DE10025521A1 - Tumor-associated antigen B132 - Google Patents

Abstract

The invention relates to the tumor-associated antigen B132, to the immunogenic peptides derived therefrom and to the DNA molecules encoding the same. The invention further relates to the use thereof in the immunotherapy of cancer diseases and as a target molecule for chemotherapeutical intervention.

Description

The invention relates to the immune and Chemotherapy for 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. It has also 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 van der 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 are transferred to the MHC-I molecules. The therapeutically most efficient form of application for a Antigen is generally defined in clinical Studies determined. Recent clinical studies on the Use of recombinant and synthetic vaccines at Treatment of melanoma has recently been demonstrated in one Article summarized and discussed (Restifo and Rosenberg, 1999).

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 This can have serious, sometimes fatal consequences. Second, the antigen is said not only in the primary tumor, but also be present in the metastases. Of further it is clinical in terms of broad Application of the antigen desirable when in several tumor types in high concentration is. Another prerequisite for the suitability of a TAA is an effective component of a vaccine 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 van der 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). In further consequence was then with the help of melanoma-specific T cells identified a TAA (CAMEL) that of an alternative reading frame from LAGE-1 is derived (Corlien et al., 1999).

The object of the present invention was to develop a new preferably protein expressed by tumor cells to provide the one hand because of its Property as tumor associated antigen (TAA) in As part of an immunotherapy for cancer can and which on the other hand is a target molecule for the  Intervention using chemotherapy methods represents.

This task was solved by first using RDA ("representational difference analysis") between one Lung adenocarcinoma cell line (A549) and Normal lung tissue a cDNA subtraction library was produced. For the selection of in the tumor overexpressed antigens were then the cDNA clones obtained and sequenced with in Databases available sequences compared. Among the annotated genes contained 321 unknown, to which mostly ESTs ("expressed sequence tags") entries existed in the database. After a further qualitative PCR analysis in cDNA libraries of critical normal tissues and immune privileged Tissues as well as more detailed database research limited the number of candidate clones to 56, whose ESTs are not from critical normal tissues. Using RT-PCR, it was found that three of the 56 clones examined an expression mainly in different tumor tissues and little or no Have expression in normal tissues. The qualitative Comparison (using PCR) of the expression of one of the clones (B132) between tumor and normal tissue showed one Overexpression of the B132 cDNA in various tumors. The expression profile analyzed by Northern blot additionally showed that B132 in the examined Normal tissues have no or only weak ones Showed transcription.

The human B132 cDNA was cloned, the one obtained Sequence is shown in SEQ ID NO: 1. The  Sequence analysis of the cloned human B132 cDNA revealed that from position 160 to position 1206 (exclusive Stop codon) a continuous open reading frame is present, at the nucleotide and protein levels, in the no known sequences of the databases Has homology or identity. From the Northern Data obtained from blot experiments is to be concluded that the B132 transcript is approximately 4.1 kb in length. The cloned region of the B132 cDNA is 4057 bp (exclusive polyA region), the presence of a PolyA tail at the 3 'end of the sequence for the Completeness of the cDNA speaks in this area. Due to the fact that in the 5 'range of cloned cDNA from position 1 to 160 none continuous reading frame is present can be inferred that the ATG at position 160 is the Start codon of B132 is.

Additional information about the further upstream lying sequence of B132 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 B132 is transcribed (e.g. colon cancer tissue or from Pulmonary adenocarcinoma-derived cell lines such as A549) reverse transcribed and then with an adapter known sequence ligated. A PCR with a Adapter primer (binds specifically to the adapter 5 'end of the cDNA) and a B132-specific primer allows the amplification of corresponding B132- Fragments. These PCR products can, as in Example 4 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 B132 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 B132 Clones that can be isolated upstream from the sequence information obtained 5 'end of the cDNA e.g. B. contain the promoter region of B132.

The isolated cDNA codes for the tumor-associated Antigen (TAA) or tumor-specific protein of the Designation B132 with the one specified in SEQ ID No: 2 Amino acid sequence (B132). The sequence of B132 will defined by the start codon at position 160 of the isolated B132 cDNA.

The invention thus relates to a first aspect tumor-associated antigen of the designation B132, 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 Exchange of amino acids are required, provided that B132 derivative which is for use in a  Tumor vaccine desired immunogenic properties having.

The natural amino acid sequence of B132 (or corresponding to the sequences of the B132 cDNA) may be modified by individual Amino acids exchanged in a B132 CTL epitope to, compared to the natural B132 CTL Epitope, an increase in the affinity of B132 peptides to MHC-I molecules and thus an increased immunogenicity and ultimately increased reactivity towards To cause tumors. Modifications in the area of B132 epitopes can be found on the total B132 protein (this is from the APCs to the corresponding peptides processed) or on larger B132 protein fragments or on B132 peptides (see below).

In another aspect, the present concerns Invention of immunogenic fragments and peptides derived from B132 are derived. The latter are referred to below as Denoted "B132 peptides". A first group are B132 peptides that have a humoral immune response (induction of antibodies). Such peptides are selected sections of B132 (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.

B132 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, B132 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 has to bind to an MHC molecule, the to patient treating the MHC molecule in his Repertoire. The determination of the MHC Subtype of the patient thus represents, with regard to the Triggering a cellular immune response, one of the essential requirements for effective application of a peptide on this patient.

The sequence of a therapeutic B132- Peptide is generated by the respective MHC molecule given with regard to anchor amino acids and length. Ensure defined anchor positions and lengths that a peptide is in the peptide binding groove of the the respective MHC molecule of the patient. this has with the result that the immune system is stimulated and a cellular immune response is generated, which, in In case of using one of a tumor antigen derived peptide, against the tumor cells of the Patient judges.  

Immunogenic B132 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 B132 peptides that represent CTL epitopes due to B132 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 matched 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, 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 B132 peptides is suitable. 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 with great reliability Predict peptide epitopes that respond to a bind specific 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 identifies those who are expected to attend bind the corresponding HLA molecules and therefore represent immunogenic CTL epitopes; the determined Peptides are listed in Table 2. Similar ones Ways can, if necessary, using other Algorithms that have different characteristics the peptides (hydrophobicity, charge, size) or Requirements for the peptides, e.g. B. the 3D structure of the HLA molecule, take into account other potential  Peptide epitopes can be determined; This also applies to Peptide epitopes of other types of HLA.

After selecting B132 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; van der 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 B132 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.

Such modified peptides are called "Heteroclitic peptides" referred to. You can go after following methods can be obtained:

First, the epitopes of MHC-I or MHC-II Ligands or their variation z. B. after that of Rammenee et al., 1995, described principle performed. The length of the peptide corresponds in the case its matching to MHC-I molecules, preferably one Minimal 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., 1996, 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 B132 peptides consist of screening peptide Libraries with CTLs that are naturally on tumors recognize occurring B132 peptides as described by Blake et al., 1996; in this context the Use of combinatorial peptide libraries proposed to design molecules derived from MHC-I restricted CTLs recognized tumor epitopes imitate.

The B132 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 B132 peptides conventional methods can be used, e.g. B. in Commercially available automatic peptide synthesizers.

As an alternative to natural B132 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 B132 peptides or B132 peptide equivalents.

The TAA of the designation B132 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 B132- positive tumors are used, especially in Renal cell, lung, colon and breast cancer.

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 B132 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 B132 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.

B132 (fragments) or B132 peptides can also be used to trigger a CTL response ex vivo. An ex vivo CTL response to a tumor expressing B132 is induced by looking at the CTL progenitor cells together with APCs and B132 peptides or B132 protein incubated. Then you leave the activated CTLs expand, after which it is administered to the patient again become. Alternatively, APCs can be loaded with B132 peptides become what leads to efficient cellular activation Immune responses to B132 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 B132 peptides or B132 peptide equivalents applied. In another Embodiment are B132 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. 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 predict the risk of a patient with a B132-associated tumor or to characterize the course of the disease of a B132-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 (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 B132 or for fragments of it.

In one aspect, the present invention 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.

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

The DNA molecules or fragments thereof according to the invention code for (poly) peptides with the designation B132 amino acid sequence shown in SEQ ID NO: 2 or for protein fragments or peptides derived therefrom; DNA molecules that are encoded by the Degeneration of the genetic code deviations from the sequence shown in SEQ ID NO: 2.

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 B132 derivative or Fragments or peptides with the for use as Tumor vaccine desired immunogenic properties encode.

The B132 DNA molecules of the present invention or the corresponding RNA molecules, too The present invention relates to how the encoded (poly) peptides for which Cancer immunotherapy used.

In one embodiment of the invention DNA molecules coding for the natural B132- Polypeptide or used for fragments thereof. As an alternative to the natural B132 cDNA or fragments  modified derivatives can be used. These include sequences with modifications for a protein (fragment) or peptide with a stronger one Encode immunogenicity, being for the modifications at the DNA level, the same considerations apply as for the peptides described above. Another type of Modification is the lining up of numerous Sequences coding for immunologically relevant Peptides, like a string of pearls ("string of beads "; Toes et al., 1997). The sequences can also be modified by adding auxiliary elements, e.g. B. Features that make delivery more efficient and Ensure immunogen processing (Wu et al., 1995). For example, by adding a Localization sequence in 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 B132-DNA contains.

The B132 DNA molecule of the present invention can preferably in recombinant form as a plasmid, 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 B132-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 you can synthetic carriers for nucleic acids, such as cationic Lipids, microspheres, microspheres or liposomes for the in vivo administration of nucleic acid molecules, coding for B132 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 B132 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 B132 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-B132 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-B132 monoclonal antibody is screened. For therapeutic or diagnostic use in Humans can use these animal antibodies optionally chimerized in a conventional manner (Neuberger et al., 1984, Boulianne et al., 1984) or humanized (Riechmann et al., 1988, Graziano et al., 1995).

Human monoclonal anti-B132 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-B132 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 B132-specific antibodies to selectively add any substances to or into a tumor bring that expresses B132. Examples of such Substances are cytotoxic or radioactive agents Nuclides, the effect of which is to redden the tumor to harm. Because of the relatively tumor-specific Expression of B132 are only slight Expected side effects. In another aspect can with the help of B132 antibodies substances for Visualization of tumors expressing B132 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-B132 antibodies come are described in WO 95/33771.

The TAA of the designation B132 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 B132- positive tumors are used, especially in Renal cell, lung, colon and breast cancer.

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 B132 mutations  is determined, expediently with the help of probes of the isolated cDNA according to the invention, the B132 cDNA cloned one or more different tumors and the sequences obtained with normal tissue B132 cDNA compared. It is expected that tumor B132 peptides from a mutant compared to normal tissue B132 Sequence section in comparison to normal tissue-B132- Peptides from the corresponding section one have increased immunogenicity. To confirm, that any mutations are tumor specific Antibodies against these areas are generated and Tumor cells on expression of possible mutations to be examined.

The present invention thus relates to one further aspect B132 peptides derived from areas of a tumor-expressed 8132, the tumor-specific Have mutations.

In addition to immunotherapeutic approaches, there is the targeted one Chemotherapy plays an essential role in treatment from cancer to. Chemotherapy means that Administration of substances by intervention in Metabolism, signal transduction and Cell division processes of malignant cells either act cytostatically or cytotoxically-cytolytically. Chemotherapy drugs are used depending on the way they affect specific targets in the tumor cell, with which cellular processes they interact and which cell cycle phase they influence in divided into different categories.  

Provide upregulated genes in tumor tissues Points of attack and thus potential target structures ("Targets") for chemotherapy.

Due to the preferred expression of B132 in Tumor cells can be thought to be this protein has an important function for the tumor, e.g. B. for Emergence, infiltration and growth and thus a Target for chemotherapy intervention represents.

With regard to its use as a target in the Targeted chemotherapy is getting closer to B132 characterized to the appropriate strategy for that Develop intervention with this function.

As a first step in the so-called "down-stream" Functional analysis of B132 is best performed in one first step through a bioinformatic analysis that for the experimental validation of B132 as Target is leading the way.

For this analysis, they are based on similarity and modular structure based bioinformatics concepts an essential basis. Established bioinformatic tools for the detection of Similarities are BLAST (http: \ \ www.ncbi.nlm.nih.gov/BLAST, Altschul et al., 1997) or FASTA (Pearson & Lipman, 1988), the specialized databases such as Pfam (http: \ \ www.sanger.ac.uk/Pfam, Bateman et al., 2000) and SMART (http: \ \ smart.embl-heidelberg.de, Schultz et al., 2000), which consider domain structures. Applications such as  Clustal (http: \ \ www2.ebi.ac.uk/clustalw, Higgins et al., 1996) HMMer (http: \ \ hmmer.wustl.edu), PSI-BLAST (Altschul et al., 1997) and the PROSITE database (http: \ \ www.expasy.ch/prosite, Hofmann et al., 1999) be used. Statistical analysis methods that do not rely on homologies, allow prediction further structure and function relevant Properties like the secondary structure and the Occurrence of transmembrane segments and helix turn Helix motifs. Methods for predicting the Secondary structure of proteins are available; Jpred is particularly noteworthy (http: \ \ barton.ebi.ac.uk/servers/jpred.html, Cuff et al., 1998). The secondary structure prediction can Support functional hypotheses, such as when the structure of the suspected homologue is known.

Subsequently, B132 becomes a biochemical and subjected to biological analysis.

The bioinformatics analysis of B132 points to this conclude that using this protein Nucleic acid interaction is related. To the direct and / or indirect interaction of B132 with Methods such as "mobility shift", South-Western, UV crosslinking etc. be carried out for their implementation from the Standard methods known in the literature (e.g. Ausubel et al., 1994) can be used.

The next step is the function of B132 clarified for the tumor occurrence; e.g. B. by Proliferation assays in vitro or in animal models that  overexpress the B132 gene under investigation (constitutive or inducible) and as a control either express in deleted (inactive) form or down regulate via antisense (see e.g. Grosveld and Kollias, 1992).

B132 can be used in screening assays Identify substances that affect the activity of this Modulate, in particular inhibit, proteins. In a Embodiment such an assay can e.g. B. in it consist of the B132 protein, or an active fragment of which, in cells that respond to the activity of B132 Proliferation react, bring in or the corresponding B132 cDNA in the cell for expression bring, and cell proliferation in the presence and to determine in the absence of a test substance.

An example of test cells are cells with lower Division rate, e.g. B. primary cells that are not endogenous B132 have. To determine the suitability of the cells for one Screening assay, B132- cDNA transformed, grown and with standard assays, e.g. B. thymidine incorporation, on their proliferation ability tested. Because of a B132 expression significant increase in their proliferation ability they can be used as test cells, e.g. B. in High Throughput Screening Proliferation Assays. Examples of proliferation assays in high throughput Format, e.g. B. based on the MTS assay are in the WO 98/00713.

Substances with an anti-proliferative effect can for the treatment of tumors with strong B132 expression  be used, especially in kidney cell, Lung, colon and breast cancer.

Figure overview

Fig. 1 RT-PCR analysis of various tumors,

Fig. 2A Northern blot analysis of various normal tissues and tumor cell lines with a cDNA fragment of B132,

FIG. 2B Northern blot analysis of various normal tissues and squamous lung with a cDNA fragment of B132,

Fig. In situ hybridization with B132 antisense and B132 sense probes in different tissue sections. The s-sample (sense) always corresponds to the negative control.

example 1 RDA ("Representational Difference Analysis") from the human lung adenocarcinoma cell line (A549) and normal lung tissue

The human lung adenocarcinoma cell line A549 (CCL 185) obtained from ATCC was grown up in T150 cell culture flasks. MEM was used as the nutrient medium with 10% heat-inactivated, fetal calf serum and 2 mM L-glutamine. Every 3 to 4 days the cells were cleaved by trypsinization 1: 5 to 1:10 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) was used to harvest the cells. The 4 ml were transferred into a 15 ml falcon tube, mixed with 8 ml PBS, centrifuged at 1200 rpm in a Haereus table centrifuge (Megafuge 2.0R) for 5 min at 4 ° C., the cell pellet with 1 ml lysis buffer (10 mM TrisHCl pH8 , 140 mM NaCl, 1.5 mM MgCl ₂ , 0.5% NP40), shaken vigorously and centrifuged in a 2 ml Eppendorf tube at 12,000 rpm and 4 ° C. for 5 min in a Sigma table centrifuge (Sigma 202 MK). The supernatant was transferred to a new Eppendorf tube and, after addition of 55 μl of 20% SDS solution, extracted twice with double the volume of a CHCl ₃ / phenol (1: 1 v / v) mixture and once with the single volume of CHCl ₃ . The aqueous RNA-containing phase was mixed with 1/10 volume of 3 M NaAc (pH5) and twice the volume of 96% EtOH and the RNA was precipitated at -20 ° C. overnight. Starting with 1 mg of total RNA, the PolyATtract Kit (Promega) was used to isolate poly-A (+) RNA in accordance with the manufacturer's protocol. The A549 poly-A (+) RNA with a concentration of 1 mg / ml in DEPC-treated H ₂ O was stored in aliquots at -80 ° C.

To carry out the representative difference analysis (RDA; Hubank and Schatz, 1994; Diatchenko et al., 1996), the poly-A (+) RNA of the lung adenocarcinoma cell line A549 was used as a "tester", which is derived from normal lung tissue (1 mg / ml; Clontech, Palo Alto; # 6524-1) used as "driver". The RDA was performed using the PCR-select ^™ kit (Clontech, Palo Alto) according to the manufacturer's protocol, with the exception that a modified primer / adapter-2 oligonucleotide system was used: adapter-2-alt-1 ( SEQ ID NO: 3) and nested PCR primer-2-alt (SEQ ID NO: 4) and adapter-2-alt-2 (SEQ ID NO: 5). The newly generated primer / adapter sequences enable the presence of three new restriction enzyme interfaces (Kpn I, Sac I and Xho I) in the sequence of the nested PCR primer-2-alt after cloning the subtracted cDNA fragments in the pPCRII- Vector a subsequent cutting out of the respective cDNA fragments. The design of a primer / adapter sequence with several available restriction enzyme interfaces was necessary because point mutations could often be observed especially in the primer sequences due to the PCR amplification steps.

After the synthesis of double-stranded cDNA by means of oligo-dT, the cDNA obtained by "tester" and "driver" was digested with RsaI (RsaI is a 4-base-recognizing restriction enzyme and provides a statistical average of 256 bp cDNA fragments). Equal parts of "tester cDNA" were ligated either with adapters 1 or 2 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 1 and 2, respectively. For further enrichment, an aliquot of this reaction was subjected to a second PCR with specific "nested" primers. The exponentially amplified cDNA fragments resulting from this reaction were ligated directly into the pCRII vector (Invitrogen; "TA-cloning vector") and then a third of the ligation mixture was transfected into competent E. coli (OneShot ^™ , Invitrogen).

712 positive transformants (blue-white selection) were obtained and in 96-well blocks in LB-Amp medium (1.3 ml per well) cultured at 37 ° C for 48 h. Per bowl 750 ul of the E. coli suspensions for the Preparation of the plasmid DNA used (96-well Mini preparation method from QIAgen according to the instructions of the Manufacturer). The remaining bacterial cultures were stored as gycerin stock cultures at -80 ° C.

A cDNA consisting of 712 individual clones was Subtraction library obtained, both in the form of E. coli glycerol stock cultures as well purified plasmids was present.

Example 2 DNA sequencing and annotation of TAA candidates

The isolated plasmid DNA of all 712 clones (see Example 1) was sequenced according to the Sanger method on an ABI-Prism device. The sequences obtained were annotated using the BioScout software (LION, Heidelberg) and subjected to database comparisons (Genbank). From 712 clones, 678 could be sequenced and annotated. The rest (34) had either only poly (A) sequences as an insert or corresponded to a religious vector or could not be sequenced. Of the 678 annotable sequences, 357 proved to be genes with a known function. The remaining 321 represented clones encoding genes with unknown function; 59 of them did not even have entries in the human EST database. Known genes were not further treated. The expression profile was estimated for those unknown genes for which an EST entry was available: all those ESTs with <95% identity (BLAST) that belonged to the experimentally determined sequence of the subtraction libraries were checked. The annotation was divided into a) critical normal tissues, b) fetal, "dispensable" and immune-privileged tissues and c) tumors and tumor cell lines. On the basis of this "virtual mRNA profile"("virtual Northern blot"), 200 clones for which no ESTs were found in group a) were selected for further experimental analyzes (including the 59 clones for which no EST entry) Template). To further narrow the candidate clones, oligonucleotide primer pairs were designed and synthesized from the sequences determined from the 200 selected clones. 8 different human tissue-derived cDNA libraries (GibcoBRL "SUPERSCRIPT ^TM "), which are directionally cloned into pCMV-SPORT, were first tested for the presence of the respective candidates by means of qualitative PCR. The cDNA libraries used here came from tissue from the heart (# 10419-018), liver (# 10422-012), leukocytes (# 10421-022), kidney (# 10420-016), lung (# 10424-018), Testis (# 10426-013), brain (# 10418-010) and fetal brain (# 10662-013). The PCR conditions were as follows: 20 μl total volume per PCR mixture contained 1 × TaqPol buffer (50 mN KCl, 10 mM TrisHCl pH9, 0.1% Triton X-100), 1.5 mM MgCl ₂ , 0, 2 mM dNTPs (Promega), 0.025 U / µl Taq DNA polymerase (Promega), 5 pM each of specific oligonucleotide primers (SEQ ID NO: 6 and 7) or (SEQ ID NO: 8 and 9) and 100 ng of each plasmid DNA to be examined. As a control, specific primers for GAPDH (SEQ ID NO: 10 and 11) were used. To check the selective detection, the respective 8132 specific primer pairs of oligonucleotide primers (SEQ ID NO: 6 and 7) and (SEQ ID NO: 8 and 9) were also tested in parallel for the isolated plasmid with the B132 "original fragment" (originally isolated cDNA Fragment of B132). The detectability of fragments of the expected length with a strong signal in one of the critical normal tissues (heart, liver, lung, kidney and leukocytes), but not in the cDNA libraries from immune-privileged tissues (brain, fetal brain and testis) under these PCR Conditions (1 cycle: 3 '94 ° C; 35 cycles: 1' 94 ° C - 1 '55 ° C - 1' 72 ° C; 1 cycle: 7 '72 ° C) was defined as the elimination criterion. This qualitative PCR analysis reduced the number of candidates to 56; Clone B132 was in this pre-selected group of candidates.

Example 3 Analysis of the expression profile of B132 at the RNA level using qualitative RT-PCR and quantitative TaqMan analysis

The RNA was isolated from frozen tissue with Trizol according to Gibco manufacturer's protocol. For Removal of any contaminating DNA was the prepared RNA digested with DNAase I as follows: 3 µg Total RNA was mixed with 20 ul 5 × AMV buffer (Promega), 1 µl RNasin (Promega) and 2 µl DNase I (Boehringer Mannheim) in a total volume of 80 µl for 15 minutes incubated at 37 ° C. 120 µl phenol: chloroform: isoamyl alcohol (25: 24: 1) were added on a Vortexer mixed and centrifuged briefly. The aqueous phase was removed, with 120 ul chloroform: isoamyl alcohol (24: 1) offset and as before centrifuged. The purified RNA was ethanol precipitated and dissolved in water.

The total RNA was then reversed with AMV Transcriptase rewritten in cDNA: To 3 µg total RNA 1 ul Oligo dT primer (Promega) was added and with Water brought to a final volume of 10 ul. To an incubation of 5 minutes at 70 ° C Solution cooled for 5 minutes at room temperature. 5 µl AMV reaction buffer (5 ×, Promega), 2.5 µl dNTPs (each 10 mM, Boehringer Mannheim), 1 µl RNAsin (10 U / µl,  Promega), 1.5 µl AMV-RT (10 U / µl, Promega) and 5 µl Water was added and 1 hour at 42 ° C incubated and the reaction by 3 minutes at 95 ° C completed.

To create a cDNA pool of a particular Tissues or tumor types were 3 to 10 different Single preparations from different patients in mixed in equal proportions.

For the quantitative determination of the two household genes GAPDH and tubulin in cDNA pools were as follows proceeded:

A) GAPDH-TaqMan PCR (Perkin Elmer)

For details on the principle of the TaqMan method, see manufacturer information (Perkin Elmer). A TaqMan PCR run included samples of GAPDH control plasmid with 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies / µl (Perkin Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified. All samples were analyzed in triplicate. For a 25 µl reaction mixture, 1 µl cDNA, 2.5 µl 10 × buffer A (Perkin Elmer), 4 µl MgCl ₂ (25 mM, (Perkin Elmer)), 0.5 µl per nucleotide (10 mM dATP, dCTP, dGTP; 20 mM dUTP), 0.125 µl TaqMan probe (20 µM; TaqMan probe for GAPDH (SEQ ID NO: 12; fluorescence-labeled at the 5 'end with tetrachlorofluorescein; at the 3' end with carboxymethylrhodamine), 1 µl per primer (each 20 µM, Perkin Elmer GAPDH forward: pos. 1453-1486 from J04038 (SEQ ID NO: 13)); GAPDH reverse: pos. 3764-3741 from J04038 (SEQ ID NO: 14), 0.25 µl AmpErase uracil N- glycosylase "UNG" (1 U / µl, Perkin Elmer), and 0.125 µl AmpliTaq gold (5 U / µl, Perkin Elmer) mixed, transferred to MicroAmp Optical Tubes (Perkin Elmer) and sealed with MicroAmp Optical Caps. The PCR was carried out as follows carried out: a cycle with 2 minutes 50 ° C for the UNG reaction, a cycle 10 minutes 95 ° C to activate the AmpliTaq, 40 cycles with 15 seconds each 95 ° and 1 minute 60 ° C. The samples were then at 25 ° C G The evaluation of the data takes place with the program "Sequence Detection System 1.5b1" (PE Applied Biosystems), whereby in principle the fluorescence signals of the cDNA samples to be quantified were compared with the signals of the control plasmid dilutions of known concentration.

B) Tubulin-SybrGreen PCR (Perkin Elmer)

Principle of the SybrGreen PCR see manufacturer information (Perkin Elmer). A SybrGreen PCR run contained samples of tubulin control plasmid with 10 ² , 10 ³ , 10 ⁴ , 10 ⁵ and 10 ⁶ copies / µl (Perkin Elmer) to determine the standard curve, a negative control without DNA and the cDNA pools to be quantified. All samples were analyzed in triplicate. For a 25 µl reaction mixture, 1 µl cDNA, 2.5 µl 10 × SybrGreen buffer (Perkin Elmer), 3.5 µl MgCl ₂ (25 mM, Perkin Elmer)), 0.5 µl per primer (20 µM, Perkin Elmer, Tubulin forward (SEQ ID NO: 15); Tubulin reverse (SEQ ID NO: 16)), 0.25 µl AmpErase uracil N-glycosylase "UNG" (1 U / µl, Perkin Elmer), and 0.25 µl AmpliTaq gold (5 U / µl, Perkin Elmer) mixed, transferred to MicroAmp Optical Tubes (Perkin Elmer) and sealed with MicroAmp Optical Caps. The PCR was carried out as follows: a cycle with 2 minutes 50 ° C for the UNG reaction, a cycle 10 minutes 95 ° C to activate the AmpliTaq, 40 cycles with 15 seconds each 95 ° and 1 minute 60 ° C. The samples were then kept at 25 ° C. The data are evaluated with the program "Sequence Detection System 1.5b1" (PE Applied Biosystems), in principle the fluorescence signals of the cDNA samples to be quantified being compared with the signals of the control plasmid dilutions of known concentration.

To check for possible contamination of the PCR with cDNA pools with genomic DNA was performed Primers specific for an intron region of the GAPDH Genes performed (GAPDH insert forward, 200 ng / µl pos. 1531-1553 of J04038 (SEQ ID NO: 17); GAPDH insert reverse, 200 ng / µl, pos. 1962-1939 by J04038 (SEQ ID NO: 18)). Genomic DNA served as a positive control (6ng / µl, Perkin Elmer).

For a 25 µl PCR approach, 1 µl of the cDNA pool or genomic DNA with 2.5 µl 10 × Taq buffer (Promega), 1.5 µl MgCl ₂ (25 mM, Promega), 0.5 µl dNTPs (each 10 mM, Boehringer Mannheim), 1 µl primer mixture (20 µM each), 0.15 µl Taq polymerase (Promega) mixed in water. The PCR was carried out as follows: 1 × 94 ° C. for 3 minutes; 30 x 94 ° C 30 seconds, 55 ° C 30 seconds, 72 ° C 1 minute; keep at 4 ° C. The PCR was analyzed on a 1.5% agarose gel.

In order to ensure good comparability of the RT-PCR results in different tissues, the cDNA pools were set to a certain number of copies of household genes. In detail, the mean value of the tubulin and GAPDH copy number was formed for each pool and adjusted to a value of 2.1 × 10 ⁴ copies / μl by dilution with water. These set cDNAs were used to create the expression profile of B132.

To determine the expression profile of B132 at the mRNA level, the RT-PCR with the primers (SEQ ID NO: 6 and 19) was carried out in 17 different cDNA pools. The B132 plasmid DNA of the original cDNA fragment served as a positive control. 1 µl cDNA each was mixed with 2.5 µl Taq buffer (10 ×, Promega), 1.5 µl MgCl ₂ (25 mM, Promega), 0.5 µl dNTPs (10 mM each, Boehringer Mannheim), 0.5 µl per primer (20 µM), 1.25 µl Taq polymerase (5 U / µl, Promega) and water added to 25 µl. The following PCR program was carried out: 1 × 94 ° C. for 3 minutes; 35 x 94 ° C 15 seconds, 55 ° C 30 seconds, 72 ° C 1 minute; keep at 4 ° C. The PCR products were then detected on a 1.5% agarose gel using ethidium bromide staining; the RT-PCR analysis is shown in Fig. 1 and (in comparison with other data) in Tab. 1A and 1B:

Fig. 1

M size marker (Boehringer Mannheim Marker 6)
P plasmid control with B132 insert (cDNA fragment)
1, 10 testis
2, 11 lung adenocarcinoma
3, 12 lung squamous cell carcinoma
4, 13 colon carcinoma
5, 14 breast cancer
6, 15 renal cell carcinoma (RCC)
7, 16 Hodgkin's lymphoma
8, 17 melanoma
9, 18 SW60 (colon carcinoma cell line).

Samples 1-9 were with 35 cycles, 10-18 with 40 cycles amplified.

The quantitative TaqMan PCR analysis of B132 was like described for the two household genes; only with B132 specific primers (SEQ ID NO: 6 and 19) (200 ng / µl) and a B132 specific probe (SEQ ID NO: 20; fluorescently labeled at the 5 'end with fluorescein, at the 3 'end with carboxymethylrhodamine) (20 µM). As B132 plasmid DNA of known copy number became standard used. The results are in Tab. 1A and 1B listed.

Table 1A

Summary of the data from the PCR analysis (PCR) used in situ hybridization (ISH) and quantitative PCR (TaqMan) of B132 in various normal tissues.

Table 1B

Summary of the data from the PCR analysis (PCR) used in situ hybridization (ISH) and quantitative PCR (TaqMan) of B132 in various tumor tissues.

Fabrics in bold indicate the use of various pooled tissue samples; those not in bold indicate individual tissue samples.
+++ extremely positive
++ strongly positive
+ positive
(+) weakly positive
- negative

Free boxes indicate that the corresponding test was not carried out in the present case.

The candidate gene B132 shows strong signals in pooled samples of colon carcinoma, hodgekin lymphoma, Lung adenocarcinoma; Signals in renal cell carcinoma (RCC) are not as strong as in the pools of the tumor samples listed above, but constant in Evidence of all examined RCC samples (Tab. 1B). Of the normal tissues prove above all Cerebellum, skin, testis and spleen due to PCR, ISH and TaqMan analysis as positive. Regarding the TaqMan data (but not the PCR data) must possibly also in the bladder with a B132 Transcript.

In the further course the candidate B132 became more precise evaluated.

Example 4 B132 transcription profile in tumor and Normal tissues

For the Northern blot analysis, human multiple tissue Northern blots (Clontech and Invitrogen) with the [α- ³² P] dCTP (NEN, Boston) labeled 209 bp long B132 PCR product of the primers (SEQ ID NO: 21 and 22) at 68 ° hybridized for 2 h. The visualization was carried out by standard autoradiography (Hyperfilm, Amersham). . Figs. 2A and 2B show the result of this analysis: from 24 normal tissues (pancreas, adrenal medulla, thyroid, adrenal cortex, testes, thymus, small intestine, stomach, brain, heart, skeletal muscle, colon, spleen, kidney, liver, placenta , Lungs, leukocytes, tonsils, appendix, lymph nodes, gallbladder, prostate, ovaries), 8 cell lines (HL-60, HeLa S3, K-562, MOLT-4, Burkitt's Lymphoma, SW480, A549, G361) and 3 squamous cell carcinomas of the lungs . For B132, a transcript at 2.4 kb and one at 4.4 kb was found in the tumor tissue of the squamous cell carcinoma of the lung. In normal tissues, a 2.4 kb length transcript is found in Testis. Of two normal lung tissue samples tested, only one shows transcripts at 2.4 kb and 4.4 kb ( Fig. 2B). However, this sample comes from the normal lung tissue of a tumor patient near the tumor, in which no tumor growth can be determined histologically, but which may already be in a transition stage to the disease (transformation). The second normal lung tissue sample and all other normal tissues show no expression of this gene. In the HeLa S3, K-562 and MOLT-4 cell lines a transcript of 4.4 kb in length can be recognized ( Fig. 2B).

The presence of the TAA B132 in cell lines and in Squamous cell carcinoma of the lungs is in good Agreement with that originally for the RDA used starting material (cell line of a Squamous cell carcinoma of the lungs). Based on this study corresponds to the 4.4 kb transcript of the tumor-associated form of B132.  

Example 5 Cloning of the B132 cDNA

The cloning of the human B132 cDNA was carried out as follows proceeded: With the primer pair (SEQ ID NO: 6 and 7) Rapid-Screen cDNA Library Panel: Human Testis Master Plate (OriGene) under the following PCR Conditions screened: 35 cycles (1'94 ° C, 1'59 ° C, 1 '72 ° C). The subplate became the positive bowl ordered and also under those described above PCR conditions screened. Bacteria from the positive Wells were plated and 2 × 96 colonies were screened.

6 positive clones were identified. The Sequencing some of these clones with the primers (SEQ ID NO: 6, 7, 23, 24, 25, 26, 27, 28, 29, 30, 31 and 32) gave a 4077 nt long cDNA (SEQ ID NO: 1), which complete at the 3 'end between bases 3756 and 3978 overlapped with the B132 "original" fragment. she owns an open reading frame, which is for a 349 AS long Protein encoded (SEQ ID NO: 2) that has homology to the C. elegans hypothetical 34.6 kDa protein AH9.2 in Chromosome X precursor, Acession Number: Q10905 shows.

Example 6 In situ hybridization with B132 antisense and B132 Sense probes in different tissue sections

To label RNA with digoxigenin, the Reagents from Boehringer's DIG RNA Labeling Kit Mannheim used. The original cDNA fragment from  B132 (item number 3756-3978 in SEQ NO ID: 1) was cloned into the vector pBluescript and the Plasmid DNA with the restriction enzyme BamHI or HindIII linearized. Synthesis of the RNA was depending on Restriction enzyme with T3 or T7 RNA polymerase carried out and thus a sense (negative control) and an antisense RNA probe (for detecting the RNA in the Fabric). In detail, 1 µg of plasmid DNA with 2 µl NTP labeling mixture (10 mM ATP, 10 mM GTP, 10 mM CTP, 6 mM UTP and 3.5 mM DIG-UTP), 2 µl Transcription buffer, 1 µl RNase inhibitor (20 U / µl) and 2 µl T3 or T7 RNA polymerase in a final volume of 20 µl mixed and incubated for 2 hours at 37 ° C. The labeled probe was removed by centrifugation cleaned over a Sephadex G50 column, precipitated, dried under vacuum and in 100 µl DEPC water dissolved.

The labeling efficiency of probes for in situ Hybridization was compared by comparison with a DIG labeled control RNA determined via "dot blot". A series of dilutions of the control RNA in 1: 5 dilution steps from 100 ng / µl to 6.4 pg / µl manufactured; the sample was made according to the same scheme diluted. 24 of each dilution level were on Nylon membrane (Hybond N +) applied, air dried and immobilized on the membrane by UV light. To a 30 minute block in ISH buffer 2 (Boehringer Mannheim) at room temperature, the Membrane in an alkaline solution for 30 minutes Phosphatase conjugated anti-DIG antibody 1: 5000 diluted in ISH buffer 2, twice each 15 minutes in ISH buffer 1 (Boehringer Mannheim)  washed and in ISH buffer 3 (Boehringer Mannheim) Equilibrated for 2 minutes. For color development the membrane in a solution of 100 µl NBT / BCIP (Boehringer Mannheim) in 10 ml ISH buffer 1 in the Given darkness and after sufficient coloring in Washed water. By comparing the color intensity the control with the sample became the concentration labeled RNA determined in the sample.

Shock-frozen tissue samples were used on a Cryo microtome (Jung CM 1800) at -20 ° C frozen sections 6 µm thick and on slides transferred. The cuts were made at room temperature Air dried for 15 minutes and then 10 minutes fixed in DEPC-PBS with 4% paraformaldehyde. To was washed twice in DEPC-PBS for three minutes the sections were acetylated with stirring for 10 minutes a solution of 0.5% acetic anhydride and 0.1 M Tris, pH 8. After washing again twice for 3 minutes each in DEPC-PBS, dehydration in one ascending alcohol series (30%, 50%, 75%, three times 96% and chloroform). The cuts were made with surrounded by a fat pen (DAKO-pen), directly to the Hybridization used or until use in Stored at -70 ° C.

Shortly before hybridization, the cuts were made in a moist chamber (DEPC water) for 30 min at 52 ° C re-hydrogenated. About 100 ul hybridization solution per Sections were carefully pipetted onto the tissue. Sense and antisense probes were run in parallel successive cuts used. RNA was Denatured on a hotplate at 95 ° C for 4 minutes and  then in a humid chamber overnight 55 ° C hybridized without a cover slip. The slides were briefly washed in 2 × SSC at room temperature, twice for 10 minutes in 2 × SSC at 52 ° C in Shake water bath and once for 10 minutes in 1 × SSC Room temperature. After rinsing in DEPC water, the Cuts in blocking solution (ISH buffer 2 from Boehringer Mannheim, 10% FCS, 3% goat serum) blocked. A was used to detect the digoxigenin 1 hour incubation step with anti-DIG antibody conjugated to alkaline phosphatase 1: 500 in Blocking solution carried out at room temperature. Unbound antibodies were washed twice removed in DEPC water. For color development, the Sections with the chromogenic substrate BCIP / NBT (200 µl in ISH buffer 3 from Boehringer Mannheim) at 4 ° up to kept for a week. With longer ones The development time was changed to the enzyme substrate. After color development was completed, the Sections briefly rinsed in water, with hematoxylin Counterstained for 10 seconds, washed in tap water and with coverslips covered with Entellan mounting medium were provided, covered.

Show z. B. cardiac muscle, striped muscle and Normal kidney (only normal cells in normal kidney weak positive) no signal during the RCC tissue sections are highly positive.

Fig. 3 shows the in situ hybridization with B132 antisense (as) and B132 (s) samples in various tissue sections. The s-sample always corresponds to the negative control.
A and B: striped muscle
C and D: heart
E and F: normal kidney
G and H: renal cell carcinoma (RCC)
I: RCC 1604, 10x magnification
J: RCC 1604; 40x magnification

Fabrics in pictures A, C, E, G, I and J were made with hybridized as B132 probe; in B, D, F, and H with s-B132 sample (corresponds to negative control) hybridizes.

Example 8 Potential MHC binding peptides in the coding Region of B132

Potential peptide epitopes within the coding Region of B132 according to SEQ ID NO: 2 were determined using the by Parker et. al., 1994 described algorithms based on known motifs (Rammenee et al. 1995). For the main HLA types, especially for HLA-A1, -A * 0201, -A3, -B7, -B14 and -B * 4403, 9-mer candidate peptides were identified which is expected to be sent to the bind corresponding HLA molecules and therefore immunogenic Represent CTL epitopes; the peptides found are in Table 2 listed. Using an analogous approach other potential peptide epitopes for other HLA types or 8- and 10-mer peptides can be determined.  

Table 2

Immunogenic B132 peptide candidates

Example 10 Bioinformatics analysis Possible function of B132

B132 has two conserved sequence motifs: an N-terminal poly (A) -specific RNA binding motif and a C-terminal 3'-5 'exonuclease domain. The exonuclease domain was identified by a search against the Pfam database (Bateman et al., 2000) (Pfam-A HMM 00929, E value: 0.076); In addition, a BLAST search (Altschul et al., 1997) provides for B132 homologs Thermotoga maritima DNA polymerase III (E value: 0.042), which contains a proofreading exonuclease. The poly (A) -specific RNA binding motif was detected by characterizing the poly (A) -binding proteins from Mus musculus and H. sapiens (Blast E values for homology to B132: 0.12 and 0.36) using Pfam in the area the sections homologous to B132 (Pfam-B HMM 9063, motif specific for poly (A) -binding proteins, E values: 6.10 ^-37 or 2.9.10 ^-36 ).

3'-5 'exonucleases have three distinctly conserved ones Sequence sections on that as ExoI, ExoII and ExoIII be designated and five for the enzymatic Activity required to contain amino acid residues (Moser et al., 1997). This is the active center Forming residues can be found in B132 (D134, E136, D234, H293, D298). It is therefore expected that is an active 3'-5 'exonuclease. Proteins containing 3'-5 'exonuclease domains recognize DNA or RNA substrates and are in the DNA Replication, repair and recombination or in the  RNA processing and translation initiation involved. The exonuclease domain in combination with the poly (A) binding motif in B132 allows mRNA Expect deadenylation as a likely function. The stability and Controlled translation competence of the mRNA.

The functions are in Saccharomyces cerevisiae Poly (A) binding and poly (A) specific exonuclease two separate proteins localized (PAB and PAN2) (Boeck et al., 1996; Sachs & Deardorff, 1992). A PSI-BLAST search (Altschul et al., 1997) PAN2 as a relative of B132. Since PAN2 is the translation controlled by mRNA substrates, was expected to For its part, PAN2 is a target molecule for signal transduction represents (Sachs & Deardorff, 1992). Indeed are at least two human proteins, one Exonuclease domain (and no other domains) contain, hormone-regulated: The expression of ISG20 is described by Interferone (Gongora et al., 1997) and the of HEM45 induced by estrogen (Pentecost, 1998). These proteins are also identified by a PSI-BALST search found with B132. HEM45 is also in Tumor cell lines expressed (Pentecost, 1998). Further human proteins involved in diseases and contain an exonuclease domain are Werner syndrome protein (premature aging) and PM-SCL (Polymyositis scleroderma autoantigen) (Moser et al., 1997; Mushegian et al., 1997).  

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SEQUENCE LOG

221 WI = 75 TI = UDF <

Claims (21)

1. tumor-associated antigen of the designation B132, with that specified in SEQ ID NO: 2 Amino acid sequence. 2. Immunogenic protein fragment or peptide, thereby characterized in that it is of one in claim 1 defined tumor-associated antigen is. 3. Immunogenic (poly) peptide according to claim 1 or 2, that triggers a humoral immune response. 4. Immunogenic (poly) peptide according to claim 1 or 2, the or its degradation products by 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: 33-113. 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 induced expressing B132. 7. Pharmaceutical composition for the parenteral, topical, oral or local Administration, characterized in that they as an effective component one or more immunogenic (poly) peptides according to one of the claims Contains 1 to 5.   8. Pharmaceutical composition according to claim 7, characterized in that they are different from B132 contains derived immunogenic peptides. 9. Pharmaceutical composition according to claim 8, characterized in that they have one or more Peptides derived from B132 mixed with von other tumor-associated antigens Contains peptides. 10. Pharmaceutical composition according to claim 8 or 9, characterized in that the peptides bind to at least two different HLA types. 11. Isolated DNA molecule coding for a protein with the immunogenic properties of an in Claim 1 defined tumor-associated antigen or for fragments of it. 12. DNA molecule according to claim 12, coding for a immunogenic polypeptide of the designation B132 with the amino acid sequence shown in SEQ ID NO: 2 or for protein fragments derived therefrom or Peptides. 13. DNA molecule according to claim 12, characterized characterized that it is a polynucleotide with the sequence shown in SEQ ID NO: 1 or 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 11 to 13.   15. DNA molecule according to one of claims 11 to 14, for cancer immunotherapy, where the B132- expressed by the DNA molecule (Poly) peptide has an immune response against tumor cells of the patient induced expressing B132. 16. Pharmaceutical composition containing as effective ingredient one or more of the in one of claims 11 to 15 defined DNA Molecules. 17. Use of cells that in claim 1 express defined tumor-associated antigen, for the production of a cancer vaccine. 18. Antibody against in one of claims 1 to 5 defined (poly) peptide. 19. Antibody according to claim 18, characterized characterized that it is monoclonal. 20. Antibody according to claim 18 or 19 for the Therapy and diagnosis of cancer that are associated with the expression of B132. 21. Method of identifying a substance with anti-proliferative effect on tumor cells, characterized in that the B132 protein, or an active fragment thereof, in cells that on the activity of B132 with proliferation react, bring in or the corresponding B132 expresses cDNA in the cell, and the Proliferation of cells in the present and in Absence of the test substance determined.