DE19909503A1 - Tumor Associated Antigen - Google Patents

Abstract

The invention relates to a tumor-associated antigen, to immunogenic peptides derived therefrom, to DNA molecules coding said tumor-associated antigen and to the use of said DNA molecules in the immunotherapy of cancers.

Description

The invention relates to the immunotherapy of Tumor diseases.

The immune system's task is to protect the organism from a variety of different microorganisms or to actively combat them. The importance of an intact immune system is particularly evident in inherited or acquired immunodeficiencies. In many cases, the use of prophylactic vaccine programs has proven to be an extremely effective and successful immunological intervention in the fight against viral or bacterial infectious diseases. Furthermore, it has been shown that the immune system is also significantly involved in the elimination of tumor cells. The detection of tumor-associated antigens (TAAs) by components of the immune system plays an important role. In the broadest sense, any component (peptide or non-peptide) of a tumor cell that is recognized by an element of the immune system and that stimulates an immunological response can act as an immunogenic tumor antigen. Of particular importance are those tumor antigens that not only cause an immunological reaction, but also cause rejection of the tumor. The identification of defined antigens that can cause such an immunological reaction is an important step for the development of a molecularly defined tumor vaccine. Although it is not yet clear which elements of the immune system are responsible for rejection of the tumor, there is a consensus on this that CD8-expressing cytotoxic T lymphocytes (CTLs) play a major role (Coulie, 1997). Especially in those types of tumors (e.g. melanoma and kidney carcinoma) that have a relatively high spontaneous remission rate, a correlation between clinical course and the increased occurrence of CD8 ⁺ and CD4 ⁺ T cells was found (Schendel et al., 1993; Mackensen et al., 1993; Halliday et al., 1995; Kawakami et al., 1995; Kawakami et al., 1996; Wang, 1997; Celluzzi and Falo, 1998). Specific CTL clones were obtained either from tumor infiltrating lymphocytes (TIL) or peripheral mononuclear blood cells (PBMC) after cocultivation with mostly autologous tumor cells and cytokine stimulation in vitro. In both animal models and in vitro cultivated human cell culture systems, the T cell response against tumor cells could be increased by transfecting the tumor cells with cytokines (by Elsas et al., 1997; Gansbacher et al., 1990; Tepper et al., 1989 ; Fearon et al., 1990; Dranoff et al., 1993).

Due to the correlation between remission and involvement of CD8 ⁺ T cells, the identification of tumor-associated antigens (TAA), which are recognized by CD8-positive CTLs, is a declared main goal on the way to the development of a tumor vaccine (Pardoll, 1998; Robbins and Kawakami , 1996). Whether other cell types of the immune system such. B. CD4 ⁺ T helper cells play an important role is still unclear; some studies with MAGE-3 / HLA-A1 peptides in melanoma patients suggest this (Marchand et al., 1995; Boon et al., 1998). A number of TAAs recognized by CTLs have been identified in recent years (Boon et al., 1994; von den Eynde and von Bruggen, 1997).

T cells recognize antigens as peptide fragments that recognize Cell surfaces of MHC molecules ("major histocompatibility complex ", in humans" HLA "=" human leukocyte antigen "). There are two Classes of MHC molecules: MHC-I molecules are emerging most cells with nucleus before and present Peptides (usually 8-10-mers) that pass through proteolytic degradation of endogenous proteins arise (so-called antigen processing, "antigen processing"). Peptide: MHC-I complexes are derived from CD8-positive CTLs recognized. MHC-II molecules only come on so-called "professional antigen presenting cells" (APC) and present peptides of exogenous proteins that are found in Recorded and processed by APC during endocytosis become. Peptide: MHC-II complexes are generated by CD4 helper T Cells detected. Through an interaction between T cell receptor and peptide: MHC complex can various effector mechanisms are triggered that lead to apoptosis of the target cell in the case of CTLs. The happens when either the MHC (e.g.% in the case of Graft rejection), or the peptide (e.g. in the case intracellular pathogens) is recognized as foreign. However, not all peptides presented meet the structural and functional requirements for an effective interaction with T cells (as from Rammenee et al., 1995 and described below).  

For the use of TAAs in a tumor vaccine basically several application forms possible: Das Antigen can either be used as a recombinant protein suitable adjuvants or carrier systems, or as cDNA encoding the antigen in plasmid (DNA Vaccine; Tighe et al., 1998), or viral vectors (Restifo, 1997) can be applied. Another Possibility exists in the use of recombinant Bacteria (e.g. Listeria, Salmonella) that the human Recombinantly express and by their antigen additional components have an adjuvant effect (Paterson, 1996; Pardoll, 1998). In all of these Is a processing and presentation of the Antigen by so-called "professional antigen- presenting cells "(APC) necessary. Another One possibility is the use of synthetic peptides (Melief et al., 1996), the corresponding T cell epitopes of the antigen correspond and the either loaded onto the APC from outside (Buschle et al., 1997; Schmidt et al., 1997) or by the APC recorded and intracellularly on the MHC-I molecules be transferred. The most therapeutically efficient Application form for a defined antigen is in the generally determined in clinical trials.

To those recognized by the tumor specific CTLs Antigens or their epitopes count molecules that can come from all protein classes (e.g. transcription factors, receptors, enzymes; for For an overview, see Rammenee et al., 1995; Robbins and Kawakami, 1996). These proteins don't have to necessarily located on the cell surface be like this with detection by antibodies  is required. In order for detection by CTLs as to act on tumor-specific antigen or for the The proteins have to be used in therapy meet certain conditions: first, that Antigen expressed only by tumor cells are or in so-called "critical" normal tissues only in lower concentration than in tumors. Critical normal tissues are essential tissues; a immune response against them serious, sometimes fatal consequences. Second, should the antigen not only in the primary tumor, but also in the metastases. Furthermore, it is in the With a view to a broad clinical application of the Antigen is desirable if it is found in several tumor types is present in high concentration. Another Precondition for the suitability of a TAA as more effective Part of a vaccine is the presence of T cell epitopes in the amino acid sequence of the antigen; TAA-derived peptides are said to be in vitro lead to vivo T cell response ("immunogenic" peptide). On another selection criterion for a clinically broad Applicable immunogenic peptide is the frequency with which of the antigen in a given patient population can be found.

The immunogenic tumor associated antigens (TAAs) from most of whom have already been shown that they T cell epitopes can be divided into several Classify categories, u. a. viral proteins, mutated Proteins, overexpressed proteins, by chromosomal Fusion proteins formed translocation, Differentiation antigens, oncofetal antigens (Van den  Eynde and Brichard, 1995; from the Eynde and from the Bruggen, 1997).

The methods of identification and characterization of TAAs that are the starting point for development of a tumor vaccine are based on the one hand the use of CTLs already induced in patients (cellular immune response) or antibodies (humoral Immune response), or are based on the creation differential transcription profiles between tumors and normal tissues. In the first case, the immunological Approach, patient CTLs for screening eukaryotic tumor cDNA expression libraries, the present the CTL epitopes via MHC-I molecules, used (Boon et al., 1994), while by means high affinity patient antisera prokaryotic cDNA Expression libraries, directly via an immunoblot Analysis of the individual plaques for the presence of TAAs are examined (Sahin et al., 1995). A Combination of CTL reactivity and protein chemical Process represents the isolation of MHC-I isolated peptides from tumor cells that are over Reactivity with patient CTLs were preselected. The peptides are washed out of the MHC-I complex and identified using mass spectrometry (Falk et al., 1991; Woelfel et al., 1994; Cox et al., 1994). The approaches that characterize CTLs of antigens are due to the required cultivation and activation of CTLs associated with considerable effort or not always successful.  

Methods for identifying TAAs based on the Comparison of the transcription profile of normal with Tumor tissues are diverse; these include the differential hybridization, the creation of Subtraction cDNA banks ("representational difference analysis "; Hubank and Schatz, 1994; Diatchenko et al., 1996) and the use of DNA chip technology or the SAGE method (Velculescu et al., 1995). In contrast to the immunological method mentioned above with the help of patient CTLs when using molecular biological methods are shown that the potential antigen candidates found therewith are tumor-specific (tumor-associated) and indeed T cell epitopes possess a cytotoxic T cell Can trigger response. In at least one case (NY-ESO / LAGE-1) was an antigen by both Use of patient sera as well as through RDA identified (Chen et al., 1997; Lethe et al. 1998), also CTL epitopes of this antigen and a simultaneous spontaneous humoral and T cell response in in one patient (Jager et al., 1998).

The object of the present invention was to create a new one to provide tumor associated antigen (TAA).

This task was solved by first using RDA ("representational difference analysis") between Renal cell carcinoma and normal kidney tissue a cDNA Subtraction library manufactured and this with a testis-specific cDNA library was hybridized, to select tumor / testis type antigens.  

The cDNA clones obtained were sequenced and labeled with in Databases available sequences compared. Among the genes identified were found to be 29 unknown, to which ESTs ("expressed sequence tags") - Entries in the database existed. Of these genes those 16 whose ESTs were not considered were examined in more detail critical normal tissue. Using RT-PCR found that five of the sixteen clones examined a clear overexpression in one Renal cell carcinoma versus normal kidney tissue exhibit. The quantitative comparison (using PCR) of the Expression of one of the clones (9D7) between tumor and Normal tissue showed a clear overexpression of the 9D7 cDNA in the tumor. The Northern Blot additionally analyzed expression profile showed that 9D7 none or only one in the normal tissues examined showed weak transcription. The in situ Hybridization of renal cell carcinoma and Normal kidney tissue with a 9D7-specific labeled RNA probe confirmed by RT-PCR, quantitative PCR and Northern blot obtained Results.

The human 9D7 cDNA was completely cloned sequence obtained is shown in SEQ ID NO: 1. The Sequence of 9D7 shows, at the nucleotide and protein level, a high homology to SM20, a through Growth factors in smooth muscle cells of the Rat induced "immediate early gene" that as specific marker for cells of the smooth aortic Muscles have been described (Wax et al. 1994, 1996). The sequence analysis of the human 9D7 cDNA in comparison with SM20 showed that the first 323 bp clearly one  have lower identity than the rest of the cDNA (40% compared to 96% at the derived level Amino acid sequence). Because the human cDNA is at the transition between higher and lower identity with SM20 Item 324 has the first ATG, it can be assumed that it is the start codon of the human 9D7 gene. Since the upstream from position 324 lying sequence also a continuous Has a reading frame that, albeit a smaller one, does shows significant identity with SM20 (40% on the Level of the deduced amino acid sequence) is not rule out a start codon further upstream is available.

The information about the further upstream Sequence can be determined by molecular biological Standard methods are obtained, e.g. B. using 5'-RACE ("rapid amplification of cDNA ends"). At this The method is RNA, preferably mRNA, from cells or Tissues in which 9D7 is transcribed (e.g. RCC Tissue or cell lines derived from RCC such as 786-0, see Example 8) reverse transcribed and then ligated with an adapter of known sequence. A PCR with an adapter primer (binds specifically to the Adapter at the 5 'end of the cDNA) and a 9D7-specific Primer (e.g. SEQ ID NO: 21, 19, 17, 16, 14, 12, 11, 4) allows the amplification of corresponding 9D7 fragments. These PCR products can, as in Example 1 described, cloned using standard methods and, characterized in particular by DNA sequencing become.  

An alternative method to characterize the 5'-end is the screening of cDNA libraries Hybridization with DNA probes specific for 9D7 or Antisera.

Performs screening of cDNA libraries based on methodological restrictions, e.g. B. inefficient reverse transcription due to pronounced Secondary structures of RNA, not to the desired goal, genomic libraries can be examined by e.g. B., as when screening cDNA libraries Hybridization with DNA probes specific for 9D7 Clones that can be isolated upstream from the obtained 5 'end of the cDNA Sequence information e.g. B. the promoter region of 9D7 contain.

The isolated cDNA codes for the tumor-associated Antigen (TAA) of the designation 9D7 with the in SEQ ID NO: 2 given amino acid sequence. This sequence will defined by the start codon at position 324 the isolated 9D7 cDNA.

The present invention relates in a first Aspect the tumor-associated antigen of the designation 9D7 with the amino acid sequence given in SEQ ID NO: 2.

The amino acid sequence shown in SEQ ID NO: 2 can Have deviations, e.g. B. those by conservative exchange of amino acids are necessary, provided that the 9D7 derivative is suitable for use in a Tumor vaccine desired immunogenic properties having.  

In a further aspect, the invention relates to a Polypeptide that contains the 9D7 sequence as a partial sequence contains. This polypeptide faces that in SEQ ID NO: 2 indicated 9D7 sequence an extension on N-terminal end, its sequence is defined by an upstream, possibly in the V direction start codon of position 924 of the 9D7 cDNA.

The amino acid sequence (or correspondingly the sequence the 9D7 cDNA) can optionally be modified, by adding single amino acids in a 9D7 CTL epitope to be exchanged to, compared to natural 9D7 CTL epitope, an increase in the affinity of 9D7 peptides to MHC-I molecules and thus an increased Immunogenicity and ultimately increased reactivity against tumors. Modifications in Range of 9D7 epitopes can be seen on total 9D7 protein (This is from the APCs to the corresponding Peptides processed) or on larger ones 9D7 protein fragments or on 9D7 peptides (see below) be made.

In another aspect, the present concerns Invention immunogenic fragments and peptides derived from 9D7 are derived. The latter are referred to below as Denoted "9D7 peptides". A first group are 9D7 peptides that have a humoral immune response (induction of antibodies). Such peptides are selected sections of 9D7 (at least 12 to 15 amino acids), which are Algorithms ("prediction algorithms") such. B. the "Surface probability blot" (Emini et al., 1985), the "hydrophobicity blot" (Kyte and Doolittle, 1982) and the  "antigenic index" (Jameson and Wolf, 1988) determined can be.

9D7 peptides are made directly or in modified form (e.g. coupled to KLH = "keyhole limpet hemocyanine") administered and the formation of antibodies by means of common immunological assays, e.g. B. by means of ELISA, certainly.

Further, within the scope of the present invention preferred 9D7 peptides are those by MHC molecules are presented and a cellular one Effect immune response. There are two classes of MHC molecules, namely MHC-I molecules, that of CD8 positive CTLs and MHC-II molecules made by CD4-positive T helper cells are recognized.

For a peptide to trigger a cellular immune response, must bind it to an MHC molecule; being the to molecule must have in his repertoire. The determination of the The patient's MHC subtype thus presents, with regard to triggering a cellular immune response, one of the essential requirements for effective application of a peptide on this patient.

The sequence of a peptide to be used therapeutically is determined by the respective MHC molecule Anchor amino acids and length specified. Defined Anchor positions and length ensure that a peptide into the peptide binding groove of each MHC molecule of the patient fits. As a consequence, that the immune system is stimulated and a cellular Immune response is generated, which, in the case of  Use of one derived from a tumor antigen Peptide against the patient's tumor cells.

Immunogenic 9D7 peptides can be prepared by known methods one of the foundations for this is the Relationship between MHC binding and CTL induction.

So since the sequence of immunogenic peptides due to their Peptide binding motif can be predetermined 9D7 peptides that represent CTL epitopes due to 9D7 protein sequence identified and synthesized become. Various methods are suitable for this for the identification of CTL epitopes from known ones Protein antigens have been used; e.g. B. from Stauss et al., 1992, for the identification of T cell Epitopes described in human papillomavirus Method.

The allele-specific requirements of each MHC-I allele product for a peptide that binds to and is presented by the MHC molecule have been summarized as a motif (eg Falk et al., 1991). A large number of both MHC peptide motifs and MHC ligands are known to date. A method suitable for the purposes of the present invention for searching for epitopes of a known protein which fits into a particular MHC-I molecule has been described in a review article by Rammenee et al., 1995. It includes the following steps:
the protein sequence is first examined for sections which correspond to the anchor motif, with certain variations in terms of peptide length and anchor occupation being possible. If e.g. B. a motif prescribes a 9-mer with Ile or Leu at the end, 10-mer with a corresponding C-terminus can also be considered, as can peptides with other aliphatic residues, such as Val or Met at the C-terminus. In this way a number of peptide candidates are obtained. These are examined for the presence of as many anchor residues as possible, which they have in common with known ligands, and / or for whether they have "preferred" residues for various MHC molecules (according to the table by Rammenee et al., 1995). In order to exclude weakly binding peptides, binding assays are expediently carried out. If the requirements for peptide binding for certain MHC molecules are known, the peptide candidates can also be examined for non-anchor residues which have a negative or positive effect on the binding or which make this possible (Ruppert et al., 1993). With this approach, however, it should be considered that the peptide binding motif is not the only decisive factor in the search for natural ligands; other aspects, e.g. B. the enzyme specificity during antigen processing, contribute - in addition to the specificity of the MHC binding - to the identity of the ligand. A method which takes these aspects into account and which is suitable in the context of the present invention for the identification of immunogenic 9D7 peptides has been used, inter alia, by Kawakami et al., 1995, to generate gp100 epitopes based on known HLA-A * 0201 motifs to identify.

The peptides can also be used with regard to their Binding ability can be selected to MHC-II molecules.  

The MHC-II binding motif spanning nine Amino acids indicates a higher degree Degeneration in the anchor positions on as the MHC-I Tie motif. Recently, starting from the X-ray structure analysis of MHC-II molecules, methods developed the exact analysis of MHC-II Attachment motifs, and based on that, variations of Allow peptide sequence (Rammenee et al., 1995, and the original literature cited there). Peptides that participate MHC-II molecules bind to the CD4 T cells typically from dendritic cells, macrophages or B cells. The CD4 T cells in turn then activate CTLs directly by z. B. Cytokine release and enhance the efficiency of the Antigen presentation by APC (dendritic cells, Macrophages and B cells).

Recently databases and prediction algorithms have been released available, the prediction with great reliability of peptide epitopes that are linked to a specific Bind MHC molecule.

Within the scope of the present invention, under Use of the one described by Parker et al., 1994 Algorithm, for the most important HLA types, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, candidate peptides identified from which to is expected to pass the appropriate HLA Binding molecules and therefore immunogenic CTL epitopes represent; the peptides found are in Table 3 listed. Similarly, if necessary using other algorithms that the different characteristics of the peptides  (Hydrophobicity, charge, size) or requirements the peptides, e.g. B. the 3D structure of the HLA molecule, Take into account further potential peptide epitopes for other HLA types can be determined.

After selecting 9D7 peptide candidates using the The methods mentioned are their MHC binding by means of Peptide binding assays tested. Next is the Immunogenicity of the peptides with good ones Binding properties determined (stability of the peptide MHC interaction correlates with in most cases Immunogenicity; von Burg et al., 1996). To the Immunogenicity of the selected peptide or peptide To determine equivalents, methods such. B. from Sette et al., 1994, in combination with quantitative MHC binding assays can be used. Alternatively, the immunogenicity of the selected one Peptide via known in vitro CTL induction Methods (as below for ex vivo CTL induction described) can be tested. The principle of in method for the Selection of peptides that trigger a is capable of cellular immune response WO 97/30721, the disclosure of which is hereby expressly stated Reference is described. A general one Strategy for obtaining efficient immunogenic peptides, which is suitable in the context of the present invention, was also described by Schweighoffer, 1997.

Instead of using the original peptides included in the Binding groove of MHC-I or MHC-II molecules fit, peptides derived from 9D7 unchanged, can be based on the  Original peptide sequence specified minimum requirements variations in anchor positions and length be made, provided that through these variations effective immunogenicity of the peptide itself is made up of its affinity to the MHC molecule and its ability to target T cell receptors stimulate, not only is not impaired, but is preferably reinforced. In this case become artificial peptides or peptide equivalents used according to the requirements of Binding ability to an MHC molecule are designed.

Peptides modified in this way are referred to as "heteroclitic peptides". They can be obtained using the following methods:
First, the epitopes of MHC-I or MHC-II ligands or their variation z. B. made according to the principle described by Rammenee et al., 1995. The length of the peptide in the case of its adaptation to MHC-I molecules preferably corresponds to a minimum sequence of 8 to 10 amino acids with the required anchor amino acids.

If necessary, the peptide can also on the C and / or on N-terminus can be extended if this extension the ability to bind to the MHC molecule is not impaired or the extended peptide on the Minimal sequence can be processed cellularly.

The modified peptides are then applied to their Detection by TILs (tumor-infiltrating lymphocytes, on CTL induction and on enhanced MHC binding and  Immunogenicity tested as described by Parkhurst et al., 1996, and Becker et al., 1997.

Another within the scope of the present invention suitable method for finding peptides with stronger immunogenicity than that of natural ones 9D7 peptides consist of screening peptide libraries with CTLs that are naturally occurring on tumors Recognize 9D7 peptides as described by Blake et al., 1996, described; in this context the use combinatorial peptide libraries proposed to To design molecules that were restricted by MHC-I Imitating CTLs recognized tumor epitopes.

The 9D7 polypeptides of the present invention or immunogenic fragments or peptides derived therefrom can be recombinant or by means of peptide synthesis are produced as described in WO 96/10413, the disclosure of which is hereby incorporated by reference. For the recombinant production becomes the corresponding one DNA molecule into one using standard methods Expression vector inserted into an appropriate one Host cell transfected, the host under appropriate Expression conditions cultured and the protein cleaned. For the chemical synthesis of 9D7 peptides conventional methods can be used, e.g. B. in Commercially available automatic peptide synthesizers.

Alternative to natural 9D7 peptides or heteroclitic peptides can be substances that such Pretend peptides, e.g. B. "Peptidomimetic" or "retro inverse peptides "can be used. To test these Molecules with a view to therapeutic  Usability in a tumor vaccine will be the same Methods applied as above for the natural ones 9D7 peptides or 9D7 peptide equivalents.

The TAA of the designation 9D7 according to the present Invention and the protein fragments derived from it Peptides or peptide equivalents or peptidomimetics can be used in cancer therapy, e.g. B. um to induce an immune response against tumor cells that express the corresponding antigen determinants. They are preferably used for the therapy of tumors with strong 9D7 expression used, especially in Renal cell, lung, colon and breast cancer as well in Hodgkin's lymphoma.

The immune response in the form of an induction of CTLs can can be effected in vivo or ex vivo.

For the in vivo induction of CTLs a pharmaceutical composition containing as effective Component the TAA 9D7 or derived from it Fragments or peptide (s) administered to a patient, of a tumor disease associated with TAA suffers, whereby the amount of TAA (peptide) must be sufficient, for an effective CTL response to the antigen-bearing To achieve tumor.

The invention thus relates in a further aspect a pharmaceutical composition for the parenteral, topical, oral or local administration. The parenteral composition is preferably used Administration, e.g. B. for subcutaneous, intradermal or intramuscular use. The 9D7 TAAs / peptides are in a pharmaceutically acceptable, preferably  aqueous, carrier dissolved or suspended. The Composition can also include common excipients such as Buffers, etc. included. The TAAs / peptides can be used alone or in combination with adjuvants, e.g. B. incomplete Freund's adjuvant, saponins, aluminum salts or, in a preferred embodiment, polycations such Polyarginine or polylysine can be used. The Peptides can also attach to components that make up the Support CTL induction or activation, be bound, e.g. B. on T helper peptides, lipids or Liposomes, or they are shared with these Substances and / or together with immunostimulating Substances, e.g. B. cytokines (IL-2, IFN-γ) administered. Methods and formulations used to manufacture and Administration of the pharmaceutical according to the invention Composition are suitable are in WO 95/04542 and WO 97/30721, the disclosure of which is hereby incorporated by reference is described.

9D7 (fragments) or 9D7 peptides can also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumor expressing 9D7 is induced by looking at the CTL progenitor cells together with APCs and 9D7 peptides or 9D7 protein incubated. Then the activated CTLs are left expand, after which it is administered to the patient again become. Alternatively, APCs can be loaded with 9D7 peptides become what leads to efficient cellular activation Immune responses to 9D7 positive tumors can result (Mayordomo et al., 1995; Zitvogel et al., 1996). A suitable method for peptides on cells, e.g. B. Charging dendritic cells is described in WO 97/19169 revealed.  

In one embodiment of the invention, a Combination of several different 9D7 peptides or 9D7 peptide equivalents applied. In another Embodiment are 9D7 peptides with from other TAAs derived peptides combined. The selection of Peptides for such combinations are considered encountered the detection of different MHC types, in order to cover the widest possible patient population, and / or it is as wide as possible Indication spectrum turned off by multiple peptides different tumor antigens can be combined. The Number of peptides in a pharmaceutical Composition can vary over a wide range fluctuate, typically includes one clinically applicable vaccine 1 to 15, preferably 3 to 10 different peptides.

The peptides according to the invention can also be used as diagnostic reagents are used.

For example, the peptides can be used to test a patient's response to the humoral or cellular immune response elicited by the immunogenic peptide. This makes it possible to improve a treatment protocol. For example, depending on the dosage form (peptide, total protein or DNA vaccine) of the TAA, the increase in precursor T cells in the PBLs which are reactive to the defined peptide epitope can be investigated (Robbins and Kawakami, 1996 and references cited therein) . In addition, the peptides or the total protein or antibodies directed against the TAA can be used to predict the risk of a patient with a 9D7-associated tumor, or to characterize the course of the disease of a 9D7-positive tumor (e.g. by immunohistochemical analyzes of primary tumor and metastases). Such a strategy has proven successful several times, e.g. B. Detection of the estrogen receptor as a decision-making basis for endocrine therapy in breast cancer; c-erbB-2 as a relevant marker in the prognosis and course of therapy for breast cancer (Raviaioli et al., 1998; Revillion et al., 1998); PSMA ("prostate specific membrane antigen") as a marker for epithelial cells of prostate cancer in serum or by using a ¹¹¹ In-labeled monoclonal antibody against PSMA in immunoscintigraphy for prostate cancer (Murphy et al., 1998 and included references); CEA ("carcinoembryonic antigen") as a serological marker for the prognosis and course in patients with colorectal cancer (Jessup and Loda, 1998).

The present invention relates in a further Aspect of isolated DNA molecules encoding a Protein with the immunogenic properties of 9D7 or for fragments of it.

The present invention preferably relates to a isolated DNA molecule containing a polynucleotide with the contains sequence shown in SEQ ID NO: 1 or the contains a polynucleotide associated with a polynucleotide the sequence shown in SEQ ID NO: 1 below stringent conditions hybridized.

The DNA molecule according to the invention or fragments thereof code for an immunogenic (poly) peptide of  Designation 9D7 with that shown in SEQ ID NO: 2 Amino acid sequence or for those derived therefrom Protein fragments or peptides; with that are DNA molecules included by the degeneration of the genetic Codes deviations from that shown in SEQ ID NO: 1 Have sequence.

The invention also relates to DNA molecules that pass through conservative exchange of amino acids conditional Deviations from that shown in SEQ ID NO: 1 Sequence if they are for a 9D7 derivative or Fragments or peptides with the for use as Tumor vaccine desired immunogenic properties encode.

The DNA molecules according to the invention can, in comparison to the molecule shown in SEQ ID NO: 1, at the 5 'end up to one, possibly upstream from position 324 located start codon to be extended.

The 9D7 DNA molecules of the present invention or the corresponding RNAs, which are also the subject of present invention, like those thereof encoded (poly) peptides, for the immunotherapy of Cancer used.

In one embodiment of the invention DNA molecules encoding the natural 9D7 used. As an alternative to the natural 9D7 cDNA or Fragments thereof can use modified derivatives become. These include sequences with modifications, with a protein (fragment) or peptides encode stronger immunogenicity, whereby for the Modifications at the DNA level have the same considerations  apply as for the peptides described above. A another type of modification is the stringing together numerous sequences coding for immunological relevant peptides, like a string of pearls ("string-of-beads"; Toes et al., 1997). The sequences can also be done by adding auxiliary elements be modified, e.g. B. Functions that a more efficient delivery and processing of the immunogen ensure (Wu et al., 1995). For example by adding a localization sequence to the endoplasmic reticulum ("ER targetting sequence") the processing and thus the presentation and ultimately the immunogenicity of the antigen can be increased.

The present invention relates in a further Aspect of a recombinant DNA molecule, the 9D7-DNA contains.

The DNA molecules of the present invention can preferably in recombinant form as plasmids, directly or as part of a recombinant virus, or Bacteria are administered. In principle, everyone can gene therapy method for the immunotherapy of Cancer based on DNA ("DNA vaccine") on 9D7-DNA are used, both in vivo and ex vivo.

Examples of in vivo administration are direct injection of "naked" DNA, either intramuscularly or using a gene gun from who has shown that they are used to form CTLs against tumor antigens. Examples of recombinant Organisms are vaccinia virus, or adenovirus  Listeria monocytogenes (an overview was provided by Coulie, 1997). Furthermore, synthetic Carriers for nucleic acids, such as cationic lipids, Microspheres, microspheres or liposomes for the in vivo administration of nucleic acid molecules, coding for 9D7 peptide can be used. Similar to For peptides, various excipients can be used Enhance immune response, be co-administered, e.g. B. Cytokines, either in the form of proteins or for them coding plasmids. The application can optionally using physical methods, e.g. B. Electroporation.

An example of ex vivo administration is Transfection of dendritic cells, such as Tuting, 1997, or other APCs described as cellular Cancer vaccine are used.

The present invention thus relates to one Another aspect is the use of cells that are 9D7 express, either on its own or, in modified form, if necessary, after transfection with the corresponding coding sequence for which Production of a cancer vaccine.

The invention relates in a further aspect Antibodies against 9D7 or fragments thereof. Polyclonal Antibodies can be carried out in a conventional manner Immunization of animals, especially rabbits, by injection of the antigen or fragments thereof, and subsequent purification of the immunoglobulin be preserved.  

Anti-9D7 monoclonal antibodies can be used Standard protocols according to those of Köhler and Milstein, 1975, principle can be obtained by Animals, especially mice, then immunized antibody-producing cells of the immunized animals be immortalized, e.g. B. by fusion with Myeloma cells, and the supernatant obtained Hybridomas using standard immunological assays anti-9D7 monoclonal antibody is screened. For the therapeutic or diagnostic use in humans can, if necessary, on these animal antibodies chimerized in a conventional manner (Neuberger et al., 1984, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al., 1995).

Human anti-9D7 monoclonal antibodies (fragments) can also be obtained from so-called "phage display libraries" (winter et al., 1994, Griffiths et al., 1994, Kruif et al., 1995, Mc Guiness et al., 1996) and by means of transgener Animals (Brüggemann et al., 1996, Jakobovits et al., 1995).

The anti-9D7 antibodies according to the invention can be used in immunohistochemical analysis for diagnostic purposes be used.

In a further aspect, the invention relates to Use of 9D7-specific antibodies to selectively add any substances to or into a tumor bring that expresses 9D7. Examples of such Substances are cytotoxic or radioactive agents Nuclides, the effect of which is to localize the tumor to harm. Because of the tumor-specific expression  of 9D7 are little or no Expected side effects. In another aspect can with the help of 9D7 antibodies substances for Visualization of tumors expressing 9D7 be used. This is for the diagnosis and the Evaluation of the course of therapy is useful.

Therapeutic and diagnostic applications of Antibodies in question for anti-9D7 antibodies come are described in WO 95/33771 for.

Figure overview

Fig. 1 transcription of 9D7 in normal tissues and RCC:
Semi-quantitative RT-PCR of RNA from kidneys, heart, leukocytes, liver, lungs, testis and four renal cell carcinomas,

Fig. 2 transcription of 9D7 in normal tissues:
Northern blot analysis of mRNA from 16 normal tissues,

Fig. 3 sections in situ hybridization of 9D7 RNA with renal cell carcinoma and normal kidney tissue.

example 1 RDA (Representational Difference Analysis) by Renal cell carcinoma and normal kidney tissue

Biopsies of human renal cell carcinomas of the clear cell type (RCC, renal cell carcinoma) and normal kidney tissue from the same patient were shock-frozen in liquid N ₂ immediately after the surgical removal and stored at -80 °. For the isolation of RNA, serial 20 µm sections were made at -20 ° in a cryomicrotome (Jung CM1800, Leica) and dissolved directly in 4 M guanidinium thiocyanate / 1% β-mercaptoethanol. This sample was subjected to ultracentrifugation over a CsCl gradient (Sambrook, 1989). Starting with 60 µg total RNA from renal cell carcinoma RCC6 and 350 µg total RNA from the autologous normal kidney N6 and another normal kidney tissue (N3), RDA (Diatchenko et al.; Hubank and Schatz,) was used using the PCR-select ^TM kit (Clontech, Palo Alto) according to the manufacturer's protocol: RNA from RCC ("tester") and normal kidney tissue ("driver") was used to isolate poly-A (+) RNA using the PolyATtract Kit (Promega) used according to the manufacturer's protocol. After the synthesis of double-stranded cDNA using oligo-dT, it was digested with RsaI. The same parts of "tester cDNA" were ligated either with the adapters A or B and then hybridized separately with an excess of "driver cDNA" at 65 ° C. The two approaches were then combined and subjected to a second hybridization with freshly denatured "driver cDNA". The enriched "tester" -specific cDNAs were then amplified exponentially by PCR with primers specific for adapters A and B, respectively. For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific "nested" primers. The product resulting from this reaction was ligated into the pCRII vector (Invitrogen) and then transfected into competent E. coli (OneShot ™, Invitrogen). 1920 positive transformants (blue-white selection) were cultivated in 96-well blocks in LB-Anip medium for 24-48 h at 37C. 200 µl aliquots of the E. coli suspensions were heated to 100 ° C for 10 minutes. After briefly centrifuging off the bacterial residues, 140 µl clear supernatant (~ 1 µg plasmid DNA) was taken up in 60 µl 20 × SSC and immobilized on equilibrated nylon membranes (Hybond-N, Amersham) according to the standard methods for producing DNA dot blots (Sambrook , 1989). The remaining bacterial cultures were stored as gycerin stock cultures at -80 ° C.

It became a 1920 cDNA subtraction library Individual clones obtained, both as E. coli glycerol Stock cultures as well as immobilized plasmids DNA dot blots.

Example 2 Selection of "Tumor Testis" Antigens by Hybridization of the cDNA subtraction library with a Testis-specific cDNA library

"Human Testis-Specific PCR-Select ™ cDNA" (Clontech, Palo Alto) was amplified according to the manufacturer's instructions using 11 cycles of PCR. Aliquots were labeled according to the "random priming" method (HiPrime Kit, Boehringer Mannheim) with [α- ³² P] dCTP (NEN, Boston) and according to standard instructions (Sambrook, 1989) under stringent conditions (65 ° C) with the under Example 1 described DNA dot blots hybridized. Clones that hybridize with the "Human Testis-Specific PCR-Select ™ cDNA" were visualized using standard autoradiography (Xomat DR film, Kodak). The result of Example 2 allowed the selection of 150 clones, which are predominantly transcribed in renal cell carcinoma RCC6 and in Testis. These clones are candidates for the class of "tumor testis" antigens.

Example 3 DNA sequencing and annotation of "tumor testis" Antigen candidate

Plasmid DNA from 62 clones, based on Example 2 results obtained had been selected isolated according to the manufacturer's instructions (QIAgen) and according to the Sanger method on an ABI-Prism device sequenced. The sequences determined in this way were using the BioWorkBench software (GeneData, Basel) annotated and database comparison (genebank) subjected. This allowed the identification of 33 known and 29 unknown genes. For the latter only EST entries existed. Known genes were not treated further. For the 29 unknown Gene was an estimate of the expression profile carried out: ESTs were made for everyone in databases with <95% identity (BLAST) to experimental determined sequence the starting tissue for the corresponding cDNA library checked. there has been a Subdivision into i) critical normal tissue, ii) fetal, "dispensable" and immune privileged tissues and  iii) tumors and cell lines made. On the base of this "virtual mRNA profile" were 16 clones for that no ESTs were found in group i) for selected another experimental analysis.

Example 4 Transcriptional analysis of the candidate clones in Renal cell carcinoma and normal kidney

Between 2 and 5 µg total RNA from tumor or Normal tissues were analyzed using SuperscriptII (Gibco) or AMV-RT (Promega) according to the manufacturer recommendations reverse transcribed. For every individual RNA sample was a second approach without reverse transcriptase as a control for contamination performed by chromosomal DNA. Alternatively, were Plasmid cDNA libraries from human heart, leukocytes, Kidney, liver, lungs and testis (Gibco) used.

Quality and quantity of the cDNAs was determined by PCR β-actin specific primers (SEQ ID NO: 29 and 30) 20, 25 and 30 cycles (40 "94 ° C, 45" 55 ° C, 1'30 " 72 ° C, from cycle 11 with 30 "55 ° and an extension checked by 3 "55 ° for each further cycle).

9D7 cDNA was generated by 30 cycles of the same program the 9D7-specific primers according to SEQ ID NO: 3 and 4 amplified. Analogously, the other 15 candidate Clones examined with specific primers. The PCR products were analyzed using agarose gel electrophoresis and ethidium bromide staining. For 5 of the 16 examined clones could be clearly identified by RT-PCR Overexpression in RCC6 renal cell carcinoma compared  to the autologous normal kidney tissue N6. As additional positive control became that for all clones Occurrence in Testis cDNA examined.

Example 5 Quantitative comparison of 9D7 mRNA Expression between tumors and normal tissues

Pools of 9 µg total RNA from 3 tumor or Normal tissues were obtained after pretreatment with DNase I (Boehringer Mannheim) and subsequent Phenol extraction using oligo-dT and AMV-RT (Promega) reverse transcribed. The number of copies of the 9D7 cDNA or Glyceraldehyde-3-phosphate dehydrogenase cDNA (GAPDH) as a reference for the quantity and quality of the entire cDNA was performed using a 5 'nuclease PCR assay (TaqMan®PCR, PE Applied Biosystems). As a primer or labeled TaqMan probes for cDNA quantification served the primers according to SEQ ID NO: 3 and 4 and SEQ ID NO: 31 (at the 5 'end with 6-carboxy-fluorescein and at 3'-end marked with 6-carboxy-tetramethyl-rhodamine) or for GAPDH the primers according to SEQ ID NO: 25 and 26 and the probe according to SEQ ID NO: 32 (at the 5 'end with Tetrachloro-6-carboxy-fluorescein and at the 3 'end with 6-carboxy-tetramethyl-rhodamine labeled). The PCR (40 cycles {15 "95 ° C, 1 '60 ° C} for 9D7 or 40 cycles {15 "95 ° C, 1 '66 ° C} for GAPDH) and the online Fluorescence measurements were made with an ABI PRISM 7700 Sequence detection system (PE Applied Biosystems).

Example 5 shows that 9D7 in pools of Renal cell carcinoma and lung carcinoma (SCC) round Is transcribed 1.5 to 6.5 times more than GAPDH. In other types of tumors, the transcription rate fluctuates  between 0.25-0.6 times GAPDH.

Normal liver and testis shows a value of about 10% the GAPDH transcription while the others examined normal tissue practically no transcription of 9D7 show (Tab. 1).  

Example 6 Expression profile of 9D7 in tumor and normal tissues

One of the 15 candidates (designation: 9D7) showed strong RT-PCR transcription in all tested renal cell carcinoma and several others Tumor types. In most normal tissues however, no transcription was found.

Fig. 1 shows a comparison between four normal kidney (N1, 2, 4, 5) and four renal cell carcinomas (RCC1, 2, 3, 6); In addition, cDNA from cardiac muscle (he), leukocyte (leu), kidney (ki), liver (left), lung (left) and testis (tes) were tested in this experiment, and a control with β-actin primers was carried out. These results were confirmed in independent experiments with three different primer pairs (SEQ ID NO: 3 and 4, SEQ ID NO: 5 and 10 and SEQ ID NO: 11 and 15). For Northern blot analysis, human multiple tissue Northern blots (Clontech, Palo Alto) were hybridized with the [α- ³² P] dCTP (NEN, Boston) labeled 170 bp 9D7 PCR product at 65 ° for 16 h. The visualization was carried out using standard autoradiography (Xomat AR film, Kodak).

Fig. 2 shows the result of this analysis: from 16 normal tissues (1: leukocytes, 2: colon, 3: Small intestine, 4: ovary, 5: test, 6: prostate, 7: thymus, 8: spleen, 9: pancreas, 10 : Kidney, 11: skeletal muscle, 12: liver, 13: lung, 14: placenta, 15: brain, 16: heart muscle), only a transcript of ~ 2.2 kb in size was shown in the heart muscle. However, the low intensity of the signal makes an immunologically relevant expression seem unlikely.

The result of all experiments regarding the mRNA profile of 9D7 (compiled from RT-PCR, quantitative RT-PCR and Northern blot analysis) is summarized in Table 2. Example 6 shows that 9D7 in a high percentage of tumors of various indications strongly is transcribed while examined in all Normal tissues have no or only weak transcription was found.

Example 7 in situ hybridization of a 9D7-specific RNA probe with renal cell carcinoma and normal kidney tissue

A 280 bp NotI fragment with 220 bp sequence information from 9D7 was subcloned into pBluescript and then competent E. coli for the transformation DH5-α used. The RNA probes were synthesized through in vitro transcription with T3 or T7 RNA Polymerase (sense: Sac I, T3; antisense: BamH I, T7) with the addition of digoxigenin UTP according to the Manufacturer recommendations (DIG RNA labeling kit, Boehringer Mannheim). After cleaning the transcripts (Sephadex G50 quick spin columns, Boehringer Mannheim) the labeling efficiency by means of dot blot certainly. The in situ hybridization was made after Standard procedures carried out (Boehringer Mannheim, Nonradioactive In Situ Hybridzation Application Manual, 1996): 5 µm frozen sections were obtained Paraformaldehyde fixation, acetylation and de- or Re-hydrated and denatured overnight at 52 ° C hybridized in a humid chamber. After the  usual washing steps were unspecific Protein binding sites 15 min with 10% FCS and 3% goat serum (DAKO) saturated. The visualization the probe hybridized to 9D7 mRNA was performed by Incubation with an α-DIG-AP conjugate together with NBT / BCIP (Boehringer Mannheim) Development at 4 ° C in the dark.

Fig. 3 shows the result of an in-situ hybridization of renal cell or normal kidney tissue with a 9D7-specific digoxigenin-UTP labeled RNA probe ( "antisense"): the tumor tissue shows strong and homogeneous staining, whereas the normal tissue does not react; the specificity of the color is shown by the negative control ("sense").

Example 7 shows that 9D7 in tumor cells from Renal cell carcinoma tissues, but not in any Cell type is transcribed from normal kidney tissue and thus confirms those described in Examples 4-6 Results.

Example 8 Cloning 9D7

Clone 9D7 owns between those through the RDA introduced a 221 bp insert of a unknown human gene. Database comparisons showed homology from 9D7 to rat SM20, 69 bp being the C-terminal coding region (accordingly 23 amino acids), the stop codon and 149 bp 3'-untranslated region from SM20 to 221 bp from 9D7 correspond in "antisense" orientation. The  23 amino acids show only 2 exchanges between Rat and human sequence. SM20 was considered a through Immediate early gene growth factors induced and specific marker for smooth aortic cells Muscles described (Wax et al.,; Wax et al.,). For complete cloning of the human sequence proceeded as follows: a UniGene analysis (National Center for Biotechnology Information) revealed the following 9D7 homologous ESTs: AA234127, AA233899, AA917421, AA878927. Total RNA from tumor cell lines 786-0 (ATCC # CRL-1932) or A549 (ATCC # CCL 185) isolated using TRIzol (Gibco) and using Superscript II MMLV-RT (Gibco) and an adapter oligo-dT primer (SEQ ID NO: 27) according to the manufacturer's instructions reverse transcribed. PCR amplification of this cDNA with the primers according to SEQ ID NO: 5, 6, 7 and 10 resulted in the original primers (SEQ ID NO: 3 and 4) and with each other according to the homologous sequences expected fragments. The longest fragment of 804 bp was achieved by combining the primers according to SEQ ID NO: 10 and won 5. The primers of the sequence SEQ ID NO: 8 and 9 are in a sequence region of EST-AA234127, that show no homology to SM-20 consequently with none of the other primers Amplification product.

Cloning of the 3 'end: 10 µg total RNA from the renal cell carcinoma cell line 786-0 were reverse transcribed and an aliquot of this cDNA was subjected to a PCR, the program of which begins with high annealing temperatures, so that only the gene-specific primer binds to the cDNA (T _m ~ 93 ° C, SEQ ID NO: 22), while the second primer (T _m ~ 53 ° C, SEQ ID NO: 28) only binds to the newly synthesized DNA template at a lower temperature. This so-called "touch-down PCR" (Mastercycler Gradient, Eppendorf) was carried out under the following conditions:
20 cycles {15 "95 ° C, 30" 80 ° C (reduced by 1 ° C per cycle), 2 '72 ° C} and 20 cycles {15 "95 ° C, 30" 50 ° C, 2' 72 ° C}.

Cloning of the 5 'end: 1 µg total RNA from the Renal cell carcinoma cell line 786-0 was developed after one modified SMART ™ protocol (SMART ™ PCR cDNA Synt.hesis Kit, Clontech) using the primers SEQ ID NO: 4 and Smart-II (SEQ ID NO: 23) revers transcribed: the cDNA was compared with the 9d7-specific primers according to SEQ ID NO: 4 and below Addition of RNasin (Promega) synthesized. The 9D7-specific primers according to SEQ ID NO: 11 were found in a final concentration of 0.4 pN, the SMART-PCR primer (SEQ ID NO: 24) with 0.1 µM. The PCR amplification was carried out with 40 cycles each (15 "95 ° C, 30" 65 ° C, 2 '72 ° C).

Aliquots of the PCR batches were made directly in the pCR2.1 vector (Invitrogen) ligated and then in competent E. coli (OneShot ™, Invitrogen) transformed. Positive clones were classified as blue and white Selection sequenced. The sequencing revealed 50 additional nucleotides labeled with EST-AA878927 matched to the 3 'end and confirmed until then 870 additional nucleotides to the previously confirmed 5 'end. PCR amplification of cDNA from tumor cell lines (786-0, A549) and tissue biopsies from Renal cell carcinomas with the primers according to SEQ ID NO: 14  to 21 resulted with the original primers as well among themselves the expected after cloning Fragments. The identity of all PCR products was verified Sequencing verified. When sequencing several clones with or without an exon of 282 bp (Pos. 399-680) identified. The presence of both Splice variants were made using RT-PCR with the primers according to SEQ ID NO: 11 and 15 or 13 in several Tumor samples (renal cell carcinoma, lung carcinoma / SCC, Colon carcinoma / AC) detected.

Example 9 Potential MHC binding peptides in the coding Region of 9D7

Potential peptide epitopes within the coding Region of 9D7 according to SEQ ID NO: 2 were determined using the by Parker et. al., 1994 algorithms described below Based on known motifs (Rammenee et al. 1995) carried out. For the main HLA types, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, candidate peptides have been identified by which is expected to match the appropriate HLA molecules bind and therefore immunogenic CTL epitopes represent; the peptides found are in Table 3 listed. By analogous procedure, others can potential peptide epitopes for other HLA types be determined.  

Table 1

Transcription of 9D7 in tumor and normal tissues relative to GAPDH

Table 2

mRNA expression profile of 9D7

Table 3

Immunogenic 9D7 peptide candidates

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SEQUENCE LOG

Claims (20)

1. tumor-associated antigen of the designation 9D7, characterized in that it has the amino acid sequence defined in SEQ ID NO: 2. 2. polypeptide, characterized in that it is the in SEQ ID NO: 2 defined sequence as partial sequence contains. 3. Immunogenic protein fragment or peptide, thereby characterized in that it is of that in claim 1 defined tumor-associated antigen is. 4. Immunogenic (poly) peptide according to one of the claims 1 to 3, which triggers a humoral immune response. 5. Immunogenic (poly) peptide according to one of the claims 1 to 3, the or its degradation products MHC molecules are presented and a cellular one Trigger immune response. 6. The immunogenic peptide of claim 5 selected from the group of peptides according to SEQ ID NO: 34 to 58. 7. Immunogenic (poly) peptide according to one of the claims 1 to 6 for the immunotherapy of Cancers in vivo or ex vivo, where the (Poly) peptide has an immune response against tumor cells of the patient that express 9D7.   8. Pharmaceutical composition for the parenteral, topical, oral or local Administration, characterized in that it is considered active component one or more immunogenic (Poly) peptides according to one of claims 1 to 6 contains. 9. Pharmaceutical composition according to claim 8, characterized in that they are different from Contains 9D7 derived immunogenic peptides. 10. Pharmaceutical composition according to claim 9, characterized in that they have one or more peptides derived from 9D7 mixed with von other tumor-associated antigens Contains peptides. 11. Pharmaceutical composition according to claim 9 or 10 that the peptides on at least two bind different HLA types. 12. Isolated DNA molecule coding for a protein with the immunogenic properties of in Claim 1 defined tumor-associated antigen or for fragments of it. 13. DNA molecule according to claim 12, coding for a immunogenic polypeptide of the designation 9D7 with the amino acid sequence shown in SEQ ID NO: 2 or for protein fragments derived therefrom or Peptides.   14. DNA molecule according to claim 13, characterized characterized in that it is a polynucleotide with the sequence shown in SEQ ID NO: 1 is or this contains or that it is a polynucleotide or contains this with a polynucleotide the sequence shown in SEQ ID NO: 1 below stringent conditions hybridized. 15. Recombinant DNA molecule containing a DNA Molecule according to one of claims 12 to 14. 16. DNA molecule according to one of claims 12 to 15, for cancer immunotherapy, where the 9D7- expressed by the DNA molecule (Poly) peptide has an immune response against tumor cells of the patient that express 9D7. 17. Use of cells that in claim 1 or Express 2 defined tumor-associated antigen, for the production of a cancer vaccine. 18. Antibodies against one of claims 1 to 6 defined (poly) peptide. 19. Antibody according to claim 18, characterized characterized as being monoclonal. 20. Antibody according to claim 18 or 19 for the Therapy and diagnosis of cancer that are associated with the expression of 9D7.