DE19936563A1 - Tumor Associated Antigen - Google Patents

Abstract

The invention relates to tumor-associated antigens, immunogenic peptides derived therefrom and the DNA molecules coding therefor as well as to their utilization in cancer immunotherapy.

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. Both in animal models and in vitro cultivated human cell culture systems, the T cell response against tumor cells could be enhanced 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).

Because of 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 cases is a processing and presentation of the antigen through so-called "professional antigen-presenting Cells "(APC) is necessary. Another possibility is the use of synthetic peptides (Melief et al., 1996), which corresponds to the corresponding T cell epitopes of the Antigen and correspond either to the outside APC loaded (Buschle et al., 1997; Schmidt et al., 1997) or recorded by the APC and intracellularly to which MHC-I molecules are transferred. The therapeutically most efficient form of application for a Antigen is generally defined in clinical Studies determined.

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 are mainly expressed by tumor cells and not or only in so-called "critical" normal tissues in a lower concentration than in tumors. Critical normal tissues are essential tissues; a immune response directed against them could be under Serious and sometimes fatal consequences to have. Secondly, the antigen should not only Primary tumor, but also present in the metastases his. Furthermore, it is broad clinical use of the antigen desirable if it in several types of tumors in high concentration is available. Another prerequisite for the Suitability of a TAA as an effective part of a Vaccine is the presence of T cell epitopes in the amino acid sequence of the antigen; derived from the TAA Peptides are said to result in an in vitro / in vivo T cell response lead ("immunogenic" peptide). Another one Selection criterion for a clinically widely applicable immunogenic peptide is the frequency with which 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; Wölfel 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 one derived from a pancreatic carcinoma patient Cell line and a pool of 11 different Normal tissues a cDNA subtraction library was produced. To generate the for the subtractive hybridization necessary cDNA fragments of "tester" and "driver" was deviated from  original protocol (Diatchenko et al., 1996) a Mixture of 6 different restriction enzymes used. The use of a mixture of different Restriction enzymes, the 6 base pairs as Need recognition sequence compared to original protocol (Diatchenko et al., 1996) the following advantages: a) by choosing two each Restriction enzymes, the recognition sequences of which Combination of 6 bases A / T (e.g. Ssp I: AATATT) or C / G (e.g. Nae I: GCCGGC) or A / C / G / T (e.g. EcoR V: GATATC) are shown, both GC and Regions of a gene rich in AT equally cut, creating a homogeneous representation of the entire gene region as restriction fragments becomes; b) it is also possible in statistical mean larger cDNA fragments of the Obtain candidate gene (approximately 800 bp), which in turn in the subsequent analysis (sequencing and Annotation) and the cloning of the "full-size" cDNA from is a big advantage. In the original protocol (Diatchenko et al., 1996) was only a 4-base recognizing Restriction enzyme (Rsa I) used that leads to a average fragment length of 256 bp leads and specifically CG or AT-rich regions cannot process. Around by the longer insert cDNA fragments to meet changed hybridization kinetics, the PCR protocol was described as in Example 2 changed.

For the selection of antigens overexpressed in the tumor the cDNA clones obtained were first separated and each one a glycerol stock culture, one Plasmid preparation and an insert  Collection of PCR fragments in 96-well plate format established. Initially, 50 were selected at random cDNA fragments of the 345 clones of the subtractive cDNA Library of pancreatic carcinoma for selection of im Tumor overexpressed antigens sequenced and with in Databases available sequences compared. Among the there were 12 unknown genes annotated which ESTs ("expressed sequence tags") entries in the Database existed. A clone, R11, indicated by its preferred presence in fetal tissues with a view suitable for use as a TAA EST Profile on. Further investigations using semi quantitative RT-PCR and Northern blot analysis confirmed the preferred expression in various Tumor (breast, kidney and pancreatic carcinoma) as well immune privileged tissues (testis, placenta and Adrenal gland) and little or no expression in Normal tissues. It is also from Northern Blot Experiments conclude that the R11 Transcript has a length of approximately 7.5 kb and possibly in the adrenal splice variants or homologous genes exist.

The human R11 cDNA was cloned from Testis, which sequence obtained is shown in SEQ ID NO: 1. The Sequence of R11 points to both nucleotide and no identity or homology at the protein level a known gene. The under the present The R11 cDNA obtained by the invention has one for 356 Continuous reading frames encoding amino acids, from which to assume based on the results obtained is that it is the C-terminal portion of the represents complete R11 antigen. The in the course  of the present invention has cloned R11 cDNA a length of 514 bp, the presence of a polyA tail at the 3 'end of the sequence for the Completeness of the cDNA speaks in this area.

Because of the present invention received data can not be excluded that Another ATG 5 'from the sequenced cDNA is present, which represents the start ATG; in this case encodes the present cDNA for the C-terminal Section of R11. Information about the 5 'end and a possibly further upstream coding DNA sequence can by standard molecular biological methods are obtained, e.g. B. using 5'-RACE ("rapid amplification of cDNA ends "). This method uses RNA, preferably mRNA, from cells or tissues in which R11 is transcribed (e.g. breast, kidney or Pancreatic carcinoma tissue) reverse transcribed and then with an adapter of known sequence ligated. A PCR with an adapter primer (binds specific to the adapter at the 5 'end of the cDNA) and an R11-specific primer (e.g. SEQ ID NO: 25) allows the amplification of corresponding R11 fragments. These PCR products can, as in Example 6 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 R11 or  the analysis of cDNA expression libraries with 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 R11 Clones that can be isolated upstream from the sequence information obtained 5 'end of the cDNA e.g. B. contain the promoter region of R11.

In the course of complete cloning of the R11 cDNA can to determine whether the in the area of the present cDNA section received an open reading frame Continues in the 5 'range and / or whether alternative reading frames are available.

The cDNA isolated in the context of the present invention has the nucleotide sequence given in SEQ ID NO: 1 on; it can be assumed (see above) that for the C- terminal portion of the tumor associated antigen (TAA) of the designation R11.

A protein expressed from the isolated CDNA has the amino acid sequence shown in SEQ ID NO: 2. The invention thus relates to a first aspect tumor-associated antigen of the designation R11, which the amino acid sequence given in SEQ ID NO: 2.  

In another aspect, the present concerns Invention of a tumor-associated antigen called R11, which is the one specified in SEQ ID NO: 2 Contains amino acid sequence as a partial sequence (this applies in the event that there is an extension in the 5 'direction of this open reading framework).

For the protein with that specified in SEQ ID NO: 2 Sequence is a product made by one approx. 7.5 kb transcript is translated, or that of the transcripts with a size of approx. 3.8 kb and 2.3 kb is translated, that of splice variants of the 7.5 kb transcripts as found in the tissue of the adrenal gland can be found or from transcripts of the homologous genes are derived.

The amino acid sequence shown in SEQ ID NO: 2 can Have deviations, e.g. B. those by Exchange of amino acids are required, provided that R11 derivative which is for use in a tumor vaccine has desirable immunogenic properties.

The natural amino acid sequence of R11 can may be modified by individual Amino acids exchanged in an R11 CTL epitope be compared to the natural R11 CTL Epitope, an increase in the affinity of R11 peptides to MHC-I molecules and thus an increased immunogenicity and ultimately increased reactivity towards To cause tumors. Modifications in the area of R11 epitopes can be found on the total R11 protein (this is from the APCs to the corresponding peptides  processed) or on larger R11 protein fragments or on R11 peptides (see below).

In another aspect, the present concerns Invention of immunogenic fragments and peptides derived from R11 are derived. The latter are referred to below as Denoted "R11 peptides". A first group are R11 peptides that have a humoral immune response (induction of antibodies). Such peptides are selected sections of R11 (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.

Also included are all those peptides that are made up of if necessary in the course of further cloning obtained N-terminal region are derived from R11.

It is known that tumor-associated antigens can have tumor-specific mutations that lead to an immunological distinction between tumor and Normal tissues (Mandruzzato et al., 1997; Hogan et al., 1998; Gaudi et al., 1999; Woelfel et al., 1995). The presence of tumor-specific R11 mutations is determined, expediently with the help of probes the cDNA according to the invention isolated from Testis, which R11 cDNA from one or more different Tumors cloned and the sequences obtained with Normal tissue R11 cDNA compared. It is expected that tumor R11 peptides from a normal tissue  R11 mutated sequence section compared to Normal tissue R11 peptides from the corresponding Section have increased immunogenicity. The The present invention thus relates to another Aspect R11 peptides derived from areas of a tumor-expressed R11, the tumor-specific mutations exhibit.

In therapeutic use, R11 peptides directly or in a modified farm (e.g. to KLH "keyhole limpet hemocyanine" coupled) administered and the formation of antibodies using common immunological assays, e.g. B. determined by ELISA.

Further, within the scope of the present invention preferred, R11 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, it must bind to an MHC molecule, the to patient treating the MHC molecule in his repertoire must have. Determination of the MHC subtype of Patients thus poses, in terms of triggering a cellular immune response, one of the essential ones Requirements for the effective application of a Peptide on this patient.

The sequence of a therapeutically used R11 peptide is by the respective MHC molecule given with regard to anchor amino acids and length.  

Defined anchor positions and lengths ensure that a peptide in 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 R11 peptides can be made 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 R11 peptides that represent CTL epitopes due to R11 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 to a peptide that binds to the MHC molecule and presented by this were motif summarized (e.g. Falk et al., 1991). So far a large number of both MHC peptide motifs also known from MHC ligands. One under the suitable method for searching for the present invention Epitopes of a known protein that are in a specific MHC-I molecule, was in one Review article by Rammenee et al., 1995,  described. It includes the following steps: First, the protein sequence is divided into sections examined that correspond to the anchor motif, where certain variations in peptide length and Anchorage are possible. If e.g. B. a motif 9-mer with Ile or Leu at the end can also 10-mers with a corresponding C-terminus into consideration pulled, as well as peptides with others aliphatic residues such as Val or Met at the C-terminus. In this way, a number of peptide candidates receive. These are based on the existence if possible many anchor remains that they already share with have known ligands, examined and / or then whether they are for different MHC molecules have "preferred" residues (according to the table of Rammenee et al., 1995). To weakly binding peptides to rule out binding assays carried out. If the requirements for the peptide Binding for certain MHC molecules are known the peptide candidates also on non-anchor residues be examined that are negative or positive affect the bond or make it possible (Ruppert et al., 1993). With this procedure is however, to consider that the peptide binding Motive for the search for natural ligands not alone is decisive; other aspects, e.g. B. the enzyme specificity during antigen processing, wear - in addition to the specificity of the MHC binding - contribute to the identity of the ligand. A method of doing this Aspects taken into account, and those within the present invention for the identification of immunogenic R11 peptides is suitable, u. a. of  Kawakami et al., 1995, applied to based on known HLA-A * 0201 motifs gp100 epitopes too 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 method described by Parker et al., 1994 and Rammenee et al., 1995 described algorithm for which most important HLA types, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, candidate peptides  of the C-terminal fragment of R11 identified by which is expected to match the appropriate HLA molecules bind and therefore immunogenic CTL epitopes represent; the peptides found are in Table 1 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 be determined; this also applies to peptide epitopes other HLA types.

After selecting R11 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 R11 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 checked as by Parkhurst et al., 199, and Becker et al., 1997, described.

Another within the scope of the present invention suitable method for finding peptides with stronger immunogenicity than that of natural ones R11 peptides consist of screening peptide libraries with CTLs that are naturally occurring on tumors Recognize R11 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 R11 polypeptide 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 R11 peptides  conventional methods can be used, e.g. B. in Commercially available automatic peptide synthesizers.

Alternative to natural R11 peptides or heteroclitic peptides can be substances that such Pretend peptides, e.g. B. "Peptidomimetics" 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 R11 peptides or R11 peptide equivalents.

The TAA of the designation R11 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 R11- positive tumors used, especially in the breast, Renal cell and pancreatic carcinoma.

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 R11 or fragments derived from it or peptide (s) administered to a patient who is a tumor associated with TAA, the amount of TAA (peptide) must be sufficient to produce one effective CTL response to the antigen-bearing tumor achieve.  

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 R11-TAA / 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 R11-TAA / peptides can alone or in combination with adjuvants, e.g. B. incomplete friend's adjuvant, saponins, Aluminum salts or, in a preferred Embodiment, polycations such as polyarginine or Polylysine. The peptides can also Components that are CTL induction or CTL activation support, be bound, e.g. B. 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 for Production and administration of the invention pharmaceutical composition are suitable are in WO 95/04542 and WO 97/30721, on their disclosure hereby referred to.

R11 fragments or R11 peptides can also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumor expressing R11 is induced by looking at the CTL progenitor cells together with APCs and R11 peptides or R11 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 R11 peptides become what leads to efficient cellular activation Immune responses to R11 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 97/1919 revealed.

In one embodiment of the invention, a Combination of several different R11 peptides or R11 peptide equivalents applied. In another Embodiment are R11 peptides with from other TAAs derived peptides combined. The selection of Peptides for such combinations will be examined 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 that 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 characterize the course of the disease of an R11-positive tumor (eg 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 (Ravaioli 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 R11 or for fragments of it.

In another aspect, the present concerns Invention an isolated DNA molecule that the in SEQ ID NO: 1 nucleotide sequence shown or  a polynucleotide that interacts with this DNA molecule stringent conditions hybridized.

Under "stringent conditions" z. B. understood:
Incubation overnight at 65 ° C-68 ° C with 6 × SSC (1 × SSC = 150 mM NaCl, 15 mM trisodium citrate), 5 × Denhardt's solution, 0.2% SDS, 50 µg / ml salmon sperm DNA, followed by Wash twice for 30 min with 2 × SSC, 0.1% SDS at 65 ° C, once for 30 min with 0.2 × SSC, 0.1% SDS at 65 ° C and if necessary, finally rinse with 0.1 × SSC, 0.1% SDS at 65 ° C.

In another aspect, the present concerns Invention of an isolated DNA molecule containing a Polynucleotide of the sequence shown in SEQ ID NO: 1 contains as a partial sequence or a polynucleotide contains that with a polynucleotide of this sequence hybridized under stringent conditions.

The DNA molecules or fragments thereof according to the invention code for (poly) peptides with the designation R11, consisting of or containing those in SEQ ID NO: 2 amino acid sequence shown, or for thereof derived protein fragments or peptides; with that DNA molecules, which are caused by the degeneration of the genetic codes deviations from that in SEQ ID NO: 1 have shown sequence.

The invention also relates to DNA molecules that pass through Mutation in an exchange of amino acids in the SEQ ID NO: 2 lead protein sequence shown, if they are for an R11 derivative or fragments or  Peptides with those for use as tumor vaccines encode desired immunogenic properties.

The R11 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 coding for natural R11 polypeptides used. As an alternative to the natural R11 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 that contains the R11 DNA contains.

The R11 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 R11 DNA applied, 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 R11 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 have R11 express, either on its own or, in possibly modified farm, after transfection with the corresponding coding sequence for which Production of a cancer vaccine.

The invention relates in a further aspect Antibodies against R11 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-R11 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-R11 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-R11 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-R11 antibodies according to the invention can be found in immunohistochemical analysis for diagnostic purposes be used.

In a further aspect, the invention relates to Use of R11-specific antibodies to selectively add any substances to or into a tumor bring that expresses R11. 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 R11 are little or no Expected side effects. In another aspect can with the help of R11 antibodies substances for Visualization of tumors expressing R11 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-R11 antibodies come are described in WO 95/33771.

The TAA of the designation R11 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 R11- positive tumors used, especially in the breast, Renal cell and pancreatic carcinoma.

Figure overview

Fig. 1 transcription of R11 in tumor and normal tissues: semiquantitative RT-PCR.

Fig. 2 Transcription of R11 in tumor tissues and normal tissues: Qualitative PCR.

Fig. 3 Northern Blot Analysis of R11 in normal tissues.

Fig. 4 Transcription of R11: Qualitative RT-PCR from RNA from human tumor cell lines.

Fig. 5 Modified region of the pCR3.1 (+) vector.

example 1 Cell culture of the cell line MZ.PC2 m7 # 1 B7.1 # 3 derived from a human pancreatic carcinoma and isolation of the poly A ⁺ RNA

The cell line MZ.PC2 m7 # 1 B7.1 # 3 is derived from human pancreatic carcinoma (MZ.PC2); it was obtained as follows: First, the tumor cells were passaged once in the mouse and a clone was selected for further studies (MZ.PC 2m7 # 1). This clone was developed under standard conditions (Ausubel et al., 1994) with a eukaryotic vector (pEE-BOS; promoter comes from the human EF-1alpha gene, selection marker: Puromycin; Mizushima and Nagata, 1ß90), which contains the cDNA of human B7.1 Gens (Selvakumar et al., 1992) contains, transfected. A clone MZ-PC2 m7 # 1 B7.1 # 3 was selected and grown up in T150 cell culture flasks. RPMI 1640 (Gibco plus 4 g / L glucose) with 10% heat-inactivated, fetal calf serum and 2 mN L-glutamine served as the nutrient medium. Every 3 to 4 days, the cells were cleaved by trypsinization 1: 5 for propagation. After approximately 80% confluence had been reached, 4 ml of a trypsin solution (data per liter: 8 g NaCl, 0.2 g KCl, 1.13 g Na ₂ HPO ₄ -hydrous, 0.2 g KH ₂ PO ₄ , 100 ml 2.5% trypsin solution, 1 g EDTA-Na salt; pH 7.2-7.4) were used to harvest the cells. A total of 2 × 10 ⁷ cells were used to isolate the RNA according to the manufacturer's protocol (RNeasy Minikit, QIAgen). Starting from approx. 100 µg total RNA, the Oligotex Kit (QIAgen) was used to isolate polyA ⁺ RNA in accordance with the manufacturer's protocol. Subsequently, starting from approx. 0.5 mg polyA + RNA, the cDNA synthesis was carried out according to the manufacturer's instructions (Clontech Marathon protocol).

Example 2 Representative Difference Analysis ("Representational Difference Analysis "; RDA) der Pancreatic carcinoma cell line MZ.PC2 m7 # 1 B7.1 # 3 versus a pool of 11 normal tissues

Starting from approx. 0.5 µg poly-A (+) the Pancreatic tumor cell line MZ.PC2 m7 # 1 B7.1 # 3 and one Pool of 2.5 µg poly-A (+) RNA from 11 normal tissues (Clontech) bone marrow, heart, kidney, liver, lungs, Pancreas, skeletal muscle, spleen, thymus, small intestine and The RDA study (Diatchenko et al.,; Hubank and Schatz,) using the PCR-select ™ kit (Clontech, Palo Alto) according to the manufacturer Protocol carried out: the RNA of the Pancreatic tumor cell line as "tester", the des Normal tissue pools as "driver" according to the Manufacturer protocol used. In contrast to original protocol was after the synthesis of double-stranded cDNA using oligo-dT with the cDNA 6 restriction enzymes: EcoRV, NaeI, NruI, ScaI (Promega), SspI, StuI (TaKaRa) in Promega buffer A2 Hours at 37 ° C and after increasing the NaCl Concentration on 150 mM for a further 2 hours at 37 ° C cut. The use of this mixture of 6 different restriction enzymes allowed Generation of approximately 800 bp long cDNA fragments were used for the representative difference analysis.

Equal parts of "tester-cDNA" were ligated either with the adapters A or B and then hybridized separately with an excess of "driver-cDNA" at 68 ° 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 exponentially amplified by PCR with primers of the kit specific for the adapters A or B with a 2 minute elongation time at 72 ° C., 27 cycles (10 “94 ° C., 30“ 66 ° C., 2 '72 ° C). For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific nested primers of the kit with an elongation time of 2 minutes at 72 ° C., 10 cycles (10 “94 ° C., 30“ 66 ° C., 2 '72 ° C). The product resulting from this reaction was converted into 3 different specially modified pCR3.1 (+) vectors (InVitrogen): vector (1.ORF), vector (2.ORF) and vector (3.ORF) ( FIG. 5: CMV Cytomegalovirus; BGH Bovine Growth Hormone; ORF Open Reading Frame)
ligated and then transformed into competent E. coli (OneShot ™, Invitrogen). These vectors enable expression in eukaryotic cells in 3 different reading frames.

For the construction of the 3 vectors the pCR3.1 (+) - vector (InVitrogen) with NheI and HindIII (Promega) cut and each with a dsDNA Oligomer by annealing two ssDNA Oligomers (SEQ ID NO: 3 and 4; vector ORF1) or (SEQ ID NO: 5 and 6; Vector ORF2) or (SEQ ID NO: 7 and 8; Vector ORF3) was produced according to Standard protocols ligated (e.g. Ausubel et al., 1994; Sambrook et al. 1989). The 3 vector types have one Start codon and a cloning site for the Expression in one of the other two Vectors different reading frames.  

The three approaches (vector 1.ORF, 2.ORF and 3.ORF) performed transformation of competent E. coli (OneShot ™, Invitrogen) with the cDNA of the subtractive cDNA library yielded approximately 9600 clones. These were by means of PCR analysis for the presence and length of the Insert cDNA checked. It was as follows proceeded: the 9600 clones were in 96-well blocks cultivated in LB-Amp medium for 48 h at 37 ° C. Then 5 ul aliquots of E. coli Suspensions in 500 µl TE buffer for 10 minutes Heated at 100 ° C and 1.5 µl of it as a template for a PCR used with the insert of the vector flanking primers (SEQ ID N0: 9 and 10) amplified was, 35 cycles (1 '99 ° C, 1' 55 ° C, 2 '72 ° C). The PCR products were analyzed using agarose gel electrophoresis and ethidium bromide staining. The remaining bacterial cultures were considered Glycerol stock cultures stored at -80 ° C.

This made a cDNA subtraction library 350 individual clones obtained as E. coli glycerol Stock cultures and their insert length by the Agarose gel electrophoresis was known. Could as expected, an average length of the inserted cDNA Fragments of approximately 800 bp can be detected.

Example 3 DNA sequencing and annotation of clones of the subtractive cDNA library of the pancreatic tumor cell line MZ.PC2 m7 # 1 B7.1 # 3

The plasmid DNA of 50 from the subtracted cDNA Library was selected clones arbitrarily 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 BLAST-Search (National Center for Biotechnology Information) annotated and EST database comparison subjected. This allowed the identification of 38 known and 12 unknown genes. For the latter only EST entries existed. For the 12th unknown genes has been estimated Expression profile made: for everyone in Databases ESTs with <95% identity (BLAST) for experimentally determined sequence the starting tissue checked for the corresponding cDNA library. It was divided into i) critical normal tissues, ii) fetal, "dispensable" and immune privileged Tissue and iii) tumors and tumor cell lines. On the basis of this "virtual mRNA profile" 4 Clones (R2, R8, R11 and R12) for another experimental analysis selected.

Example 4 Transcriptional analysis of the candidate clones in Tumor and normal tissues

Between 2 and 5 µg total RNA from tumor or normal tissues were reverse transcribed using SuperScriptII (GibcoBRL) or AMV-RT (Promega) according to the manufacturer's recommendations. For each individual RNA sample, a second approach without reverse transcriptase as a control for contamination by chromosomal DNA was carried out. The quality and amount of the cDNAs were determined by PCR with β-actin-specific primers (SEQ ID NO: 13 and 14) and GAPDH-specific primers (SEQ ID NO: 15 and 16) after 30 and 35 cycles (1-95 ° C, 1 ' 55 ° C, 1 72 ° C), checked. The 4 candidate genes were analyzed analogously with specific primers. The PCR products were detected by agarose gel electrophoresis and ethidium bromide staining. One candidate, designated "R11", showed a relatively specific tumor / testis transcription profile after 35 cycles with R11-specific primers (SEQ ID NO: 11 and 12); the semi-quantitative RT-PCR of RNA from breast cancer, lung adenocarcinoma, lung squamous cell carcinoma, kidney carcinoma, colon carcinoma, heart, lung, liver, kidney, colon, spleen and testis is shown in FIG. 1. Another qualitative PCR of cDNA from patient tissue from 3 human pancreatic tumors with the same R11-specific primers (SEQ ID NO: 11 and 12) showed the expression in human pancreatic tumors ( FIG. 2). Furthermore, an additional qualitative PCR of cDNA from different tumor cell lines of human lung (LC 6, 16), gall bladder (GB 1) and pancreatic tumors (PC 1, 2) as well as two melanomas (Mel 2, 7) with the same R11-specific were Primers (SEQ ID N0: 11 and 12) carried out, which gave a clear expression in all tumor cell lines ( Fig. 4). This analysis was carried out according to the protocol of Perkin Elmer (GeneAmp RNA PCR Kit, # N808-0017) (RT reaction: (1 ×) 15 '/ 42 ° C-5' / 99 ° C-5 '/ 4 ° C; PCR reaction: (35 ×) 2 '/ 95 ° C-1' / 95 ° C-1 '/ 60 ° C and (1 ×) 7' / 72 ° C-4 ° C ( Fig. 4) As described above, the PCR products were detected by agarose gel electrophoresis and ethidium bromide staining using a 1kb size marker from Gibco BRL.

Example 5 R11 transcription profile in normal tissues

For Northern blot analysis, "Human Multiple Tissue Northern Blots" (Clontech, Palo Alto and Invitrogen) were hybridized with the [α- ³² P] dCTP (NEN, Boston) labeled approximately 1000 bp R11 PCR product at 68 ° for 2 h . The visualization was carried out by standard autoradiography (Hyperfilm, Amersham). Fig. 3 shows the result of this analysis: from 19 normal tissues (pancreas, adrenal medulla, thyroid, adrenal cortex, testis, thymus, small intestine, stomach, brain, heart, skeletal muscle, colon, spleen, kidney, liver, placenta, lung, leukocytes). For R11, a prominent transcript of 7.5 kb in length is found in the placenta, adrenal medulla, adrenal cortex and in testis. A very weak band of 7.5 kb can also be detected in the brain. Since all of these normal tissues have an immune privileged status (Streilein, 1995), an attack by CTL can be ruled out in immunotherapy based on this antigen.

Additional transcripts of 3.8 kb and 2.3 kb, which may represent splice variants of the 7.5 kb transcript or are derived from a homologous gene, were identified in the adrenal medulla and the adrenal cortex ( FIG. 3).

Example 6 Cloning of R11 cDNA

The cloning of the human R11 cDNA was carried out as follows proceeded: a BLAST analysis showed that to the in Example 3 R11 obtained by sequencing "Original Sequence" (796 bp) an overlapping fragment AF038197 and a variety of overlapping ESTs, such as e.g. B. N42343, W69539, H82474, H51766, N28313, could be identified. Starting from the sequence AF038197 was using the EstExtractor on TigemNet (http: // gcg.tigem.it/cgi-bin/uniestass.pl) one with the Clone R11 overlapping contig found. The overlap des Contigs and the 796 bp R11 "original sequence" by PCR aplification with an R11 "original sequence" specific primer and one on the contig Primer (SEQ ID NO: 17 and 18) from a SuperScript ™ Human Testis cDNA Library (GibcoBRL) and subsequent Sequencing to be verified. From the SuperScript ™ Human Testis cDNA Library could be analyzed by PCR an R11 specific primer (SEQ ID NO: 19) and one Vector-specific primers (SEQ ID NO: 20) further Fragments belonging to R11 using the Advantage cDNA PCR Kit (Clontech) and the one described there Standard protocol are amplified. The knowledge these new sequences in turn enabled PCR approaches with R11 specific primers (SEQ ID NO: 21 and 22) and the vector specific primer (SEQ ID NO: 20).  

A was used for the further extension of the R11 cDNA human testis rapid screening cDNA library panel (OriGene Technologies, Inc) with specific for R11 Primers (SEQ ID NO: 23 and 24) among those from the manufacturer the specified standard PCR conditions. Van den an aliquot was used as a template for positive cells a PCR with an R11 specific and one for the Vector specific primers (SEQ ID NO: 25 and 26) below Use of the Advantage cDNA PCR Kit (Clontech) and amplified the standard protocol described there. Aliquots of the PCR batches were used for the sequence analysis ligated directly into the pCR2.1 vector (Invitrogen) and then in competent E. coli (OneShot ™, Invitrogen) transformed and, as in Example 3 described, sequenced.

The cloned region of the R11 cDNA is 5714 bp, the presence of a polyA tail on 3 'end of the sequence for completeness of the cDNA in this area speaks. A continuous reading frame is through the start codon at position 570 and that Stop codon (TAA) represented at position 1638. To this gene no database entries from known Genes exist, no statement can be made about the function to be hit. An analysis of the protein profile (http: // www.expasy.ch/prosite/) resulted in addition to the two obvious proline-rich sections (position # 128-141 and 330-351 in SEQ ID NO: 2), potential Motifs for two N-glycosylation sites (104-107 and 250-253), a protein kinase C phosphorylation site (108-110), four casein kinase II Phosphorylation sites (99-102, 165-168, 198-201,  273-276), two N-myristoylation sites (21-26, 309-314) and a region similar to the active one Center of eukaryotic and viral aspartate Proteinases (16-27).

Example 7 Potential MHC binding peptides in the for C-terminal part of R11 coding region

Potential peptide epitopes within the coding Region of the C-terminal fragment of R11 according to (SEQ ID NO: 2) would be determined using the method described by Parker et. al., Algorithms described in 1994 on the basis known motifs (Rammenee et al. 1995). For the most important HLA types, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, became 9-mer Candidate peptides identified from which to be expected is that they bind to the corresponding HLA molecules and therefore represent immunogenic CTL epitopes; the The peptides found are listed in Table 1. By analogous procedure can further potential peptide Epitopes for other HLA types or 8- and 10-mer peptides be determined.

Table 1

Immunogenic peptide candidates of the C-terminal fragment of R11 (356 amino acids)

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SEQUENCE LOG

Claims (19)

1. Tumor-associated antigen of the designation R11, characterized in that it has the amino acid sequence defined in SEQ ID NO: 2 or contains it as a partial sequence. 2. Immunogenic protein fragment or peptide, thereby characterized in that it is of that in claim 1 defined tumor-associated antigen is. 3. Immunogenic (poly) peptide according to claim 2, which a triggers humoral immune response. 4. Immunogenic (poly) peptide according to claim 2, which or its degradation products through MHC molecules are presented and a cellular immune response trigger. 5. The immunogenic peptide of claim 4 selected from the group of peptides according to SEQ ID NO: 27 to 70. 6. Immunogenic (poly) peptide according to one of the claims 1 to 5 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 R11. 7. 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 5 contains. 8. Pharmaceutical composition according to claim 7, characterized in that they are different from Contains R11 derived immunogenic peptides. 9. Pharmaceutical composition according to claim 8, characterized in that they have one or more peptides derived from R11 mixed with von other tumor-associated antigens Contains peptides. 10. Pharmaceutical composition according to claim 7 or 8 that the peptides on at least two bind different HLA types. 11. Isolated DNA molecule coding for a protein with the immunogenic properties of in Claim 1 defined tumor-associated antigen or for fragments of it. 12. DNA molecule according to claim 11, coding for a immunogenic polypeptide of the designation R11, which the amino acid sequence contained in SEQ ID NO: 2 has or contains them, or for them derived protein fragments or peptides. 13. DNA molecule according to claim 12, characterized characterized in that it is a polynucleotide with the sequence shown in SEQ ID NO: 1 is or contains this sequence or that it is a Polynucleotide is or contains this that with a polynucleotide that is shown in SEQ ID NO: 1  sequence shown under stringent Conditions hybridized. 14. Recombinant DNA molecule containing a DNA Molecule according to one of claims 12 to 13. 15. DNA molecule according to one of claims 11 to 14, for cancer immunotherapy, where the R11 expressed by the DNA molecule (Poly) peptide has an immune response against tumor cells of the patient that express R11. 16. Use of cells that in claim 1 express defined antigen for which Production of a cancer vaccine. 17. Antibodies against one of claims 1 to 5 defined (poly) peptide. 18. Antibody according to claim 17, characterized characterized as being monoclonal. 19. Antibody according to claim 17 or 18 for the Therapy and diagnosis of cancer that are associated with the expression of R11.