mixture of the above 9mer peptides (10µg / peptide) and (b) with a mixture of the above 10mer peptides (10µg / peptide). The T cells were restimulated weekly for 6 weeks. In each case 10 ⁷ T cells were cocultivated with 2 × 10 ⁶ peptide-loaded T2 cells in 24 perforated plates.

The reactivity to the peptide-loaded T2 cells was determined weekly, starting on day 0 of the experiment, by performing an IFN-γ Elispot analysis. On day 28, reactivity was observed by the mixture of (a) (400 specific cells per million cells). The main reactivity was directed against the peptide 199 (1000 specific cells / 1,000,000 cells). Less activity was observed against the mixture of (b) (150 specific cells / 1,000,000 cells). Here the peptide 33 showed the highest reactivity (600 specific cells / 1,000,000 cells).

Thus it became clear that CD8 ⁺ T cells activated against the KOC protein can be stimulated.

(B) Lysis of carcinoma cells expressing the KOC protein

After a further restimulation, the activated CD8 ⁺ T cells were incubated with the HLA A0201 + pancreatic carcinoma cell panels, which express the KOC protein. The kidney cells HEK293 were used as controls 48

det that do not express the KOC protein. 10 ⁶ panel cells were labeled with ⁵¹ Cr (100μCi) for 1 h at 37 ° C and co-cultivated with increasing numbers of activated CD8 ⁺ T cells for 3 hours. The specific lysis of the panel cells was determined by the amount of radioactivity released in the supernatant.

It was shown that panel cells are lysed by the activating CD8 ⁺ T cells, but not the control cells HEK293.

Patient- KOC-RT- Histo / NR. PCR Cyto HISTO / CYTO ok lmmunhisto: CEEA pos, Cytoker18 pos, poorly differentiated squamous cell carcinoma ok Histology: poorly differentiated mucus adenocarcinoma wπ SVi-s Crohn's disease ok Cytology: malignant epithelial neoplasia ok Cytology: malignant epithelial neoplasia ok

11 Cytology: poorly differentiated epithelial. neoplasia; PaCa metastasis

12 ok cytology: malignant epithelial neoplasia

13 ++ ok histo negative! Cyto: negative, laparascopic histo: follicular. lymphoma

131! ok IHisto negative! Cyto: negative, laparascopic histo: follicular. lymphoma

14 ok 1. Histo: normal, cytology: dysplastic liver parenchyma, HCC

Ψm. m no malignancy

16 TTT ok Qsophageal carcinoma

17 ok Cyto: inconspicuous, CT-controlled puncture: epithelial neoplasia in the liver, necrotic disintegration no malignancy

19 ++ ok rectal cancer

20 ok, s.B. Cyto: inconspicuous, inoperable BronchialCA with liver space requirement

21 ++ ok Cyto: malignant epithelial neoplasia, mucus-forming AdenoCA

22 ok epithelial malignant neoplasia, ColoCA compatible, metastasis

23 +++ ok colon cancer

24 (+) _____ Hemogioma, heavily bloody aspirate

25 ++ ok cytology: malignant epithelial neoplasia no malignancy

27 ++ CEA strongly positive, diagnosis currently questionable

28 ok Cyto: malignant epithelial neoplasia, little differentiated

29 ok 1. Histo: negative, 2. Cyto: moderate. differentiated adenocarcinoma

Table 1

Table 1 (continued)

Claims

50Patentansprüche

1. DNA sequence of the KOC promoter, comprising the nucleic acid sequence shown in FIG. 9 or a different nucleic acid sequence, the biological function of which essentially corresponds to that of the KOC promoter.

2. Medicament containing the DNA sequence according to claim 1 or a DNA sequence which codes for an RNA-binding protein KOC with the amino acid sequence shown in Figure 1.

3. Medicament according to claim 2, wherein the DNA sequence contains the nucleic acid sequence shown in Figure 1.

4. Medicament containing a DNA sequence which codes for a protein with the biological properties of an RNA-binding protein KOC encoded by the DNA sequences according to claim 2 or 3:

(a) which differs from a DNA sequence of claim 3 in the codon sequence due to the degeneration of the genetic code;

(b) hybridizing with a DNA sequence of claim 3 or 4 (a); or

(c) which is a fragment, an allelic variant or another variant of the DNA sequence of claim 3, 4 (a) or 4 (b).

5. Medicament containing an expression vector which comprises a DNA sequence according to any one of claims 1 to 4.

6. Medicament containing the RNA-binding protein KOC or a protein 51

with its biological activity, which is encoded by the DNA sequence according to any one of claims 2 to 4.

7. Medicament according to one of claims 2-6, wherein the protein and / or the DNA sequence are present in an amount suitable for the immunization of an individual.

8. Diagnostic method for the detection of a tumor associated with the expression of KOC, in which a sample is used with a probe suitable for specific hybridization with a probe transcribed from the DNA sequence according to one of claims 2 to 4 or a primer or a KOC protein according to claim 6 or an antibody against the KOC protein defined in claim 6 or a fragment thereof in contact and then directly or indirectly determines whether the concentration of KOC, KOC mRNA, KOC protein and / or anti-KOC antibody in the sample compared to a control sample.

9. Diagnostic method according to claim 8 for differential diagnosis between chronic pancreatitis and pancreatic carcinoma.

10. Diagnostic method according to claim 8 for the diagnosis of premalignant lesions in unclear masses.

11. Diagnostic method according to claim 8 for risk stratification in premalignant lesions.

12. Diagnostic method according to claim 8 for evaluating therapeutic success. 52

13. Diagnostic kit for carrying out the method according to claim 7 or 8, which is suitable for specific hybridization with a probe which is transcribed from the DNA sequence according to one of claims 2 to 4, or a primer or a KOC protein according to claim 6 and / or contains an anti-KOC antibody or a fragment thereof.

14. Use of a compound defined in one of claims 2 to 6 for the artificial induction of the pluripotency of body cells.

15. Use of a compound defined in one of claims 2 to 6 for the production of tissues or organs via the differentiation of stem cell populations.

16. Use of a compound defined in one of claims 2 to 6 for high-dose chemotherapy.

17. Use of a compound defined in one of claims 2 to 6 for the improvement of the ex vivo expansion of hemotopoietic stem cells.

18. Use of a compound defined in one of claims 2 to 6 for the improvement of the engraftment in allogeneic bone marrow transplants.

19. Use of a compound defined in one of claims 2 to 6 for slowing down and / or reversing aging processes induced by physiological or physical noxae.

20. Use of a compound defined in one of claims 2 to 6 for producing a prophylactic effect in chemo- or 53

Radiotherapy.

21. Use according to claim 20 for producing a prophylactic effect in the case of exposed skin.

22. Use of a compound defined in one of claims 2 to 6 for the regeneration of skin defects, skin cosmetics or for accelerated wound healing

23. Use of a compound defined in one of claims 2 to 6 for the immunization of an individual against malignant tumors and their precursors.

24. Use of the DNA sequence of the KOC promoter or a different nucleic acid sequence according to claim 1 for the isolation and / or enrichment and / or selective proliferation of stem cells.

25. Use of the DNA sequence of the KOC promoter or a sequence differing therefrom according to claim 1 for determining the degree of differentiation or degree of dedifferentiation of a cell or a tissue.

26. Use of the DNA sequence of the KOC promoter or a different nucleic acid sequence according to claim 1 for gene therapy cancer treatment.

27. A diagnostic kit for use according to claim 25, which contains a probe or a primer suitable for specific hybridization with a DNA sequence according to claim 1. 54

28. A method for identifying compounds that bind to the DNA sequence of the KOC promoter according to claim 1 and modulate its activity, the method comprising the following steps: a) contacting a DNA sequence according to claim 1 with a test compound in a cellular assay; and b) determining the modulation of the activity of the KOC promoter, where modulation indicates that the test compound is active.