EP1349871A2 - Novel marker for the diagnosis and therapy of tumours - Google Patents

Abstract

The invention relates to a novel marker for tumours, preferably CTCL. The invention further relates to the application of the above for the diagnosis and therapy of tumour-related diseases, preferably CTCL.

Description

 New markers for the diagnosis and therapy of tumors

The present invention relates to the use of new markers for the diagnosis or therapy of tumor diseases, preferably CTCL.

Cutaneous T-cell lymphomas (CTCL) represent a heterogeneous group of diseases in which CD4-T cells predominate as the malignant cell type. In most cases, the mono- or at least oligoclonal origin of the malignant cells is documented using T-cell receptor rearrangements. In addition to various other subtypes, mycosis fungoides and Sezary syndrome (SS) are the most common forms of CTCL. Both diseases are monoclonal T-helper memory lymphomas that are characterized by cutaneous plaques, tumors or erythroderma. SS is also characterized by generalized lymphadenopathy and the presence of neoplastic T cells in peripheral blood.

Therapeutic approaches include the stage-dependent selection of PUVA (psoralen and UV-A), retinoids, interferon α-2a in combination with acitretin or PUVA, various immunomodulators, electron radiation or extracorporeal photopheresis. These procedures are successful in the early stages of the disease, but not in the aggressive later stages. Possible useful future therapies for CTCL include immunological therapies, e.g. Vaccination with peptides or peptide-loaded dendritic cells as has already been used to treat melanoma.

The presence and activity of CD8 ⁺ cells in CTCL was correlated with the prognosis. It could be shown that CD8 ⁺ - reactive infiltrates are CTCL specific and lytic. Thus, immunotherapies may be a promising concept for the treatment of CTCL, but a prerequisite for such a strategy is the identification of tumor-specific antigens. In this context, the T cell receptor itself has been proposed as an antigen (similar to the idiotype immunoglobulins as a target for B cell specific T cells). In In both cases, however, one is faced with the disadvantage that the antigen T cell receptor would have to be identified for each individual patient. In summary, however, it must be stated that no tumor-associated antigens are currently known for tumor types such as CTCL, so the possibilities of specific diagnosis or therapy are severely limited.

The present invention is therefore essentially based on the technical problem of identifying and providing markers (genes or their products) which are associated with tumors, in particular CTCL, and which may be of diagnostic and / or therapeutic benefit in the context of vaccination therapy ,

The solution to this technical problem has been achieved by providing the embodiments characterized in the patent claims.

Surprisingly, a number of genes have been found, the expression of which is correlated with CTCL. In the experiments leading to the present invention, CTCL-specific antigens were identified by screening a testis cDNA bank or a cDNA bank which had been produced from tumor RNA from various cutaneous lymphomas with sera from tumor patients. About 3 x 10 ⁶ recombinants were screened with sera from patients with Sezary syndrome or mycosis fungoides. The results show that tumor antigens from CTCL tumors can be identified with antibodies derived from tumor patients. It was possible to identify positive clones belonging to 19 different genes / ORFs, including five previously unknown sequences. All tumor antigens found are specific, that is to say that only tumor patients, but not healthy individuals, form antibodies directed against these, although 13 of these tumor antigens are expressed in at least 21% of the control tissues tested. A tumor-specific antigen was also found that is only expressed in testis and tumor tissues. This is se2-1, which was found in a CTCL tumor. This gene shows Similarities to SCP-1, a mitosis-related protein. Four sera from CTCL patients reacted with different SCP-1-like clones. Thus, by means of the experiments leading to the present invention, CTCL-associated antigens (independent of the T cell receptor) could be identified for the first time, which thus represent valuable tumor markers. The identification of such antigens is of interest since the encoded proteins and peptides derived therefrom serve as target structures, for example for cytotoxic cells, and can be used as antigens for the production of diagnostic or therapeutic antibodies. For tumor therapy, the peptides or fragments thereof encoded by the nucleic acids according to the invention can either be applied directly or loaded onto antigen-presenting cells. The peptides representing antigens can also be expressed with the aid of vectors in different cells (eg dendritic cells as antigen-presenting cells). The identified nucleic acids also form the basis for the development of diagnostic tests in order to ensure a more reliable and early diagnosis of those affected in the future. In addition, functional analyzes of the proteins will undoubtedly contribute to an understanding of tumor development. The nucleic acids according to the invention are thus to be regarded as candidate genes for studies of the pathomechanisms which underlie various tumor diseases, such as, for example, CTCL.

The present invention thus relates to a diagnostic composition which has at least one

Contains nucleic acid sequence, the altered expression is associated with a tumor disease, the

Nucleic acid sequence se2-5 (Fig.l), se20-10 (Fig.), Se57-l

(Fig.3), se70-2 (Fig.4), Lgl-2 (Fig. 5), sel-1 (Fig. 6), se2-l

(Fig.7), se2-2 (Fig.8), sel4-3 (Fig.9), se20-4 (Fig.10), se20-7

(Fig.11), se20-9 (Fig.12), se33-l (Fig.13), se37-2 (Fig.14), se89-l (Fig.15), L14-2 (Fig.16) , L15-7 (Fig.17), Li9-1 (Fig.18),

Li9-4 (Fig. 19), LÜ5-2 (Fig. 20), LiilO-6 (Fig. 21), Liii4-5

(Fig. 22) or GBP-TA (Fig. 23). The present invention furthermore relates to a medicament which contains a nucleic acid sequence, the altered expression of which is associated with a tumor disease, the nucleic acid sequence se20-10 (FIG. 2), se57-l (FIG. 3), Lgl-2 ( Fig. 5), sel-1 (Fig. 6), se2-l (Fig.7), se2-2 (Fig.8), sel4-3 (Fig.9), se20-4 (Fig.10), se20-7 (Fig.11), se20-9 (Fig.12), se33-l (Fig.13), se37-2 (Fig.14), L14-2 (Fig.16), L15- 7 (Fig .17), Li9-1 (Fig.18), Li9-4 (Fig.19), LÜ5-2 (Fig. 20), LiilO-6 (Fig. 21), Liii4-5 (Fig. 22) or GBP -TA (Fig. 23).

The present invention further relates to a diagnostic composition or a medicament defined above, the nucleic acid sequence, the altered expression of which is associated with a malignant tumor disease, comprising a nucleic acid sequence,

(a) which differs from a preceding nucleic acid sequence shown in FIGS. 1 to 23 in the codon sequence due to the degeneration of the genetic code;

(b) which hybridizes with a nucleic acid sequence according to one of FIGS. 1 to 23 or according to (a); or

(c) which is a fragment, an allelic variant or another variant of one of the nucleic acid sequences defined above.

The term “hybridizing nucleic acid sequence” refers to a nucleic acid sequence which hybridizes with a nucleic acid sequence shown in the figures under usual conditions, in particular at 20 ° C. below the melting point of the nucleic acid. The term "hybridize" used in the present invention refers to conventional hybridization conditions, preferably to hybridization conditions in which 5xSSPE, 1% SDS, lxDenhar dts solution is used and / or the hybridization temperature between 50 ° C. and 70 ° C, preferably 65 ° C. After hybridization, washing is preferably carried out first with 2xSSC, 1% SDS and then with 0.2xSSC at temperatures between 50 ° C and 70 ° C, preferably at 65 ° C (for the definition of SSPE, SSC and Denhardts solution see Sa brook et al. , Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor NY (1989)). Stringent hybridization conditions, as described, for example, in Sa brook et al. , supra.

The terms "other variant" or "fragment" used in the present invention encompass nucleic acid sequences which differ from the sequences indicated in the figures or the above hybridizing sequences by deletion (s), insertion (s), exchange (s) and / or others Distinguish modifications known in the prior art or comprise a fragment of the original nucleic acid sequence, the protein encoded by these nucleic acid sequences also having one or more of the properties described above or in the examples. This also includes allele variants. The variants have a homology to the claimed sequences of at least 70%, at least 80%, preferably at least 90%, very preferably at least 95%, 96%, 97%, 98% or 99%. Methods for generating the above changes in the nucleic acid 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 nucleic acid sequence modified in this way still has the desired biological properties. Especially in connection with diagnostic applications or compositions in which one of the above nucleic acid sequences is used, the term "fragment" refers to a fragment that has a length of at least 12, preferably at least 20 and even more preferably at least 25 nucleotides.

In a preferred embodiment, the nucleic acid olefin defined above is a cDNA.

In a further preferred embodiment, the nucleic acid sequence is a genomic DNA which is preferably derived from a mammal, for example a human. Screening methods based on nucleic acid hybridization allow the Isolation of the genomic DNA molecules according to the invention from each organism or derived genomic DNA banks, using probes which contain the nucleic acid sequence shown in the figures or a fragment thereof.

The nucleic acid sequences can also be inserted into a vector or expression vector. Examples of such vectors are known to the person skilled in the art. In the case of an expression vector for E. coli, these are e.g. B. pGEMEX, pUC derivatives (e.g. pUC8), pBR322, pBlueScript, pGEX-2T, pET3b and pQE-8. For expression in yeast e.g. To name pYlOO and Ycpadl, while for expression in animal cells e.g. pKCR, pEFBOS, cDM8 and pCEV4 must be specified. The baculovirus expression vector pAcSGHisNT-A is particularly suitable for expression in especially insect cells. In a preferred embodiment, the nucleic acid sequence in the vector is functionally linked 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 A0X1 or GALl promoter in yeast, and the CMV, SV40 , RVS-40 promoter, CMV or SV40 enhancer for expression in animal cells. Further examples of suitable promoters are the metallothionein I and the polyhedrin promoter.

Suitable vectors include in particular T7-based expression vectors for expression in bacteria

(Rosenberg et al., Gene _56, (1987), 125) or pMSXND for expression in mammalian cells (Lee and Nathans, J. Biol. 263

(1988), 3521).

General methods known in the art can be used to construct vectors or plasmids containing the above nucleic acid sequences and suitable control sequences be used. These methods include, for example, in vitro recombination techniques, synthetic methods and in vivo recombination methods, as described, for example, in Sambrook et al. , supra.

Host organisms can be transformed with the vectors described above. These transformants include bacteria, yeast, insect and other animal cells, preferably mammalian cells. The E. coli strains HB101, DHl, xl776, JM101, JM109, BL21, XLlBlue and SG 13009, the yeast strain Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, HeLa and the insect cells sf9 are preferred. Methods for transforming these host cells, for phenotypically selecting transformants, and for expressing the above nucleic acid sequences using the vectors described above are known in the art.

The above-mentioned nucleic acids are particularly suitable as an antigen-coding structure for therapeutic purposes. The aim is to stimulate the immune system, to eliminate tumor cells that are identified by a nucleic acid. There are different ways, e.g. B. Injecting the naked DNA into the patient. For this purpose, a plas id with a very active promoter and at least one nucleic acid according to the invention, which in particular se20-10, se57-l, Lgl-2, sel-1, se2-l, se2-2, sel4-3, se20-7, se20-9, se33-l, se37-2, L14-2, L15-7, Li9-1, Li9-4, LÜ5-2, LiilO-6, Liii4-5 or GBP-TA, for example in the muscle or injected intradermally.

Furthermore, the nucleic acid sequence can not only be inserted into a vector for the purposes of recombinant production, but also to inject the DNA with the aid of vectors into patients, where it encodes an antigen for therapeutic purposes. The aim is that the cells take up the plasmid, produce antigens, present individual peptides via HLA molecules and thus elicit a cytotoxic T-cell immune response. This should then lead to the defense against tumor cells. In general, this procedure is described in Conry et al. , Clinical Cancer Research 4, pp. 2903-2912 (1998). a an alternative way is the "gene gun" method, which is described in Fynan et al., Proc. Natl. Acad. Be. USA 90, pp. 11478-11482 (1993). The nucleic acid is brought to the HLA presentation of the encoded protein with the aid of a vector in vivo antigen-presenting cells (APCs), for example dendritic cells. The vector containing the nucleic acid according to the invention can be injected in various ways: a) Lipid or liposome-packed DNA or RNA, for example generally described by Nabel et al. , Proc. Natl. Acad. Be. USA 93, pp. 15388-15393 (1996). b) With a bacterium as a transport vehicle for the expression vector. Suitable bacteria are, for example, (attenuated) listeria [eg Listeria monocytogenes], Salmonella strains [eg Salmonella spp.]. In general, this technique is from Medina et al. , Eur. J. Immuno1. 29., pp. 693-699 (1999) and Guzman et al., Eur. J. Immuno1. 28, pp. 1807-1814 (1998). Furthermore, WO 96/14087; Weiskirch et al., Immunological Reviews 158, pp. 159-169 (1997) and US-A-5, 830, 702. c) Using Gene-Gun (Williams et al., Proc. Natl. Acad. Sci. USA 88 / pp. 2726-2730, 1991).

In a preferred embodiment, the vector containing the above nucleic acid sequences is a virus, for example an adenovirus, vaccinia virus or an AAV virus, which is useful in gene therapy. Retroviruses are particularly preferred. Examples of suitable retroviruses are MoMuLV, HaMuSV, MuMTV, RSV or GaLV. The aforementioned viruses and fowlpox virus, canarypox virus, influenza virus or Sindbis virus are also suitable as the basis of a vaccine. Such new vaccines which confer anti-tumor immunity after administration to the patient are described, for example, by N. Restifo, Current Opinion in Immunology 8, pp. 658-663 (1996) or Ying et al. , Nature Medicine 5 (7), p. 823 ff., (1999). For the purposes of gene therapy, the above nucleic acid sequences 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). It is further preferred to generate tumor immunity, to transfect a nucleic acid sequence above into antigen presenting cells and to inject them into the patient. Here, a plasmid is placed in vitro in an antigen-presenting cell (APCs), for example dendritic cells, which then produce antigens and present individual peptides via HLA molecules. The plasmid DNA can be brought into the antigen-presenting cells in various ways:

(a) as naked DNA, e.g. using "Gene Gun" or electroporation;

(b) DNA or RNA packaged as lipid or liposomes (Nair et al., Nature Biotechnology 16, pp. 364 ff. (1998));

(c) with a virus as a vector (Kim et al., J. of Immunotherapy 20 (), pp. 276-286 (1997));

(d) with a bacterium as a transport vehicle for the expression vector (Medina et al., Eur. J. Immuno1. 29, pp. 693-699 (1999); Guzman et al., Eur. J. Immuno1. 28, p. 1807-1814 (1998))

The protein encoded by a nucleic acid above that is associated with the presence of a malignant tumor disease can also be produced. The method preferably used for this comprises culturing the host cells described above under conditions which allow expression of the protein (preferably stable expression) and obtaining the protein from the culture. Suitable methods for the recombinant production of the protein are generally known (see, for example, Holmgren, Annu. Rev. Bioche. 54 (1985), 237; LaVallie et al., Bio / Technology 11 (1993), 187; Wong, Curr.Opin.Biotech . 6 (1995), 517; Romanos, Curr.Opin.Biotech. 6 (1995), 527; Williams et al., Curr. Opin. Biotech. (1995), 538; and Davies, Curr. Opin.Biotech. 6 (1995), 543. Also suitable purification methods (for example preparative chromatography, affinity chromatography, for example immunoaffinity chromatography, HPLC etc.) are generally known. In this connection it should be mentioned that the above-mentioned protein according to the usual in the art known, can be modified. These modifications include Exchanges, insertions or deletions of amino acids that modify the structure of the protein, while essentially maintaining its biological activity. The exchanges preferably include "conservative" exchanges of amino acid residues, ie exchanges for biologically similar residues, for example the substitution of a hydrophobic residue (for example isoleucine, valine, leucine, methionine) for another hydrophobic residue, or the substitution of one polar residue for another polar residue (eg arginine against lysine, glutamic acid against aspartic acid etc.) Deletions can lead to the generation of molecules which are significantly smaller in size, ie which lack amino acids at the N or C terminus, for example.

Injections of at least one of the proteins or one or more peptides derived therefrom are also suitable for the desired anti-tumor vaccination. For this, HLA-dependent peptide fragments are determined from the sequence of the protein according to the invention either by means of appropriate computer programs or by means of experiments (e.g. phagocytotic uptake of the total protein, then analysis of the presenting peptides). These are artificially produced using methods known to those skilled in the art and then injected into the patient (possibly with factors stimulating the immune system, e.g. interferons, interleukins, etc.). The aim of this treatment is that the APCs take up the peptides, present them and thus stimulate the production of tumor-specific cytotoxic T cells in vivo. In general, this principle has been described by Melief et al., Current Opinion in Immunology 8, pp. 651-657 (1996).

As described above, instead of the vector, the above protein or fragments thereof can also be loaded onto APCs in vitro. The loaded cells are then injected into the patient's lymph nodes, for example, and directly stimulate and multiply tumor-specific cytotoxic T cells (Nestle et al., Nature Medicine 4 (3), p. 328 ff. (1998); Schadendorf et al., In: Burg, Dummer, Strategies for Immunointerventions in Dermatology, Springer Verlag, Berlin Heidelberg, pp. 399-409, 1997) For vaccination, it may be advantageous to modify individual amino acids as described above compared to the wild-type antigen, since this may result in an increase in binding and an improvement in effectiveness (Clay et al., The Journal of Immunology 162, pp. 1749-1755, 1999).

Particularly preferred for the above therapeutic measures is at least one nucleic acid sequence which contains the nucleic acid sequence se20-10 (FIG. 2), se57-l (FIG. 3), Lgl-2 (FIG. 5), sel-1 (FIG. 6) , se2-l (Fig.7), se2-2 (Fig.8), sel4-3 (Fig.9), se20-7 (Fig.11), se20-9 (Fig.12), se33-l ( Fig.13), se37-2 (Fig.14), L14-2 (Fig.16), L15-7 (Fig.17), Li9-1 (Fig.18), Li9-4 (Fig.19), LÜ5-2 (Fig. 20), LiilO-6 (Fig. 21), Liii4-5 (Fig. 22) or GBP-TA or a protein or a fragment thereof encoded.

The present invention also relates to antibodies which specifically recognize the proteins (tumor antigens) described above. The antibodies can be monoclonal, polyclonal or synthetic antibodies or fragments thereof, for example Fab, Fv or scFv fragments. These are preferably monoclonal antibodies. For the production it is favorable to immunize animals, in particular rabbits or chickens for a polyclonal and mice for a monoclonal antibody, with an above (fusion) protein or fragment (s) thereof. Further "boosters" of the animals can be carried out with the same (fusion) protein or fragments thereof. The polyclonal antibody can then be obtained from the serum or egg yolk of the animals. The antibodies according to the invention can be produced according to standard methods, the protein encoded by the above-mentioned nucleic acid sequences or a synthetic fragment thereof serving as an immunogen. Monoclonal antibodies can be produced, for example, by the method described by Köhler and Milstein (Nature 256 (1975), 495) and Galfre (Meth. Enzymol. 73 (1981), 3), wherein mouse myeloma cells are fused with spleen cells derived from immunized mammals , These antibodies can, for example, for immunoprecipitating the above discussed proteins or used to isolate related proteins from cDNA expression banks. The antibodies can be bound, for example, in liquid phase immunoassays or to a solid support. 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.

In addition to their diagnostic suitability, the antibodies can also be used therapeutically. Here, e.g. a protein encoded by the above nucleic acid sequences as a target for bispecific antibodies. For this purpose, reference is made to Kastenbauer et al., Laryngorhinootologie 78 (1), pp. 31-35 (1999) and Cao et al., Bioconj. Chem. 9 (6), pp. 635-644 (1998). The antibodies of the invention are suitable for. B. to trap overexpressed antigen in tumors and thus inhibit tumor growth, since there are indications that in some cases the presence of tumor antigens not only indicates the presence of malignant tumors, but actively promotes tumor growth.

Furthermore, the use of antisense DNA (RNA) or ribozymes can inhibit the translation of the above nucleic acid sequences, the expression of which is increased in tumors, and thus have a therapeutic effect specifically on these nucleic acid sequences or genes. RNA / DNA hybrids form in the corresponding tumor cells, which thus prevent transcription and - in the case of antisense RNA - simultaneously break down the hybrids (and thus the RNA) by RNase H (Scanion et al., The Faseb Journal 9, pp. 1288-1296, 1995)

The present invention thus relates to a medicament or a diagnostic composition which contains the nucleic acid sequences, vectors, proteins, antibodies etc. or combinations thereof described above, or to the use thereof for diagnosis and / or therapy. These are preferably used for the diagnosis or treatment of malignant tumor diseases, in particular CTCL. Is preferred the provision of a vaccination agent based on either the nucleic acid sequence or the protein / peptide id as described above. The diagnostic composition is suitable on the one hand to determine a malignant tumor disease, but also to carry out a follow-up, for example to accompany the therapy.

The above drugs may also contain a pharmaceutically acceptable carrier. Suitable carriers and the formulation of such medicaments 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, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration. The appropriate dosage is determined by the attending physician and depends on various factors, for example the age, gender, weight of the patient, the stage of the disease, the type of administration etc.

A nucleic acid sequence above can also be used as a probe to isolate DNA molecules, for example derived from another species or organism, which encode a protein with the same biological activity. For this purpose, the probe preferably has a length of at least 20, particularly preferably at least 25, bases. Suitable detection methods based on hybridization are known to the person skilled in the art, for example Southern or Northern blot. Suitable labels for the probe are also known to the person skilled in the art and include, for example, labeling with radioisotopes, bioluminescent, chemiluminescent, fluorescent labels, metal chelates, enzymes etc. In addition, this can also be done by PCR (Wiedmann et al., PCR Methods Appl. 3, pp. 551-564 (1994); Saiki et al., Nature 324, pp. 163-166 (1986)) or "Ligase chain reaction "(LCR)

(Taylor et al., Curr. Opin. Biotechnol. 6, pp. 24-29 (1995);

Rouwendal et al. , Methods Mol. Biol. Pp. 149-156 (1996)), the primers being derived from the sequence in the figures and suitable primers (in terms of length, complementarity to the matrix, the region to be amplified, etc.) according to the person skilled in the art usual procedures can be designed.

The present invention also relates to a method for

Diagnosis of malignant tumor diseases, in vitro, using the above nucleic acid sequences or fragments thereof as a probe.

For the diagnostic method, it is preferred to provide the nucleic acids and / or proteins in the form of an ELISA kit, protein chips, nucleic acid chips or a membrane loaded with DNA, RNA or protein.

In the above-mentioned method, methods known to the person skilled in the art regarding the preparation of DNA or RNA from biological samples, the restriction digestion of the DNA, the separation of the restriction fragments on size-separating gels, for example agarose gels, the preparation and labeling of the probe and the detection of the hybridization, for example via "Southern blot" or in-situ hybridization.

This diagnostic method is preferably a method which comprises the following steps:

Isolation of nucleic acid from the patient, performing an LCR or PCR with suitable primers or a hybridization analysis with one or more suitable probes based on a nucleic acid sequence of the figures,

Detection of an amplified product or hybridization as an indication of the presence (or Non-existence) of a tumor disease (depending on whether the respective nucleic acid sequence is expressed more or less (or not) in the tumor compared to control tissue.)

Primers are used which flank a nucleic acid sequence discussed above or suitable subregions. Amplification products of RNA from the tissue in question are of diagnostic importance, which differ from the amplification products of mRNA from healthy tissue with regard to the occurrence of tumor-specific, in particular CTCL-specific bands.

In an alternative preferred embodiment, a method can be used which comprises the following steps: isolation of RNA from the patient,

Performing a Northern analysis with one or more suitable probes,

Comparison of the concentration and / or length of the corresponding mRNA of the patient sample with an mRNA of a healthy person, an increased or decreased concentration of mRNA (depending on the corresponding marker; see Table 4) being indicative of compared to the control mRNA from normal tissue is a tumor, especially CTCL.

In this method, methods known to the person skilled in the art can be used with regard to the preparation of total RNA or poly (A) + RNA from biological samples, the separation of the RNAs on size-separating gels, for example denaturing agarose gels, the production and labeling of the probe and the Detection via "Northern blot" can be applied.

In a further alternative embodiment, a possible tumor disease can also be diagnosed by a method which comprises the following steps:

Obtaining a cell sample from the patient,

Bringing the cell sample thus obtained into contact with one or more of the nucleic acid sequences according to the invention encoded proteins or fragments thereof as probe (s) under conditions which allow the binding of antibodies, the presence of antibodies in the cell sample being indicative of a tumor disease, in particular CTCL.

This detection can also be performed using standard techniques known to those skilled in the art. These are also known cell disruption processes which allow the isolation of the antibodies in such a way that they can be brought into contact with the antigen. The detection of the bound antibody is preferably carried out via immunoassays, for example Western blot, ELISA, FACS or RIA or immunohistochemical methods. This is preferably done via ELISA or "dot blot". To establish an ELISA, the above nucleic acid sequences or fragments thereof can be cloned into expression plasmids and the corresponding proteins can be produced recombinantly, preferably as fusion proteins with a "His tag", which facilitates their purification. The proteins are then applied to membranes or other suitable surfaces, if necessary fixed and incubated with adequately diluted patient sera. After the usual washing steps, an incubation with a secondary labeled antibody then takes place according to routine procedures for the detection of the bound patient antibodies. The patient sera are preferably incubated with a large number of marker proteins (antigens), since the detection of the presence (or absence) of different antibodies better indicates the underlying tumor disease and may also allow classification according to the stage of the disease.

The present invention further relates to a kit for carrying out the diagnostic methods according to the invention, which contains the antibody according to the invention or a fragment thereof, a protruding protein (or peptide derived therefrom), a nucleic acid sequence according to the invention (as a probe) or one which is suitable, for example, for PCR or LCR Sequence of the nucleic acid sequences according to the invention based primer (or a pair of primers) contains, optionally in combination with a suitable detection means. Depending on the configuration of the diagnostic method to be carried out with the kit according to the invention, the compounds contained in the kit (nucleic acid molecules, proteins, antibodies or fragments thereof) can be immobilized on a suitable carrier, ie in the form of a chip or bound on a membrane.

All of the proteins mentioned above are recognized serologically only by antibodies from sera from tumor patients, but not by sera from control persons, and are therefore serologically tumor-specific. Since this specificity is not limited to a type of tumor, these proteins and antibodies are very well suited to make a distinction between malignancy and "non-malignancy". It has been found to be advantageous to use more than one of the tumor markers mentioned above, i.e. a combination of tumor markers, and depending on this choose a therapeutic approach. This means that the medicament according to the invention should also contain more than one of the nucleic acids, proteins or antibodies mentioned above.

The invention will now be further described with reference to the figures, which show:

Fig.l: nucleic acid sequence of se2-5 and an ORF derived therefrom

Fig. 2: nucleic acid sequence of se20-10 and an ORF derived therefrom

3: nucleic acid sequence of se57-1 and an ORF derived therefrom

4: nucleic acid sequence of se70-2 and an ORF derived from it

5: nucleic acid sequence of Lgl-2 and an ORF derived therefrom 6: nucleic acid sequence of sel-1 and an ORF derived therefrom

7: nucleic acid sequence of se2-1 and an ORF derived therefrom

Fig. 8: Nucleic acid sequence of se2-2 and an ORF derived therefrom

Fig. 9: Nucleic acid sequence of sel4-3 and an ORF derived therefrom

10: nucleic acid sequence of se20-4 and an ORF derived therefrom

11: nucleic acid sequence of se20-7 and an ORF derived therefrom

12: nucleic acid sequence of se20-9 and an ORF derived therefrom

Fig. 13: Nucleic acid sequence of se33-1 and an ORF derived therefrom

Fig. 14: nucleic acid sequence of se37-2 and an ORF derived therefrom

Fig. 15: Nucleic acid sequence of se89-l and an ORF derived therefrom

Fig. 16: L14-2 nucleic acid sequence and an ORF derived therefrom

Fig. 17: nucleic acid sequence of L15-7 and an ORF derived therefrom

Fig. 18: Nucleic acid sequence of Li9-1 and an ORF derived therefrom Fig. 19: Nucleic acid sequence of Li9-4 and an ORF derived therefrom

Fig. 20: Nucleic acid sequence of LÜ5-2 and an ORF derived from it

Fig. 21: Nucleic acid sequence of LiilO-6 and an ORF derived therefrom

Fig. 22: Nucleic acid sequence of Liii4-5 and an ORF derived therefrom

Figure 23: GBP-TA nucleic acid sequence and an ORF derived therefrom

Fig. 24: Localization of GBP-TA, GBP-TA _short and Lgl-2 on chromosome lp22.3. The primers used to differentiate the splicing variants are shown.

Pri erset I: tgt tgt aga tca ctt caa ggt gc (forward) cca tat cca aat tcc ctt ggt gtg ag (right) Anneal ing-Tem. 63 ° C

Primer set II: aga agg aag aaa ctc caa aca cat cc (forward) cca tat cca aat tcc ctt ggt gtg ag (right) Anneal ing temp. 48 ° C

The invention will now be described below with reference to the examples.

With regard to the methods used, in addition to the methods given in Example 1, Sambrook, J, Fritsch, E, F. and Maniatis, T. (Molecular cloning; a laboratory manual; second edition; Cold Spring Habor Laboratory Press, 1989) and Current Protocols in Molecular Biology (John Wiley and Sons, 1994-1998), using the techniques mentioned below, in particular screening of cDNA libraries, preparation of DNA or RNA, PCR, RT-PCR or Northern blot are sufficiently known to the person skilled in the art and are mastered.

Example 1: General procedures

(A) tissues and sera

Sera and tumor tissue were obtained in routine diagnostic or therapeutic procedures with the patient's consent (and the permission of the responsible ethics committee). The tissues and sera were stored at -20 ° C or -80 ° C.

(B) Preparation of the mRNA from cDNA banks was carried out from Testis samples using a kit for RNA isolation ("RNeasy midi kit"; Qiagen, Hilden, Germany) and then a kit for mRNA isolation ("oligotex mRNA kit"; Qiagen) extracted according to the manufacturer's recommendations. A total of 10.4 μg mRNA were used for the construction of the λ-ZAP expression bank (“UNI-ZAP ™ XR custom cDNA library”; Stratagene, La Jolla, CA, USA). The cDNA library consisted of 10 ⁶ primary recombinants with an insertion length of more than 0.4 kbp and was amplified to 10 ¹⁰ plaque-forming units (pfu). For the construction of the CTCL library, 4.8 μg mRNA from various samples of cutaneous T and B cell lymphomas were used. The number of primary recombinants was 6 x 10 ⁷ . The procedure was analogous to that described above at Testis-Bank.

(C) Immunscreeninα

As in Sahin et al. (PNAS USA 92. (1995), 11810-11813) and Türeci et al. (Cancer Res. 56 (1996), 4766-4772). All sera were in Tris-buffered saline (TBS with 0.2% dry milk powder, pH 7.5) and pre-absorbed with E. coli proteins (broken up mechanically or lysed by phage without insertion). E.coli transduced with recombinant λ-ZAP phages were plated on NZY agar at a concentration of 2000 plaques / plate and the expression of the recombinant proteins was induced by means of isopropyl-β-D-thiogalactoside. The plates were placed at 37 ° C above sea level. incubated and the proteins transferred to nitrocellulose membranes for 4 hours at 37 ° C. and bound. The membranes were washed with TBS containing Tween-20 ™ (0.05%), saturated with 5% dry milk powder in TBS and incubated with sera (either from patients or as a control from healthy subjects) at a final concentration of 1/100. Reactive proteins were detected with an alkaline-phosphatase-coupled secondary antibody (goat anti-hu an-IgC, Fc fragment; Dianova, Hamburg, Germany) and using 5-bromo-4-chloro-3-indolyl phosphate and nitroblue tetrazolium made visible. Positive phagemids were further examined using sera from patients with mycosis fungoides (n = 15) and Sezary syndrome (n = 3) and healthy individuals as controls (n = 10). Positive phagemids were subcloned to monoclonality and subjected to an in vivo excision of the "pBluescript" plasmid (according to the protocol of the manufacturer of the Genbank, Stratagene, La Jolla, CA, USA). DNA was isolated according to the manufacturer's protocol ("QIAprep spin miniprep"; Qiagen). The size of the inserts was analyzed by Smal / Kpnl cleavage and gel electrophoresis. The sequencing was carried out using an automatic fluorescence sequencer (model 377; Perkin-Elmer / Applied Biosystems, Forster System, CA, USA) and the dye terminator method according to the manufacturer's instructions ("ABI PRISM Big Dye Ready Reaction Terminator Cycle Sequencing Kit "; Perkin-Elmer) performed. Primers have been chemically synthesized. The sequences of the clones were completely determined on both complementary strands.

(D) Tumor tissue and cell lines

Tissue samples obtained from 17 CTCL patients served as the source for the production of tumor cDNA: 13 mycosis fungoides (Stage Ib to IVb, mainly Ilb), 2 Sezary syndromes (stage III), 1 T-zone lymphoma (stage IVb) and 1 CD30 + CTCL (stage Ilb). In addition, cDNAs were produced from the following 4 CTCL cell lines: My-La (Mycosis fungoides; Kaltoft et al., In Vitro Cell Dev. Biol. 28a (1992), 161-167), SeAx (Sezary Syndrome; Kaltoft et al. , Arch. Dermatol. Res. 280 (1988), 264-267), HH (lymphomatoid papulose; ATCC no .: CRL-2105) and HuT-78 (Sezary syndrome; ATCC no .: TIB-161). In addition, cDNA was produced from six leukemia cell lines (ARA-10, Jurkat, KGl, K562, Nalm-2 and SKW6. And 22 melanoma cell lines.

Control cDNAs were used extensively to analyze tissue distribution within normal tissues, including three fields of commercially available cDNAs (all from Clontech, CA, USA): human "multiple tissue" cDNA field I, field II and human fetal MTC -Field. In addition, various commercially available total RNAs for the production of further control cDNAs were generated using the method described above. Finally, cDNAs from three activated CD8 + T cell lines (Möller et al., British Journal of Cancer 77 (1998), 1907-1916) also served as control T cells.

(E) RT-PCR

Due to the limited amount of RNA, RT-PCR was preferred to examine the identified sequences within different normal tissues and tumor tissues. In selected cases, these studies were completed using Northern blot analyzes. RT-PCR was carried out using "MJ Research PCT-200" (Biozym, Oldendorf, Germany) with a one-minute addition at variable temperature with 35 cycles. All RT-PCR were performed in at least two independent experiments. The RNA isolation, RT-PCR and Northern blot analyzes were otherwise carried out in accordance with customary standard procedures and standard conditions. The primer sequences and annealing temperatures for the different clones are given in Table 1 below: (F) Northern blot

10 μg of total RNA are separated electrophoretically on a MOPS gel and transferred to positively charged nylon membranes. The probes are marked using the Röche High Prime Kit with α- ³² P-dCTP. The non-incorporated nucleotides are removed using the Qiagen Removal Kit (Qiagen, Hilden). The pre-hybridization is carried out at 60 ° C for 1-2 hours. Hybridization takes place at 60 ° C., preferably overnight. The pre-hybridization and hybridization solutions have the composition: 10% dextran sulfate, 1% SDS, 10x Denhardt's reagent, 3x SSC. The subsequent washing is carried out for 2 x 30 minutes with 2xSSC / 0.1% SDS at 42 ° C and then 2 x 30 minutes with 0.2xSSC / 0.1% SDS at 65 ° C. A Kodak X-Omat film is applied for an exposure time of 3-10 days.

Example 2: Screening for positive clones

Approximately 1.9 x 10 ⁶ recombinant clones from a cDNA library obtained from normal Testis tissue were screened with sera from patients with cutaneous T-line lymphoma (CTCL) including mycosis fungoides (MF) and Sezary syndrome. 28 clones representing 22 different ORFs or genes were detected and these were further investigated with regard to serological reactivity and molecular distribution. Secondary confirmation was obtained using additional sera from patients with a positive diagnosis for mycosis fungoides (MF) (n = 15) or Sezary syndrome (n = 3) and 10 control sera from healthy volunteers. The reactivity of the patient's sera was associated with the tumor stage (at most stage III) (Table 2). However, this was not statistically significant (X ² test and Mann-Whitney U test), presumably due to the low number of sera with high tumor stages. The reactivity of the patient sera ranged from 11% to 71% of recombinant clone-identifying sera. Table 2: Number of positive clones correlated with the tumor stage of the serum donor

The primary screen of a Testis cDNA bank was carried out successively with 17 individual sera, which came from patients who had tumors in the indicated stage. The number of positive and total plaques analyzed are given in the 3rd column. During the secondary screen, each positive plaque (28) was tested with up to 18 individual sera from different patients in the indicated tumor stage. ⁽¹ ^ Cumulated over all sera tested. ^ Positive clones divided by the total number of clones tested.

The num _ber and probability of all serological responses are summarized in terms of the tumor stage of the patients from whom serum was taken for screening a testis cDNA library. Although the data ahrscheinlichkeit a higher _W for the presence of antibodies against _T umor- _A ntigene (with the peak in stage III) show, these differences are not statistically significant. Table 3: Serological analyzes of the identified clones

The table shows the percentage reactivity of the sera (number n) against the tested clones in the secondary screen.

The percentage of reacting and the total number of sera (s) tested during secondary screening are given. In the case of the clones se2-l and se20-4, one of the homologous clones is additionally indicated, since these differ in their reaction pattern, presumably due to sequence differences. Five antigens represent previously unknown sequences (se2-5, se20-10, se57-l, se70-2 and Lgl-2). The RNA expression pattern of the identified antigens analyzed by RT-PCR varied between highly restricted and ubiquitous expression in 28 normal, 17 CTCL tumor tissues and 33 tumor cell lines of different origins (Tables 4 and 5).

Table 4: Expressxon analysis using RT-PCR with antigen-specific primers and cDNA from different tissues and

Cell-lines

after RT-PCR analysis on (number of tissues is in brackets; nt: not tested). ⁽¹⁾ RT-PCRs on Testis cDNA always gave positive results. In the case of Klonse2-l, Testis cDNA was the only positive sample. For the composition of the control palette, see Table 5. ⁽²⁾ Activated cytotoxic T cells. ^{3) For details on these results, see Table 5. ⁽⁴⁾ GBP-TAshort with 7%.

Table 5: RT-PCR analyzes using specific primers against differentially expressed sequences and "multiple tissue"

(MTC) cDNA

Pallets manufactured (Clontech). Each sample contained cDNA from samples from several individuals. The skin cDNA was made from a single sample. RT-PCR against GBP-TA / GBP-TAshort differentiates the two variants.

Example 3: Tumor-specific antigens

Six clones (represented by at least four different recombinants) were homologous to SCP-1, a protein related to meiosis (Türeci et al., PNAS USA 95. (1998), 5211-5216). Interestingly, the serological reactivity of different sera differed between the different SCP-1 clones: clone se2-l was detected by sera from 2/15 MF patients and 2/3 from patients with Sezary syndrome. It was demonstrated by RT-PCR that se2-l is tumor-specific. Another clone homologous to SCP-1 (se37-l) was detected by 3/9 MF sera and 1/3 Sezary syndrome sera. This could reflect different epitopes of SCP-1 because the clones differed in length. Clone se33-2 also responded with 1/5 control sera. Interestingly, this clone encoded another peptide sequence within the first reading frame that was not present in the other clones homologous to SCP-1 and could therefore be an autoantigen that is responsible for the reactivity of the control serum. The PCR analyzes were carried out with the same primers as by Türeci et al. (1998) published that also matched the SCP-1 homologous clones perfectly. Within all tested In normal tissues as well as the tumor samples and cell lines, only one Testis sample and one MF sample (patient HS) were found, which expressed SCP-1 mRNA. The positive result of the MF cDNA could be confirmed by Northern blot, which gave a band of approximately 4.3 kb. In addition, the patient's HS serum also reacted with se2-1 and one of the other clones homologous to SPC-1. Furthermore, according to Northern blot analysis, the clones se57-l and L15-7 are tumor-specific.

Example 4: (A) antigens with a restricted expression pattern and (B) ubiguously expressed antigens

13 antigens with differential or ubiquitous expression (detected by RT-PCR) are described below (see Tables 4 and 5).

(A)

For five new antigens (se2-5, se20-10, se57-l, se70-2 and Lgl-2) and two antigens with homologies to known genes (se33-l: NP220; se89-l: protein related to retinoblastoma RAP140) showed a differential expression at the molecular level. The serological reactivity against these clones (defined as the percentage of reactive sera during secondary screening) averaged 31%. A low reactivity rate was shown for the clones se20-10 and se70-2 (2/18 and 1/18 reactive sera, respectively), while 71% of the sera from CTCL patients (n = 14) reacted positively with the clone se89-l , All 6 clones showed no reaction with up to 10 control sera.

It started to quantify the RT-PCR results using Northern analyzes. Such antigens, which do not turn out to be equally expressed in normal tissues compared to tumor samples, are classified as potential therapeutic agents. RT-PCR analyzes showed that the se-2-5-specific mRNA is expressed almost ubiquitously within normal tissue, but not in activated T cells and only in 55% of the CTCL tissue samples. In contrast, Northern blot analysis revealed a restricted expression pattern even within normal tissues. Strong signals were detectable in the kidney, trachea and testis, weaker signals in the colon, small intestine, thymus, bone marrow and stomach. While three bands were detectable in all positive normal tissues (5.2, 4.2 and 3.9 kB), the only positive CTCL cell line SeAx showed a signal at 3.9 kb.

Specific mRNAs for the clones se20-10 and se57-l were found by RT-PCR in 43% and 21% of the examined control tissues. Interestingly, the expression of the mRNA specific for se57-l was very strongly downregulated in all tumor tissues and cell lines. In contrast, the expression of mRNA from clone se70-2 was upregulated (100%) compared to normal tissues (54%) within the CTCL and leukemia cell lines, while the CTCL tissues and Me 1 anomal z 11 lines showed mean expression levels (33% and 45%). Among the control tissues, all fetal tissues were positive in the RT-PCR.

Differential expression was shown for two antigens with homologies to known sequences: se33-l cDNA is homologous to NP220, a DNA-binding protein, and se89-l cDNA is homologous to RAP140, a retinoblastoma-associated clone. Clone se33-l showed 99% similarity within an overlapping distance of 3830 bp at the 3 'end of NP220, but this clone is shortened at the 5' end, which leads to a shortened ORF. The RT-PCR

(using se33-l-specific primers) demonstrated the detection of mRNA in 6/8 fetal and 16/20 normal tissues

(Table 5) as well as within CTCL tissues (12/16), activated cytotoxic T cells and in most cell lines

(Table 4).

Clone se89-l cDNA showed 98% similarity to RAP140 within an overlapping region of 3444 bp and a gap of 60 bp which lies within the ORF and leads to an amino acid gap of 20 amino acids. RT-PCR was carried out with different primers: First with the primer combination RAP140 (see Table 1 and Example 1), both RAP140 and se89-l were detected and three bands in Testis cDNA and two bands in different other cDNAs were found. To specify the expression of se89-l, a new reverse primer (see Table 1) was designed that spans the gap within se89-l. Only one band was amplified using this primer together with the forward primer against RAP140, which also detects se89-l. The frequency of positive cDNAs for the se89-l-specific primers did not differ significantly between the control tissues (79%), CTCL tissues (75%) and cell lines (CTCL and leukemia cell lines: 100%, melanoma lines: 73 %). Northern blot analyzes confirmed the presence of mRNA specific for se89-l within mRNA, which originated from the brain, kidney, colon and testis and the CTCL line SeAx.

(B)

Six antigens homologous to known sequences (sel-1, se2-2, sel4-3, se20-4, se20-9 and se37-2) were found to be ubiquitously expressed. In all control tissues (n = 28) specific mRNA was detectable by RT-PCR for these clones. In the two clones sel4-3 and se20-4, all tumor tissues and cell lines were also positive in the RT-PCR. In contrast, the clones se2-2, se20-7, se20-9 and se37-2 were only expressed in a subset of the melanoma and leukemia cell lines, while CTCL tissue and CTCL cell lines showed a higher percentage of in RT- PCR showed positive cDNAs.

The reactivity of patient sera with these clones was on average around 29%, with two extremes also being observed: the reactivity against clone sel4-3 was found in 11% (1/9) and against clone sel-1 in 50 % (5/10) CTCL sera. Two control sera (n = 10) reacted with clone se20-6, the clone se20-4 is homologous. It could be shown that se20-6 codes for a different peptide (72 aa) in the first reading frame, which was neither present in se20-4 nor in its homologous gene HRIHFB2216. Sequence analysis of these clones and comparison with their homologous counterparts revealed insertions, deletions or elongations in some cases.

It must be emphasized that all clones tested are serologically specific.

Example 6: Sequence analysis for Lgl-2

A number of other clones with great agreement to Lgl-2 were isolated, which were completed in the 3 'direction (stop codon and 3' untranslated region present). This is shown in Fig. 24. These clones could be summarized in a gene (GBP-TA), which could be assigned to the chromosome lp22.3. A distinction was made between two splicing variants: GBP-TA was assigned 12 exons, while GBP-TA _shor exon number 2 was missing.

A protein could be derived from GBP-TA, which has certain homologies with the known guanylate-binding proteins GBP-1, GBP-2 and HGBP (US-A-5, 871, 965). However, the sequence of HGBP does not include exon 2.

Example 7: Expression analyzes for GBP-TA

RT-PCR experiments were carried out to analyze the expression of GBP-TA. Two different pairs of primers (see FIG. 24) were used to differentiate the two splicing variants. A large number of control cDNAs were used, each of which was produced from a collection of tissues from different donors. While 11 control tissues were nagative for both primers, GBP-TA was _short in 5 tissues and both variants of GBP-TA in only 2 tissues (bone marrow and stomach) (Table 6). Table 6: Evidence of GBP-TA and GBP-TA'S, hoard in adults

Kontro11geweben

Contro11 fabric Result Primerset I Primersetll

Brain, colon, heart, kidney, liver, ovary, lung, PMNC, prostate testis, thymus, trachea

Placenta, small intestine, spleen, active. CD8 T cells, uterus

Bone marrow, stomach

In contrast to the control tissues, GBP-TA was detected in various tumor tissues using RT-PCR: cutaneous T-cell lymphomas (26%, n = 19), tumors from the ENT area (21%, n = 14). GBP-TA _short was found in 20% of ENT tumors (n = 15) and 9% of colon carcinomas (n = 35). Se57-1 was found in 20% of colon carcinomas (n = 35) and 57% of ENT tumors (n = 28).

Since the RT-PCR is extremely sensitive and does not allow any statement about the presence and the amount of protein, the expression was checked by Western blot and a GBP-TA specific antibody. Several of the RT-PCR positive controls could be tested as protein medleys in a Western blot: placenta, small intestine, spleen, fetal liver, stomach, testis, uterus. Like other control protein medleys tested (mammary gland, testis), these proved to be negative, while proteins obtained from tumor (CTLC) cell lines (SeAx, MyLa, Hut-78, HH; isolation via Tristar, AGS, Heidelberg) showed a clear band showed in the appropriate size. This proves the suitability of GBP-TA as a specific target structure for therapy.

Example 8: Preparation of antibodies against GBP-TA

To produce a GBP-TA-specific antibody, the insert of a clone comprising bases 539 up to and including 1991 from GBP-TA was cloned into a His vector and expressed in E-coli. The recombinant protein was purified on a nickel column, then separated in an SDS gel for further purification and the corresponding band cut out. A rabbit whose preimmune serum did not react with the antigen was immunized with the excised piece of gel.

Immunization protocol for polyclonal antibodies in rabbits

For each immunization, 600 μg of purified KLH-coupled peptide in 0.7 ml of PBS and 0.7 ml of complete or incomplete Freund ¹ s adjuvant are used.

Day O: 1. Immunization

(complete friend ¹ s adjuvant)

Day 14: 2. I m m u n i s i e r u n g (incomplete friend 's

adjuvant; icFA)

Day 28 3rd Immunization (icFA) Day 56 4th Immunization (icFA) Day 80 Bleed

The rabbit's serum is tested in an immunoblot. For this purpose, the peptide used for the immunization is subjected to SDS-polyacrylamide gel electrophoresis and transferred to a nitrocellulose filter (cf. Khyse-Andersen, J., J. Biochem. Biophys. Meth. 10, (1984), 203-209). Western blot analysis was performed as in Bock, C.-T. et al. , Virus Genes 8, (1994), 215-229. For this purpose, the nitrocellulose filter is incubated for one hour at 37 ° C. with a first antibody. This antibody is rabbit serum (1: 10000 in PBS). After several washing steps with PBS, the nitrocellulose filter is incubated with a second antibody. This antibody is an alkaline phosphatase-coupled monoclonal goat anti-rabbit IgG antibody (Dianova) (1: 5000) in PBS. After 30 minutes of incubation at 37 ° C, there are several washing steps with PBS and then the alkaline phosphatase detection reaction with developer solution (36μM 5 'bromo-4-chloro-3-indolylphosphate, 400μM nitroblue tetrazolium, 100mm Tris-HCl, pH 9.5, 100 mM NaCl, 5 mM MgCl ₂ ) at room temperature until bands become visible.

It turns out that polyclonal antibodies according to the invention can be produced.

Example 9: ELISA

The same GBP-TA insert used for antibody production (base no. 539 up to and including 1991 from GBP-TA) was cloned into a pGEX vector, expressed recombinantly and used in a GST-ELISA. The ELISA system used is based on Sehr et al. (J. of Immunol. Meth. 2001, 253, 153-162). For this purpose, glutathione-casein-coated ELISA plates were loaded with the fusion protein and then sera from CTCL patients and control persons were tested for the presence of specific antibodies. It was found that 17% of the CTCL sera (n = 60) but only 2% of the control sera (n = 99) reacted with the fusion protein GST-GBP-TA.

The ELISA is suitable for all specified marker antigens for diagnostic purposes, for prognostic assessment and for follow-up.

Claims

claims

1. Diagnostic composition which contains at least one nucleic acid sequence, the altered expression of which is associated with a tumor disease, the nucleic acid sequence se2-5 (FIG. 1), se20-10 (FIG. 2), se57-l

(Fig.3), se70-2 (Fig.4), Lgl-2 (Fig. 5), sel-1 (Fig. 6), se2-l (Fig.7), se2-2 (Fig.8) , sel4-3 (Fig.9), se20-4 (Fig.10), se20-7 (Fig.11), se20-9 (Fig.12), se33-l (Fig.13), se37-2 ( Fig.14), se89-l (Fig.15), L14-2 (Fig.16), L15-7 (Fig.17), Li9-1 (Fig.18), Li9-4 (Fig.19), LÜ5-2 (Fig. 20), LiilO-6 (Fig. 21), Liii4-5 (Fig. 22) or GPB-TA (Fig. 23).

2. Medicament which contains at least one nucleic acid sequence, the altered expression of which is associated with a tumor disease, the nucleic acid sequence se20-10 (FIG. 2), se57-l (FIG. 3), Lgl-2 (FIG. 5) , sel-1 (Fig. 6), se2-l (Fig.7), se2-2 (Fig.8), sel4-3 (Fig.9), se20-7 (Fig.11), se20-9 ( Fig.12), se33-l (Fig.13), se37-2 (Fig.14), L14-2 (Fig.16), L15-7 (Fig.17), Li9-1 (Fig.18), Li9-4 (Fig. 19), LÜ5-2 (Fig. 20), LiilO-6 (Fig. 21), Liii4-5 (Fig. 22) or GBP-TA (Fig. 23).

3. Diagnostic composition according to claim 1 or medicament according to claim 2, wherein the at least one nucleic acid sequence, the altered expression of which is related to a tumor disease, comprises a nucleic acid sequence,

(a) which differs from a nucleic acid sequence defined in claim 1 or 2 in the codon sequence due to the degeneration of the genetic code;

(b) which hybridizes with a nucleic acid sequence defined in one of claims 1, 2 or 3 (a); or

(c) which is a fragment, an allelic variant or another variant of a nucleic acid sequence defined in one of claims 1, 2, 3 (a) or 3 (b).

4. Nucleic acid sequence as defined in any one of claims 1 to 3, which is a cDNA or genomic DNA.

5. protein whose altered concentration is related to a tumor disease and which is encoded by a nucleic acid sequence according to any one of claims 1 to 4.

6. Diagnostic composition containing at least one vector containing one of the nucleic acid sequences according to claim 1 or 3, at least one protein encoded by a nucleic acid sequence according to claim 1 or 3 or at least one antibody directed against this protein.

7. Diagnostic composition according to claim 6 for the diagnosis or monitoring of a tumor disease.

8. Diagnostic composition according to claim 7, wherein it is provided in the form of an ELISA, protein chips, nucleic acid chips or a membrane loaded with DNA, RNA or protein.

9. Medicament which contains at least one vector containing one of the nucleic acid sequences according to claim 2 or 3, at least one protein encoded by a nucleic acid sequence according to claim 2 or 3 or at least one antibody directed against this protein.

10. Medicament according to claim 9 for the therapy of tumor diseases.

11. Use of a nucleic acid sequence according to one of claims 1 to 4 for the diagnosis and / or therapy of a tumor disease.

12. Use of at least one protein encoded by a nucleic acid sequence according to one of claims 1 to 4 for the diagnosis and / or therapy of a tumor disease.

13. Use of at least one antibody directed against a protein encoded by a nucleic acid sequence according to one of claims 1 to 4 for the diagnosis and / or therapy of a tumor disease.

14. Use according to claim 12 or 13 as a vaccination agent.

15. Use according to claim 12 for the production of peptide-loaded antigen-presenting cells (APC).

16. Use according to claim 12 for the generation of tumor-specific T cells.

17. Diagnostic composition according to claim 1, 3 or 6, medicament according to claim 2, 3 or 9, use according to any one of claims 11 to 17, wherein the tumor is CTCL.