HK1165461A - In tumours differentially expressed gene products and use of the same - Google Patents

Abstract

The invention relates to the identification of genetic products expressed in association with tumors and to coding nucleic acids for said products. Said invention also relates to the therapy and diagnosis of disease in which the genetic products are aberrantly expressed in association with tumors, proteins, polypeptides and peptides which are expressed in association with tumors, and to the nucleic coding acids for said polypeptides, peptides and proteins.

Description

Despite interdisciplinary approaches and stimulation of classical therapeutic modalities, cancer continues to be one of the leading causes of death. Newer therapeutic approaches aim to integrate the patient's own immune system into the overall therapeutic approach by using recombinant tumor vaccines and other specific measures such as antibody therapy. A prerequisite for the success of such a strategy is the detection of tumor-specific or tumor-associated antigens or epitopes by the patient's immune system, whose effector functions are to be enhanced interventionally.When such tumor-associated structures are recognised by the specific immune system of the tumor-carrying host, they are called tumour-associated antigens.The specific recognition of tumour-associated antigens involves cellular and humoral mechanisms, which are two functionally interconnected units: CD4+ and CD8+ T lymphocytes recognise processed antigens which are present on the molecules of the MHC major histocompatibility complex class II and I respectively, while B lymphocytes circulate antibody antibodies which directly lead to unprocessed antibodies.The potential clinical significance of tumour-associated antigen recognition stems from the fact that it can be used to detect antibodies and antibodies in the immune system by the anti-photoxic antibodies (e.g. antigenic antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antibodies, antiThe molecular nature of these antigens remained a long time enigma. It was only when appropriate cloning techniques were developed that it was possible to systematically screen cDNA expression of tumour-associated antigens by analysing the target structures of cytotoxic T-lymphocytes (CTL) (van der Bruggen et al., Science 254:1643-7, 1991) or with circulating autoantibodies (Sahin et al., Curr. Opin. Immunol. 9:709-16, 1997) as probes.to clone the respective antigens.

These approaches have led to the identification of a wide range of antigens in various neoplasms in recent years. However, the classical antigen identification methods described above use immune effector (circulating autoantibodies or CTL clone) from patients with cancer, usually already advanced, as probes. A number of data indicate that tumors can lead to T-cell tolerance and aggregation, for example, and that specific target immune effector characteristics that could be used for effective immune recognition are lost during the course of the disease.

The present invention was intended to provide target structures for the diagnosis and treatment of cancer.

This task is solved, in accordance with the invention, by the subject matter of the claims.

According to the invention, a strategy for identifying and providing tumor-associated expressed antigens and the nucleic acids coding for them is pursued based on the fact that certain genes expressed organ-specifically, e.g. exclusively in colon, lung or kidney tissue, are also expressed in the respective organs by tumor cells and furthermore in other tissues in ectopic and unauthorized reactivation in tumor cells. Data mining first creates a most complete list of all known organ-specific cancer genes and then assesses their expression in different actors by means of specific PCR analysis, but has been shown to be successful. Data mining is a method for tumor gene expression, which is based on the use of DNA DNA analysis and DNA analysis in the US, and is usually used in the databases RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTGS, RTG, RTG, RTG, RTG, RTG, RTG, RTG, RTG, RTG, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT, RT

Err1:Expecting ',' delimiter: line 1 column 265 (char 264)

A combined strategy based on two different bioinformatics scripts enabled the identification of new tumor genes, which have so far been classified as purely organ-specific. The recognition that these genes are aberrantly activated in tumor cells allows them to be assigned a substantially new quality with functional implications. The identification and provision of these tumor-associated genes and the gene products encoded by them was done independently of any immunogenic effect, according to the invention.

The tumor-associated antigens identified in accordance with the invention have an amino acid sequence encoded by a nucleic acid selected from the group consisting of (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NO: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid identified under strict conditions with the nucleic acid identified in (a) hybrid, (c) a nucleic acid identified in relation to the nucleic acid identified in (a) or (b) and (d) a nucleic acid identified in relation to the nucleic acid identified in (a), (b) or (c) as a complementary nucleic acid, a tumor-associated antigen is identified in a preferred form and a nucleic acid derivative identified in a preferred form consisting of the nucleic acid identified in (a), (b) or (c) is identified in a tumor-associated antigen, or a nucleic acid identified in (a), (b) or (c) as a complementary nucleic acid, in the group identified in (a), (b) or (c) is identified in (d) an antigen, and is identified in a tumor-associated antigen, identified in (e) group identified in (i), (i), (ii), (ii), (ii), (ii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (iii), (

The present invention relates generally to the use of tumor-associated antigens or parts or derivatives thereof, of nucleic acids coding for them or of nucleic acids directed against the coding nucleic acids, or of antibodies directed against tumor-associated antigens or parts or derivatives thereof, identified in accordance with the invention, for therapy and diagnosis, and may be used alone or in combination of several of these antigens, functional fragments, nucleic acids, antibodies, etc., in one embodiment, or in combination with other tumor-associated genes and antigens for diagnosis, therapy and control.

Diseases preferred for therapy and/or diagnosis are those in which there is selective expression or abnormal expression of one or more of the tumour-associated antigens identified in accordance with the invention.

The invention also relates to nucleic acids and gene products expressed by tumour cells.

In addition, the invention relates to gene products, i.e. nucleic acids and proteins or peptides, which are produced by altered splicing (splicing variants) of known genes or by altered translation using alternative open reading frames. In this respect, the invention relates to nucleic acids which contain a nucleic acid sequence selected from the group consisting of the sequences in accordance with SEQ ID NO: 3-5 of the sequence protocol. In addition, the invention relates to proteins or peptides which contain an amino acid sequence selected from the group consisting of the sequences in accordance with SEQ ID NO: 10 and 12-14 of the sequence protocol. The invention is therefore suitable as a drug for the diagnosis and therapy of tumours.

In particular, the invention relates to the amino acid sequence according to SEQ ID NO: 10 of the sequence protocol, which is encoded by an alternative open reading frame identified in accordance with the invention and differs from the prescribed protein sequence (SEQ ID NO: 9) by 85 additional amino acids at the N-terminus of the protein.

The development of variations in stress can be caused by a variety of mechanisms, such as: The use of variable transcription initiation sites,the use of additional exons,the complete or incomplete splitting of single or multiple exons,splice regulatory sequences altered by mutation (deleting or creating new donor/acceptor sequences),the incomplete elimination of intron sequences.

Translating a splice variant into its altered sequence domain results in a modified protein that may be significantly different from the original in structure and function. Tumor-associated splice variants may produce tumor-associated transcripts and tumor-associated proteins/antigens. These can be used as molecular markers for both tumor cell detection and therapeutic targeting of tumors. The detection of tumor cells e.g. in blood, serum, bone marrow, sputum, bronchial lymph nodes, body secretions and tissue can occur under conditions such as biopsy.The oligonucleotides described in the examples for this purpose, in particular oligonucleotides containing a sequence selected from SEQ ID NO: 34-36, 39, 40 and 107-110 of the sequence protocol, are suitable for detection. In addition to PCR, all sequence-dependent detection systems are suitable for detection. These include, for example, gene-chip/microarray systems, Northem-Blotse RNA protection assays (RDA) and others. All detection systems are designed to detect a specific hybridisation with at least one splice-variant specific nucleic acid sequence.In this respect, the invention relates in particular to peptides which contain or contain a sequence selected from SEQ ID NO: 17-19, 111-115, 120 and 137 of the sequence protocol and specific antibodies directed against it. Amino acids which exhibit significant epitope differences to the gene product variant (s) which are preferably produced in healthy cells are particularly suitable for immunisation. Detection of tumor cells with antibodies may be performed on a sample isolated from the patient or as an imaging with intravenously administered antibodies.The epitopes of the invention can be used to target therapeutically effective monoclonal antibodies or T lymphocytes. Passive immunotherapy involves the adoptive transfer of antibodies or T lymphocytes that recognize splice-variant-specific epitopes. The generation of antibodies can also be done using standard technologies (immunization of animals, paning strategies for the isolation of recombinant bodies) using polypeptides containing these epitopes. Nucleic acids encoding oligo- or polypeptides containing these epitopes can be used for immunization.The use of oligopeptides or polypeptides containing or coding for splice-variant-specific epitopes is also possible as pharmaceutically active substances in active immunotherapy (vaccination, vaccine therapy).

The invention also describes proteins which differ in the type and amount of their secondary modifications in normal and tumour tissues (e.g. Durand & Seta, 2000; Clin. Chem. 46: 795-805; Hakomori, 1996; Cancer Res. 56: 5309-18).

To detect specific O- and N-glycosidic bonds, protein lysates are incubated with O- or N-glycosides prior to denaturation by SDS (according to the respective manufacturer's specifications, e.g. p-glycosidase, endoglycosidase F, endoglycosidase H, Rounder Diagnostics). A Westem blot is then performed. When reducing the size of a target protein, a specific glycosidase pathway can be identified after injection with a glycosidase. However, the specific glycosidase pathway has not been shown to be effective in this type of tumor.

According to the invention, differential glycosylation of claudin-18 in tumors has been demonstrated. Gastrointestinal carcinomas, pancreatic carcinomas, esophageal tumors, prostate tumors, and lung tumors all exhibit a less glycosylated form of claudin-18. Glycosylation in healthy tissues masks claudin-18 protein pitopes that are exposed on tumor cells due to lack of glycosylation. Accordingly, according to the invention, ligands and antibodies can be selected to bind to these domains.

Similar to the one described above for tumor-associated splice variants, differential glycosylation can be used to distinguish between normal and tumor cells for both diagnostic and therapeutic purposes.

In one respect, the invention relates to a pharmaceutical composition comprising a substance that detects the tumour-associated antigen identified in accordance with the invention and is preferably selective for cells that exhibit expression or abnormal expression of a tumour-associated antigen identified in accordance with the invention. The substance may, in certain embodiments, induce cell death, reduce cell growth, damage the cell membrane or secrete cytokines and preferably exhibit tumor-inhibiting activity. In one embodiment, the substance is a selective antisense acid that binds selectively to the nucleic acid that codes for the tumour-associated antigen. The substance is a selective antigen that binds selectively to the tumour-associated antigen.In particular, a complement-activated or toxin-conjugated antibody that selectively binds to the tumor-associated antigen. In a further embodiment, the device includes several agents that selectively detect different tumor-associated antigens, each with at least one tumor-associated antigen being a tumor-associated antigen identified in accordance with the invention. The detection need not be directly associated with an inhibition of antigen activity or expression. In this aspect of the invention, the tumor-only antigen selectively serves as a marker to recruit mechanisms at that specific site. In a preferred embodiment, the agent is a lymphocyte antigen that detects a cytotoxic effect on an HLA molecule and lytically detects the target cell.In another embodiment, the agent is an antibody that selectively binds to the tumor-associated antigen and thus recruits natural or artificial effector mechanisms to that cell. In another embodiment, the agent is a T helper lymphocyte that enhances effector functions of other cells that specifically recognize this antigen.

In one embodiment, the invention concerns a pharmaceutical composition comprising a substance that inhibits the expression or activity of a tumour-associated antigen identified in accordance with the invention; in a preferred embodiment, the substance is an antisense nucleic acid that selectively hybridizes with the nucleic acid encoding the tumour-associated antigen; in a further embodiment, the substance is an antibody that selectively binds to the tumour-associated antigen; in a further embodiment, the substance comprises several substances, each selectively inhibiting the expression or activity of different tumour-associated antigen, whereby the tumour-associated antigen binds to at least one tumour-associated antigen identified in accordance with the invention.

The invention also relates to a pharmaceutical composition comprising a substance which, when administered, selectively increases the amount of complexes between an HLA molecule and a peptide pitop from the tumour-associated antigen identified in accordance with the invention, and which, in an embodiment, comprises one or more components selected from the group consisting of (i) the tumour-associated antigen or part thereof, (ii) a nucleic acid encoding the tumour-associated antigen or part thereof, (iii) a host cell expressing the tumour-associated antigen or part thereof, and (iv) isolated peptide complexes between a tumour-associated antigen and a tumour-associated antigen. The selective embodiment comprises a complex of at least one antigen and a peptide molecule, each containing an antigen identifying a tumour-associated antigen.

Furthermore, the invention relates to a pharmaceutical composition comprising one or more components selected from the group consisting of (i) a tumor-associated antigen or part thereof identified in accordance with the invention, (ii) a nucleic acid encoding a tumor-associated antigen or part thereof identified in accordance with the invention, (iii) an antibody binding to a tumor-associated antigen or part thereof identified in accordance with the invention, (iv) an antisense nucleic acid specifically hybridized with a nucleic acid associated with a tumor-associated antigen or part thereof identified in accordance with the invention, (v) a tumor-associated antigen or part thereof identified in accordance with the invention, and (viii) an antigen or part thereof identified in accordance with the invention, (iv) an anti-molecule and a tumor-associated antigen or part thereof identified in accordance with the invention.

A nucleic acid encoding a tumor-associated antigen or part thereof identified in accordance with the invention may be present in the pharmaceutical composition in an expression vector and functionally linked to a promoter.

A host cell in a pharmaceutical composition of the invention may secrete, surface-express, or additionally express a HLA molecule that binds to the tumor-associated antigen or part thereof. In one embodiment, the host cell expresses the HLA molecule endogenously. In another embodiment, the host cell expresses the HLA molecule and/or tumor-associated antigen or part thereof recombinantly. Preferably, the host cell is non-proliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, specifically a dendritic cell, monocyte or macrophage.

An antibody contained in a pharmaceutical composition of the invention may be a monoclonal antibody. In other embodiments, the antibody may be a chimeric or humanized antibody, a fragment of a natural antibody, or a synthetic antibody, all of which may be produced by combinatorial techniques. The antibody may be coupled with a therapeutically or diagnostically useful agent or substance.

An antisense nucleic acid contained in a pharmaceutical composition of the invention may comprise a sequence of 6-50, in particular 10-30, 15-30 or 20-30 nucleotides from the nucleic acid encoding the tumour-associated antigen identified in accordance with the invention.

In other embodiments, a tumor-associated antigen, provided by a pharmaceutical composition of the invention, either directly or by nucleic acid expression, or a portion thereof, binds to MHC molecules on the surface of cells, preferably by inducing a cytolytic reaction and/or cytokine release.

A pharmaceutical composition of the invention may include a pharmaceutically compatible carrier and/or adjuvant. The adjuvant may be selected from saponins, GM-CSF, CpG nucleotides, RNA, a cytokine or a chemokine. A pharmaceutical composition of the invention is preferentially used to treat a disease characterized by the selective expression or abnormal expression of a tumor-associated antigen.

The invention also relates to a method for the treatment or diagnosis of a disease characterised by the expression or abnormal expression of one or more tumour-associated antigens.

In one respect, the invention relates to a method for the diagnosis of a disease characterized by the expression or abnormal expression of a tumor-associated antigen identified in accordance with the invention, which includes the detection of (i) a nucleic acid encoding for the tumor-associated antigen or part thereof and/or (ii) the detection of the tumor-associated antigen or part thereof and/or (iii) the detection of an antibody against the tumor-associated antigen or part thereof and/or (iv) the detection of cytotoxic or helper T lymphocytes that are pro-tumour-associated antigen or part thereof in a specific biological patient.which binds specifically to the nucleic acid coding for the tumour-associated antigen or part thereof, to the tumour-associated antigen or part thereof, to the antibody or to cytotoxic or helper T-lymphocytes specific for the tumour-associated antigen or part thereof and (ii) the evidence of complex formation between the agent and the nucleic acid or part thereof, the tumour-associated antigen or part thereof, the antibody or cytotoxic or helper T-lymphocytes. In one embodiment, the disease is characterised by the expression or abnormal expression of several different tumour-associated antigenic agents and the detection includes a test for the presence of several tumour-associated nucleic acids coding for different tumour-associated antigenic agentsor parts thereof, the detection of several different tumour-associated antigens or parts thereof, the detection of several antibodies binding to several different tumour-associated antigens or parts thereof, or the detection of several cytotoxic or helper T-lymphocytes specific to several different tumour-associated antigens.

In another respect, the invention relates to a method for determining the regression, course or onset of a disease characterized by the expression or abnormal expression of a tumor-associated antigen identified in accordance with the invention, involving the monitoring of a sample from a patient who has or is suspected of having the disease in relation to one or more parameters selected from the group consisting of (i) the amount of nucleic acid encoding the tumor-associated antigen or part thereof, (ii) the amount of tumor-associated antigen or part thereof, (iii) the amount of antibodies binding to the tumor-associated antigen or part thereof,(iv) the amount of cytolytic T cells or helper T cells specific to a complex between the tumour-associated antigen or part thereof and an MHC molecule. The procedure preferably involves determining the parameter or parameters at a first time in a first sample and at a second time in a second sample, and determining the course of the disease by comparing the two samples. In certain embodiments, the disease is characterised by the expression or abnormal expression of several different tumour-associated antigens and monitoring includes monitoring (i) the amount of several nucleic acids coding for several different tumour-associated antigens,or parts thereof and/or (ii) the amount of several different tumour-associated antigens or parts thereof and/or (iii) the amount of several antibodies binding to several different tumour-associated antigens or parts thereof and/or (iv) the amount of several cytolytic T cells or helper T cells specific for complexes between several different tumour-associated antigens or parts thereof and MHC molecules.

The detection of a nucleic acid or a part thereof or the monitoring of the amount of a nucleic acid or a part thereof may be accomplished by a polynucleotide probe specifically hybridized with the nucleic acid or part thereof, or by selective amplification of the nucleic acid or part thereof, as described in the invention.

In certain embodiments, the tumour-associated antigen or part thereof to be detected is present intracellularly or on the cell surface. Detection of a tumour-associated antigen or part thereof or monitoring of the amount of a tumour-associated antigen or part thereof may be performed according to the invention with an antibody that specifically binds to the tumour-associated antigen or part thereof.

In other embodiments, the tumour-associated antigen or part thereof to be detected is in a complex with an MHC molecule, in particular an HLA molecule.

An antibody detection or antibody quantity monitoring may be performed according to the invention with a protein or peptide that binds specifically to the antibody.

The detection of cytolytic T cells or helper T cells or the monitoring of the amount of cytolytic T cells or helper T cells specific for complexes between an antigen or part thereof and MHC molecules may be performed according to the invention with a cell presenting the complex between the antigen or part thereof and an MHC molecule.

The polynucleotide probe, antibody, protein or peptide or cell used for detection or monitoring is preferably detectable. In certain embodiments, the detectable marker is a radioactive marker or an enzyme marker. Detection of T lymphocytes may be achieved by detecting their proliferation, cytokine production, and cytotoxic activity, which is triggered by specific stimulation with the complex of MHC and tumor-associated antigen or parts thereof. Detection of T lymphocytes may also be achieved by a recombinant MHC molecule or a complex of MHC molecules that are loaded with specific FLL or T-cell-associated immune receptors, and which can be identified by the T-cell specific receptor.

In another respect, the invention relates to a method for the treatment, diagnosis or monitoring of a disease characterized by the expression or abnormal expression of a tumor-associated antigen identified in accordance with the invention, involving the administration of an antibody that binds to the tumor-associated antigen or part thereof and is coupled to a therapeutic or diagnostic agent or substance. The antibody may be a monoclonal antibody. In other embodiments, the antibody may be a chimeric or humanized antibody or a fragment of a natural antibody.

The invention also relates to a procedure for the treatment of a patient with a disease characterized by the expression or abnormal expression of a tumour-associated antigen identified in accordance with the invention, involving (i) the removal of a sample of immune-reactive cells from the patient, (ii) the contact of the sample with a host cell expressing the tumour-associated antigen or part thereof, under conditions which favour the production of cytolytic T cells against the tumour-associated antigen or part thereof, and (iii) the introduction of cytolytic T cells into the patient in a quantity capable of lycing cells which express the tumour-associated antigen or part thereof.

In one embodiment, the host cell expresses an HLA molecule endogenously; in another embodiment, the host cell expresses an HLA molecule and/or the tumor-associated antigen or part thereof recombinantly; preferably, the host cell is nonproliferative; in a preferred embodiment, the host cell is an antigen-presenting cell, specifically a dendritic cell, monocyte, or macrophage.

In another respect, the invention relates to a procedure for the treatment of a patient with a disease characterized by the expression or abnormal expression of a tumor-associated antigen, involving (i) the identification of a nucleic acid coding for a tumor-associated antigen identified in accordance with the invention, expressed by cells associated with the disease, (ii) the transfection of a host cell with the nucleic acid or part thereof, (iii) the culture of the transfected host cell for nucleic acid expression (this is not mandatory when a high transfection rate is achieved), and (iv) the introduction of the host cells or an extract thereof into the patient in a quantity suitable for the treatment of the disease.The procedure may also involve the identification of an MHC molecule presenting the tumour-associated antigen or part thereof, whereby the host cell expresses the identified MHC molecule and presents the tumour-associated antigen or part thereof. The immune response may include a B cell response or a T cell response. In addition, a T cell response may involve the production of cytolytic T cells and/or helper T cells specific to the host cells presenting or part of the tumour-associated antigen or part thereof or specific to the tumour-associated patient or part thereof.

The invention also relates to a method for the treatment of a disease characterized by the expression or abnormal expression of a tumour-associated antigen identified in accordance with the invention, involving (i) the identification of cells from the patient expressing abnormal amounts of the tumour-associated antigen, (ii) the isolation of a sample of the cells, (iii) the culture of the cells and (iv) the introduction of the cells into the patient in a quantity suitable to induce an immune response against the cells.

Preferably, the host cells used according to the invention are non-proliferative or are made non-proliferative.

Furthermore, the present invention relates to a nucleic acid selected from the group consisting of (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3-5, a part or derivative thereof, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid which is comparable to the nucleic acid under (a), (b) or (c), and (c) a nucleic acid encoding a protein or a polypeptide containing a nucleic acid, selected from group 12 to 14, or a nucleic acid containing a derivative of SEQ or a part of the group 12 to 14, and (d) a nucleic acid which encodes a polypeptide containing a nucleic acid.

In another respect, the invention concerns promoter sequences of nucleic acids according to the invention which can be functionally linked to another gene, preferably in an expression vector, and thus ensure the selective expression of that gene in corresponding cells.

In another respect, the invention concerns a recombinant nucleic acid molecule, in particular a DNA or RNA molecule containing a nucleic acid of the invention.

The invention also relates to host cells containing a nucleic acid of the invention or a recombinant nucleic acid molecule containing a nucleic acid of the invention.

The host cell may also contain a nucleic acid that encodes for an HLA molecule. In one embodiment, the host cell expresses the HLA molecule endogenously. In another embodiment, the host cell expresses the HLA molecule and/or the nucleic acid of the invention or part thereof recombinantly. Preferably, the host cell is non-proliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, in particular a monocyt, a dendritic cell, or a macrophage.

Err1:Expecting ',' delimiter: line 1 column 225 (char 224)

In another respect, the invention relates to a protein, polypeptide or peptide encoded by a nucleic acid selected from the group consisting of (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NO: 3-5, a part or derivative thereof, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).

In another respect, the invention relates to an immunogenic fragment of a tumor-associated antigen identified in accordance with the invention, which preferably binds to a human HLA receptor or human antibody, preferably comprising a sequence of at least 6, in particular at least 8, at least 10, at least 12, at least 15, at least 20, at least 30, or at least 50 amino acids.

In this respect, the invention relates in particular to a peptide which contains or comprises a sequence selected from the group consisting of SEQ ID NO: 17-19, 90-97, 100-102, 105, 106, 111-116, 120, 123, 124 and 135-137 and a part or derivative thereof.

In another aspect, the invention concerns an antibody that selectively binds to a complex of (i) a tumor-associated antigen or part thereof identified in accordance with the invention; in a preferred embodiment, the agent is an antibody; in other embodiments, the antibody is a chimeric, humanized or combinatorial antibody or a fragment of an antibody; and in a second embodiment, the invention concerns an antibody that selectively binds to a complex of (i) a tumor-associated antigen or part thereof identified in accordance with the invention; and (ii) an MHC molecule to which the tumor-associated antigen or part thereof identified in accordance with the invention binds, whereby all antibodies (i) may be a natural or monomers or antibodies (ii) may be a chimeric or monomers or an antibody of a human or non-human origin.

In particular, the invention relates to such a device, in particular an antibody that specifically binds to a peptide, which contains or includes a sequence selected from the group consisting of SEQ ID NO: 17-19, 90-97, 100-102, 105, 106, 111-116, 120, 123, 124 and 135-137, or a part or derivative thereof.

In addition, the invention relates to a conjugate between a substance of the invention which binds to a tumor-associated antigen or part of a tumor-associated antigen identified in accordance with the invention or an antibody of the invention and a therapeutic or diagnostic substance or substance.

In another respect, the invention relates to a kit for detecting the expression or abnormal expression of a tumor-associated antigen identified in accordance with the invention, comprising means for detecting (i) the nucleic acid encoding the tumor-associated antigen or part thereof, (ii) the tumor-associated antigen or part thereof, (iii) antibodies binding to the tumor-associated antigen or part thereof, and/or (iv) T cells specific to a complex between the tumor-associated antigen or part thereof and an MHC molecule. In one embodiment, the means for detecting the nucleic acid or part thereof are selective for the amplification of the tumor-associated antigen or part thereof, and in particular comprise 20-30-50 nucleic acid sequences, comprising a complex of 10 or 30 nucleic acid sequences, in particular, of the nucleic acid sequence.

The invention relates in particular to: 1. pharmaceutical composition, including a substance that inhibits the expression or activity of a tumor-associated antigen, where the tumor-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid referred to in (a) or (b), and (d) a nucleic acid which becomes the nucleic acid referred to in (a),(b) or (c) is complementary.2. Pharmaceutical composition, including a substance with tumour-suppressing activity, which is selective for cells exhibiting expression or abnormal expression of a tumour-associated antigen, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b), and (d) a nucleic acid,which is complementary to the nucleic acid referred to in (a), (b) or (c).3. Pharmaceutical composition referred to in point 2, where the agent induces cell death, reduces cell growth, damages the cell membrane or secrete cytokines.4. Pharmaceutical composition referred to in point 1 or 2, where the agent is an antisense nucleic acid that selectively hybridizes with the nucleic acid that codes for the tumour-associated antigen.5. Pharmaceutical composition referred to in point 1 or 2, where the agent is an antibody that selectively binds to the tumour-associated antigen.6. Pharmaceutical composition referred to in point 2, where the agent is a complementary antibody that selectively binds to the tumour-associated antigen.4.7. pharmaceutical composition, including a substance which, when administered, selectively increases the amount of complexes between an HLA molecule and a tumour-associated antigen or part thereof, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b), and (d) a nucleic acid,a complementary nucleic acid as defined in (a), (b) or (c).8. Pharmaceutical composition as defined in point 7, where the product contains one or more constituents selected from the group consisting of: (i) the tumour-associated antigen or part thereof, (ii) a nucleic acid coding for the tumour-associated antigen or part thereof, (iii) a host cell expressing the tumour-associated antigen or part thereof, and (iv) isolated complexes between the tumour-associated antigen or part thereof and an HLA molecule.9. Pharmaceutical composition as defined in points 1, 2 or 7, where the agent comprises several agents, each selectively inhibiting the expression or activity of different tumour-associated anenes,are selective for cells expressing different tumour-associated antigens or increase the amount of complexes between HLA molecules and different tumour-associated antigens or parts thereof, at least one of the tumour-associated antigens having a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b), and (d) a nucleic acid,which is complementary to the nucleic acid in (a), (b) or (c).10. Pharmaceutical composition consisting of one or more constituents selected from the group consisting of: (i) a tumor-associated antigen or part thereof, (ii) a nucleic acid encoding a tumor-associated antigen or part thereof, (iii) an antibody binding to a tumor-associated antigen or part thereof, (iv) an antisense nucleic acid specifically hybridising with a nucleic acid encoding a tumor-associated antigen, (v) a host cell expressing a tumor-associated antigen or part thereof, and (vi) isolated complexes between a tumor-associated antigen or part thereof and an HLA molecule, where the tumour-associated antigen has a sequence,which is encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid in (a), (b) or (c).11.the nucleic acid is functionally linked to a promoter as defined in (ii).13. pharmaceutical composition according to point 8 or 10, where the host cell secretes the tumour-associated antigen or part thereof.14. pharmaceutical composition according to point 8 or 10, where the host cell additionally expresses an HLA molecule which binds to the tumour-associated antigen or part thereof15. pharmaceutical composition according to point 14, where the host cell expresses the HLA molecule and/or tumour-associated antigen or part thereof recombinantly.16. pharmaceutical composition according to point 14, where the host cell expresses the HLA molecule endogenously.17. pharmaceutical composition according to point 8, or 14, where the host cell is an antigen-expressing cell.18. pharmaceutical composition according to point 17, where the antigen-presenting cell is a dendritic cell or a macrophage.19. pharmaceutical composition according to one of points 8, 10 and 13-18 where the host cell is non-proliferative.20. pharmaceutical composition according to point 5 or 10, where the antibody is a monoclonal antibody.21. pharmaceutical composition according to point 5 or 10, where the antibody is a chimeric or humanized pharmaceutical organism.22. pharmaceutical composition according to point 5 or 10, where the antibody is a fragment of a natural pharmaceutical organism.23. antibacterial composition according to point 5 or 10, where the antibody is composed of a therapeutic or diagnostic drug.24. pharmaceutical composition according to point 10, or 4Antisense nucleic acid comprises a sequence of 6 to 50 nucleotides linked from the nucleic acid that encodes the tumour-associated antigen.25. Pharmaceutical composition according to one of the following 8 and 10-13 where the tumour-associated antigen or part thereof provided by the pharmaceutical composition binds to MHC molecules on the surface of cells expressing an abnormal amount of tumour-associated antigen or part thereof.26. Pharmaceutical composition according to 25, where the binding induces a cytolytic reaction and/or induces cytokine release.27. Pharmaceutical composition according to 1 to 26, where the composition of a pharmaceutical substance is a saponin/adjuvant and/or a saponin.28.Pharmaceutical composition according to heading 29 where the disease is a lung tumour, a breast tumour, a prostate tumour, a melanoma, a colon tumour, a stomach tumour, a pancreatic tumour, an ENT tumour, a renal cell carcinoma or a cervical tumour, a colon tumour or a NOQ.32. Pharmaceutical composition according to heading 29 where the disease is a tumour of the type 9 to 19, consisting of a group of antibodies:45-48, 60-66, 85, 90-97, 100-102, 105, 106, 111-116, 118, 120, 123, 124 and 135-137, a part or derivative thereof is selected.33. (i) the detection of a nucleic acid coding for the tumour-associated antigen or part thereof, and/or (ii) the detection of the tumour-associated antigen or part thereof, and/or (iii) the detection of an antibody against the tumour-associated antigen or part thereof, and/or (iv) the detection of cytotoxic or helper T-lymphocytes specific to the tumour-associated antigen or part thereof in a biological sample isolated from a patient;wherein the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (i) the contact of the biological sample with a substance;which specifically binds to the nucleic acid encoding the tumour-associated antigen or the part thereof, to the tumour-associated antigen or part thereof, to the antibody or to the cytotoxic or helper T-lymphocytes, and (ii) the detection of complex formation between the agent and the nucleic acid or part thereof, the tumour-associated antigen or part thereof, the antibody or cytotoxic or helper T-lymphocytes35. procedure referred to in paragraph 33 or 34, where the detection is compared with the detection in a comparable normal biological sample.36. procedure referred to in paragraphs 33 to 35, where the disease is detected by the expression or abnormal expression of several different tumour-associated acids and a nuclear detection of the disease,which code for several different tumour-associated antigens or parts thereof, includes the detection of several different tumour-associated antigens or parts thereof, the detection of several antibodies binding to several different tumour-associated antigens or parts thereof, or the detection of several cytotoxic or helper T-lymphocytes specific to several different tumour-associated antigens.37. Processes in one of the paragraphs 33 to 36, where the nucleic acid or part is detected by a polynucleotide probe specifically hybridised with the nucleic acid or part thereof.3839 method according to one of the points 33-36 where the nucleic acid or part thereof is detected by selective amplification of the nucleic acid or part thereof. 40 method according to one of the points 33-36 where the tumour-associated antigen or part thereof to be detected is present in a complex with an MHC molecule. 41 method according to point 40, where the MHC molecule is an HLA molecule. 42 method according to points 33-36 and 40-41 where the tumour-associated antigen or part thereof is detected by an antibody specifically binding to the tumour-associated antigen or part thereof. 43 method according to point 33-36.where the antibody is detected by a protein or peptide that binds specifically to the antibody.44. method for the determination of regression, progression or onset of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including monitoring of a sample from a patient who has or is suspected of having the disease with respect to one or more parameters selected from the group consisting of: (i) the amount of nucleic acid coding for the tumour-associated antigen or part thereof; (ii) the amount of tumour-associated antigen or part thereof; (iii) the amount of antibodies;which bind to the tumour-associated antigen or part thereof, and (iv) the number of cytolytic or cytokine-expressing T cells specific for a complex between the tumour-associated antigen or part thereof and an MHC molecule, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,The procedure described in paragraph 44 involves determining the parameter (s) at a first time in a first sample and at a second time in a second sample and determining the course of the disease by comparing the two samples. (i) the amount of several nucleic acids coding for several different tumour-associated antigens or parts thereof;(iii) the number of multiple antibodies binding to several different tumour-associated antigens or to parts thereof, and/or (iv) the number of multiple cytolytic or cytokine-expressing T cells specific for complexes between several different tumour-associated antigens or parts thereof and MHC molecules.47. procedure according to one of the points 44-46, where the amount of nucleic acid or part thereof is monitored by a polynucleotide probe specifically hybridised with the nucleic acid or part thereof.48. procedure according to point 47, where the polynucleotide sequence comprises a continuous sequence of 6 to 50 nucleotides from the nucleic acid coding for the tumour-associated antigen.46. procedure according to point 44-49, where the amount of nucleic acid or part thereof is monitored by a polynucleotide probe specifically hybridised with the nucleic acid or part thereof.48.the monitoring of the amount of nucleic acid or part thereof by selective amplification of the nucleic acid or part thereof.50. procedure in accordance with one of points 44 to 46, where the monitoring of the amount of tumor-associated antigen or part thereof is carried out with an antibody specifically binding to the tumor-associated antigen or part thereof.51. procedure in accordance with one of points 44 to 46, where the monitoring of the amount of antibodies is carried out with a protein or peptide specifically binding to the antibody.52. procedure in accordance with one of points 44 to 46, where the monitoring of the amount of cytolytic or cytokine-expressing T cells is carried out with a cell presenting the complex between the tumor-associated antigen or part thereof and the HCV-molecule.Method according to one of points 37-38, 42-43, 47-48 and 50-52 where the polynucleotide probe, antibody, protein or peptide or cell is detectably marked.54. Method according to point 53, where the detectable marker is a radioactive marker or an enzyme marker.55. Method according to point 33-54, where the sample includes body fluids and/or body tissues.56. Method according to point 56 for the treatment of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including administration of a compound as defined in points 1 to 32, where the tumour-associated antigen is selected from a group of pharmaceutical drugs, and where the sequence is coded for by a nucleic acid selected from a group ofconsisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).57 Procedures for the treatment, diagnosis or monitoring of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including the administration of an antibody which is bound to a tumour-associated antigen or part thereof, or a therapeutic agent, andwhere the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid in (a), (b) or (c).58 The procedure described in paragraphs 42, 50 or 57, where the antibody is a monoclonal antibody.59. procedures in points 42, 50 or 57, where the antibody is a chimeric or humanized antibody.60. procedures in points 42, 50 or 57, where the antibody is a fragment of a natural antibody.61. procedures for the treatment of a patient with a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including: (i) the removal of a sample of immune-reactive cells from the patient, (ii) the contact of the sample with a host cell expressing the tumour-associated antigen or part thereof under conditions which favour the production of cytolytic or cytokine-expressing T cells against the tumour-associated antigen or part thereof, and (iii) the introduction of the cytolytic or cytokine-expressing T cells into the patient in quantity,which is capable of lysing cells expressing the tumour-associated antigen or part thereof, the tumour-associated antigen having a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,procedure in point 61, where the host cell expresses a recombinant HLA molecule that binds to the tumour-associated antigen or part thereof63. procedure in point 62, where the host cell expresses an HLA molecule endogenously that binds to the tumour-associated antigen or part thereof.64. procedure for the treatment of a patient with a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including: (i) the identification of a nucleic acid expressed by cells associated with the disease, the nucleic acid being selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence consisting of the group consisting of SEQ ID NOs: 1-8,41-44, 51-59, 84, 117 and 119,a part or derivative of which is selected, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c), (ii) the transfection of a host cell with the nucleic acid or part thereof, (iii) the culturing of the transfected host cell for expression of the nucleic acid, and (iv) the introduction of the host cells or extract thereof into the patient in a quantity suitable to control the immune response of the patient against the cells associated with the disease,The procedure described in paragraph 64 shall also include the identification of an MHC molecule presenting the tumour-associated antigen or part thereof, whereby the host cell expresses the identified MHC molecule and the tumour-associated antigen or part thereof. The procedure described in paragraph 64 or 65, whereby the immune response includes a B cell response or a T cell response, shall also be increased.68 procedures as defined in points 61 to 67, where the host cells are non-proliferative. 69 procedures for the treatment of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including: (i) identification of cells from the patient expressing abnormal amounts of the tumour-associated antigen, (ii) isolation of a sample of the cells, (iii) culture of the cells, and (iv) introduction of the cells into the patient in a quantity suitable to trigger an immune response against the cells, whereby the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence,selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,(b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a),(c) a nucleic acid which is degenerated with respect to the nucleic acid in (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid in (a), (b) or (c).70 A procedure under points 33 to 69 where the disease is cancer71 A procedure to prevent the development of cancer in a patient, involving the administration of an effective pharmaceutical composition of points 1 to 32.72.where the tumour-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-19, 45-48, 60-66, 85, 90-97, 100-102, 105, 106, 111-116, 118, 120, 123, 124 and 135-137, a part or derivative thereof.73. (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3-5, a part or derivative thereof, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid referred to in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid referred to in (a) or (b) and (d) a nucleic acid which converts to the nucleic acid referred to in (a),(b) or (c) is complementary74. nucleic acid encoding a protein or polypeptide containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 12-14, a part or derivative thereof.75. recombinant DNA or RNA molecule containing a nucleic acid according to item 73 or 74. recombinant DNA molecule according to item 75, where the recombinant DNA molecule is a vector of DNA.77. recombinant DNA molecule according to item 76, where the vector is a viral vector or a bacteriophage.78. recombinant DNA molecules according to item 75-77, which also contains expression control sequences controlling the expression of nucleic acid.79. recombinant or heterologous nucleic acid controls according to item 78, which are the expression control sequences of the nucleic acid.79.Host cell containing a nucleic acid as defined in item 73 or 74 or a recombinant DNA molecule as defined in one of claims 75-79.81. Host cell of item 80, which also contains a nucleic acid encoding for an HLA molecule.82. Protein or polypeptide encoded by a nucleic acid as defined in item 73.83. Protein or polypeptide containing an amino acid sequence selected from the group consisting of SEQ NO IDs: 10 and 12-14, one or a derivative thereof.84. Immunogenic fragment of protein or polypeptide according to item 82 or 83.85. Fragment of protein or polypeptide according to item 82 or 83.83, which binds to human HLA receptor or human antibody.8.6.the protein or polypeptide is encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which under strict conditions hybridizes with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).87.consisting of SEQ ID NOs: 9-19, 45-48, 60-66, 85, 90-97, 100-102, 105, 106, 111-116, 118, 120, 123, 124 and 135-137, and any part or derivative thereof.88 means of heading 86, or 87, where the means is an antibody.89 means of heading 88, where the antibody is a monoclonal, chimeric or humanised antibody or a fragment of an antibody.90. antibody selectively binding to a complex of: (i) binds to a protein or polypeptide or part thereof and (ii) binds to an MHC molecule to which the protein or polypeptide or part thereof binds, provided that the antibody does not bind to (i) or (ii) alone and the protein or polypeptide is coded for by a nucleic acid,selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs 1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).45-48, 60-66, 85, 90-97, 100-102,105,106,111-116,118, 120, 123, 124 and 135-137, a part or derivative thereof.92. Antibodies of heading 90 or 91, where the antibody is a monoclonal, chimeric or humanized antibody or a fragment of an antibody.93. Conjugate between a product of heading 86-89 or an antibody of heading 90-92 and a therapeutic or diagnostic product.94. Conjugate of heading 93, where the therapeutic or diagnostic product is a toxin.95. Kit for detection of the expression or abnormal expression of a tumour-associated antigen, comprehensive detection of the product (i) the nucleic acid coding for the tumour-associated antigen or part thereof;(iii) antibodies binding to the tumour-associated antigen or part thereof and/or (iv) T cells specific for a complex between the tumour-associated antigen or part thereof and an MHC molecule, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs-1-8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof, (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid in (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid in (a) or (b), and (d) a nucleic acid,96. Kit according to paragraph 95, where the means of detection of the nucleic acid encoding the tumour-associated antigen or part thereof are nucleic acid molecules for selective amplification of the nucleic acid.97. Kit according to paragraph 96, where the nucleic acid molecules for selective amplification of the nucleic acid comprise a sequence of 6 to 50 interconnected nucleotides from the nucleic acid encoding the tumour-associated antigen.98. Recombinant DNA molecule, comprising a promoter region derived from a nucleic acid sequence, selected from the group consisting of the following: IDQs: 41-4118, 41-4119, 8-574, 119 and SEQs: 1-519.

Detailed description of the invention

The invention describes genes that are selectively expressed or aberrantly expressed in tumor cells and represent tumor-associated antigens.

According to the invention, these genes and/or their gene products and/or their derivatives and/or parts are preferred target structures for therapeutic approaches. Conceptually, the therapeutic approaches may aim to inhibit the activity of the selectively expressed tumor-associated gene product. This makes sense if the aberrant or selective expression is functionally of tumor pathogenetic significance and its interruption is accompanied by selective damage to the relevant cells. Other therapeutic concepts consider tumor-associated antigens as markers that selectively recruit target molecules with cell-damaging potential to tumor cells.

Err1:Expecting ',' delimiter: line 1 column 47 (char 46)

A nucleic acid is preferably a deoxyribonucleic acid (DNA) or a ribonucleic acid (RNA) according to the invention. Nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules according to the invention. A nucleic acid can be present as a single-stranded or double-stranded and linear or covalently closed circular molecule according to the invention.

Err1:Expecting ',' delimiter: line 1 column 131 (char 130)

Err1:Expecting ',' delimiter: line 1 column 100 (char 99)

Complementary nucleic acids shall have at least 40%, in particular at least 50%, at least 60%, at least 70%, at least 80%, at least 90% and preferably at least 95%, at least 98% or at least 99% nucleotide identity.

Err1:Expecting ',' delimiter: line 1 column 519 (char 518)

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

Thus, on the one hand, the tumor-associated antigens shown here can be combined with any expression control sequence and promoter, and on the other hand, the promoters of the tumor-associated gene products shown here can be combined with any other gene, allowing the selective activity of these promoters to be exploited.

Err1:Expecting ',' delimiter: line 1 column 717 (char 716)

Err1:Expecting ',' delimiter: line 1 column 329 (char 328)

The nucleic acids encoding a tumor-associated antigen identified in accordance with the invention can be used for transfection of host cells. Nucleic acids refer to both recombinant DNA and RNA. Recombinant RNA can be produced from a DNA matrix by in vitro transcription. It can also be modified prior to application by stabilizing sequences, capping and polyadenylation.

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

In cases where an HLA molecule presents a tumor-associated antigen or part thereof, an expression vector may also include a nucleic acid sequence encoding for the HLA molecule. The nucleic acid sequence encoding for the HLA molecule may be present on the same expression vector as the nucleic acid encoding for the tumor-associated antigen or part thereof, or both nucleic acids may be present on different expression vectors. In the latter case, the two expression vectors cannot be cotransmitted into a cell. If a tumor cell does not express the tumor-associated antigen or part thereof, the HLA vector can be expressed on both nucleic acids encoding for the tumor or part thereof. In the case of the HLA vector, the expression vector can be expressed on either the tumor or the part already expressed.

The invention includes kits for amplification of a nucleic acid encoding for a tumor-associated antigen. For example, such kits include a pair of amplification primers that hybridise to the nucleic acid encoding for the tumor-associated antigen. The primers preferably include a sequence of 6-50, especially 10-30, 15-30 or 20-30 consecutive nucleotides from the nucleic acid and are non-overlapping to avoid the formation of primer dimers. One primer is hybridized to a strand of the nucleic acid encoding for the hybrid tumor-associated antigen, and the other primers are hybridized to the strand in an arrangement that allows for a nuclear amplification that encodes for the tumor-associated antigen.

Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

In preferred embodiments, the antisense oligonucleotide hybridizes with an N-terminal or 5'-upstream site such as a translation initiation, transcription initiation or promoter site.

In one embodiment, an oligonucleotide of the invention consists of ribonucleotides, deoxyribonucleotides or a combination thereof, the 5' end of one nucleotide and the 3' end of another nucleotide linked together by a phosphodiester bond, which can be synthesized or recombinantly produced in a conventional manner.

Err1:Expecting ',' delimiter: line 1 column 121 (char 120)

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

Err1:Expecting ',' delimiter: line 1 column 143 (char 142)

Such proteins and polypeptides are used, for example, in the production of antibodies and can be used in an immunological or diagnostic assay or as therapeutics.

Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

Amino acid insertion variants include amino and/or carboxyterminal fusions, as well as insertions of single or multiple amino acids in a particular amino acid sequence. In single-insertion amino acid sequence variants, one or more amino acid residues are introduced at a predetermined location in an amino acid sequence, although random insertion is also possible with appropriate screening of the resulting product. Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence. Amino acid substitution variants are characterized by the replacement of at least one residue in the sequence and another residue at another location in its volume. Amino acid substitution variants are characterized by the removal of a similarly homogeneous substitution between the amino acids (such as proteins, proteins, polyphenols, and other substitutes) and by the substitution of a similarly conserved group of amino acids. 1. small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr (Pro, Gly) 2. negatively charged residues and their amides: Asn, Asp, Glu, Gln3. positively charged residues: His, Arg, Lys4. large aliphatic, nonpolar residues: Met, Leu, Ile, Val (Cys) 5. large aromatic residues: Phe, Tyr, Trp.

Three residues are placed in brackets due to their special role for protein architecture. Gly is the only remainder without a side chain, thus giving the chain flexibility. Pro has an unusual geometry that greatly limits the chain. Cys can form a disulfide bridge.

Err1:Expecting ',' delimiter: line 1 column 164 (char 163)

Err1:Expecting ',' delimiter: line 1 column 43 (char 42)

A part or fragment of a tumor-associated antigen has, according to the invention, a functional property of the polypeptide from which it is derived. Such functional properties include interaction with antibodies, interaction with other polypeptides or proteins, selective binding of nucleic acids and enzymatic activity. An important property is the ability to enter a complex with HLA and, if necessary, to generate an immune response. This immune response may be based on stimulation of cytotoxic or helper T cells. Preferably, a part or fragment of a tumor-associated antigen according to the invention includes a sequence of at least 6, especially at least 8, at least 12, at least 15, at least 20, at least 30, or at least 50 consecutive amino acids from the tumor-associated antigen.

A part or fragment of a nucleic acid encoding a tumor-associated antigen is, according to the invention, the part of the nucleic acid encoding at least the tumor-associated antigen and/or a part or fragment of the tumor-associated antigen as defined above.

The isolation and identification of genes coding for tumour-associated antigens also allows the diagnosis of a disease characterised by the expression of one or more tumour-associated antigens. These procedures include the determination of one or more nucleic acids coding for a tumour-associated antigen and/or the determination of the coded tumour-associated antigens and/or peptides derived from them. Nucleic acid determination may be carried out by conventional means, including by polymerase chain reaction or hybridisation with a labelled probe. The determination of tumour-associated antigens or peptide derived from them may be carried out by screening a patient's immune system for a tumour-associated antigen and/or a specific antigen and may also be carried out by screening the patient for the antigen/peptide on the T-cell.

The present invention also allows the isolation of proteins that bind to the tumor-associated antigens described herein, including antibodies and cellular binding partners of the tumor-associated antigens.

Err1:Expecting ',' delimiter: line 1 column 103 (char 102)

The result of the expression of a dominant negative polypeptide in a cell is a reduction in the function of active proteins.

The invention also covers substances such as polypeptides that bind to tumor-associated antigens, which can be used, for example, in screening assays to detect tumor-associated antigens and complexes of tumor-associated antigens with their binding partners, and in clearance of tumor-associated antigens and complexes of tumor-associated antigens with their binding partners, and in inhibiting the activity of tumor-associated antigens by binding to such antigens, for example.

The invention therefore includes binders such as antibodies or antibody fragments that have the ability to selectively bind to tumour-associated antigens.

The test chemical is then applied to the test chemical to determine the concentration of the active substance in the test chemical.

Err1:Expecting ',' delimiter: line 1 column 217 (char 216)

Err1:Expecting ',' delimiter: line 1 column 161 (char 160)

It is known that only a small part of an antibody molecule, the paratop, is involved in binding the antibody to its epitope (see Clark, W.R. (1986), The Experimental Foundations of Modern Immunology, Wiley & Sons, Inc., New York; Roitt, I. (1991), Essential Immunology, 7th edition, Blackwell Scientific Publications, Oxford). The pFc'- and Fc-regions are, for example, effectors of the complement cascade, but are not involved in antigen binding. An antibody from which the pFc-region has been enzymatically cleaved or produced without the pFc-fragment is called a complete antibody (Figment F2); both are part of a complete antibody. An antigen is a single antibody that is produced in a similar way. An antigen is a compound that is formed from a single antigen (Figment F) that has been isolated from a single antigen (Figment F2) and is capable of binding to the enzyme without the presence of a complex antigen (Figment F2) and is capable of producing up to ten antibodies.

Within the antigen-binding portion of an antibody are complementarity regions (CDRs) that interact directly with the antigen's epitope and scaffold regions (FRs) that maintain the paratope's tertiary structure. Both the Fd fragment of the heavy chain and the light chain of IgG immunoglobulins contain four scaffold regions (FR1 to FR4), each separated by three complementarity regions (CDR1 to CDR3). The CDRs and especially the CDR3 regions and even more the CDR3 region of the heavy chain are largely responsible for antibody specificity.

Err1:Expecting ',' delimiter: line 1 column 345 (char 344)

Err1:Expecting ',' delimiter: line 1 column 69 (char 68)

Err1:Expecting ',' delimiter: line 1 column 395 (char 394)

The invention also covers F ((ab') 2-, Fab-, Fv- and Fd-fragments of antibodies, chimeric antibodies in which the Fc and/or FR and/or CDR1- and/or CDR2- and/or light chain CDR3-regions have been replaced by homologous human or non-human sequences, chimeric F ((ab') 2-fragment antibodies in which the FR and/or CDR1- and/or CDR2- and/or light chain CD-R3-regions have been replaced by homologous human or non-human sequences, chimeric antibodies in which the FR and/or FR- and/or CD-R1- and/or CD-R2- and/or Fab- and/or CD-R3-like light chain antibodies have been replaced by homologous human or non-human sequences, and non-CD-R2- and CD-R2- and/or CD-R2-like antibodies in which the homologous antibodies have been produced by homologous human or non-human sequences.

The invention also includes polypeptides that bind specifically to tumor-associated antigens. For example, such polypeptide binders can be provided by degenerate peptide libraries, which can be easily made in solution in an immobilized form or as phage display libraries. Combinator libraries of peptides with one or more amino acids can also be made. Libraries can also be made from peptides and non-peptide synthetic residues.

Err1:Expecting ',' delimiter: line 1 column 897 (char 896)

Err1:Expecting ',' delimiter: line 1 column 886 (char 885)Err1:Expecting ',' delimiter: line 1 column 694 (char 693)

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

A biological sample of the invention may be a tissue and/or cellular sample and may be obtained in the conventional manner for use in the various procedures described herein, such as tissue biopsy, including stencipse, and collection of blood, bronchial aspirate, sputum, urine, feces or other bodily fluids.

Err1:Expecting ',' delimiter: line 1 column 55 (char 54)

Some therapeutic procedures involve a response of a patient's immune system that results in lysis of antigen-presenting cells, such as cancer cells that present one or more tumor-associated antigens. For example, autologous cytotoxic T lymphocytes specific to a complex of a tumor-associated antigen and an MHC molecule are administered to a patient with a cell abnormality. The production of such cytotoxic T lymphocytes in vitro is known. An example of a procedure for differentiating T cells is found in WO-A-9633265.

In another method for antigen-specific cytotoxic T-lymphocyte selection, fluorogenic tetramers of MHC class I molecules/peptide complexes are used to detect specific clones of cytotoxic T-lymphocytes (Altman et al., Science 274:94-96, 1996; Dunbar et al., Curr. Biol. 8:413-416, 1998). Soluble MHC class I molecules are selected in vitro in the presence of β2-microglobulin and a peptide antigen bound to the class I molecule, after purification of the MHC class I molecule/peptide complexes with biomarker. These tetramers are then sorted by the biomarker LHC class T. These cytotoxic tetramers can be detected in the blood by a 4:1 ratio of cytotoxicity to the cytotoxic T-lymphocytes (as in the case of T-lymphocytes, the cytotoxicity can be increased by a phytophotoxins, which are in contact with the cytotoxic T-LHC class T cells).

In a therapeutic procedure known as adoptive transfer (Greenberg, J. Immunol. 136 ((5):1917, 1986; Riddel et al., Science 257:238, 1992; Lynch et al., Eur. J. Immunol. 21:1403-1410, 1991; Kast et al., Cell 59:603-614, 1989), cells presenting the desired complex (e.g., dendritic cells) are combined with cytotoxic T-lymphocytes of the patient to be treated, resulting in an increase in specific cytotoxic T-lymphocytes. The increased cytotoxic T-lymphocytes are then administered to a patient with a cell abnormality characterized by certain abnormalities that are specific to the cytotoxic complex, resulting in a therapeutic effect that is achieved by the release of lymphocytic fluid.

Alternatively, the T-cell receptor itself may be transferred, which also combines cells that present the desired complex (e.g. dendritic cells) with cytotoxic T-cells from healthy individuals or from another species (e.g. mouse). This results in a proliferation of high-affinity specific T-cells when the T-cells come into contact with a host organism that has not previously had a specific receptor; these T-cells can then be transferred to another human cell by a specific T-cell receptor (TZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZTZT

The above therapeutic aspects assume that at least some of the abnormal cells of the patient present a complex of a tumor-associated antigen and an HLA molecule. Identification of such cells can be done in a way that is known in itself. Once cells presenting the complex have been identified, they can be combined with a sample from the patient containing cytotoxic T lymphocytes. If the cells presenting the complex are lysed by the cytotoxic T lymphocytes, it can be assumed that a tumor-associated antigen is present.

The adoptive transfer is not the only form of therapy that is applicable according to the invention. Cytotoxic T lymphocytes can also be produced in vivo in a known way. One procedure uses non-proliferative cells that express the complex. The cells used will be those that normally express the complex, such as irradiated tumor cells or cells transplanted with one or both genes necessary for presentation of the complex (i.e. the antigen peptide and the presenting HLA molecule). Various cell types can be used. Further vector detectors can be used that carry one or both of the V. virulens or particularly preferential bacterial genes of interest.For example, nucleic acids encoding a tumor-associated antigen or a part thereof may be functionally linked to promoter and enhancer sequences that control expression of the tumor-associated antigen or a fragment thereof in certain tissues or cell types. The nucleic acid may be incorporated into an expression vector. Expression vectors may be unmodified extrachromosomal nucleic acids, plasmids, or viral genomes into which the insertion of exogenous nucleic acids is possible. Nucleic acids encoding a tumor-associated antigen may also be incorporated into a retroviral genome, allowing the integration of the nucleic acid into the genome of the target cell or tissue.Err1:Expecting ',' delimiter: line 1 column 131 (char 130)

A similar effect can be achieved by combining the tumor-associated antigen or a fragment thereof with an adjuvant to allow incorporation into antigen-presenting cells in vivo. The tumor-associated antigen or a fragment thereof may be represented as a protein, as DNA (e.g. within a vector) or as RNA. The tumor-associated antigen is processed to yield a peptide partner for the HLA molecule, while a fragment thereof can be presented without the need for further processing.In general, an effective amount of the tumor-associated antigen can be given to a patient, e.g. by intradermal injection, but the injection can also be given intranodally into a lymph node (Maloy et al., Natl Acad Acad Acad Acad 98:3299-303, 2001). It can also be given in combination with reagents that facilitate uptake into dendritic cells.The use of the drug in the treatment of cancer is not recommended for patients with a history of cancer, but for patients with a history of cancer, such as those who have not had a spontaneous immune response.

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Adjuvants can enhance the immune response by providing a reservoir of antigen (extracellular or inrophagenic), activating a specific type of antigen and/ or stimulating certain lymphocytes. Adjuvants are known and include, but are not limited to, QMPQ, Monophosphoryl QL-L-L-L (QL-L-L-L), QMQ, QS-L-L-Q, and QS-L-Q. They are typically produced in a range of doses from approximately 1:10 to approximately 100 μl (SQ-L-L-L-L-Q, SQ-L-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-L-Q, SQ-Q-Q, SQ-Q-Q, SQ-Q-Q, SQ-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, SQ-Q-Q-Q, and D-Q-Q-Q-Q-Q. They are typically produced in a range of 1 to 10 to 10 molecules, and are produced in human blood and blood, typically in a range of approximately 1 to 10 to 10 to 100 μl (SQ-Q-Q, SQ-Q, SQ-Q, SQ, SQ-Q, SQ-Q, SQ, SQ-Q, SQ, SQ, SQ, SQ, SQ-Q, SQ, SQ, SQ, SQ, SQ, SQ, SQ, SQ, SQ, SQ, SQ, SQ, S

Other substances that stimulate a patient's immune response may also be used. For example, cytokines can be used in vaccination due to their regulatory properties on lymphocytes. Such cytokines include, for example, interleukin-12 (IL-12), which has been shown to enhance the protective effects of vaccines (see Science 268:1432-1434, 1995), GM-CSF and IL-18.

There are a number of compounds that enhance an immune response and can therefore be used in a vaccination. These include co-stimulating molecules provided in the form of proteins or nucleic acids. Such co-stimulating molecules are, for example, B7-1 and B7-2 (CD80 and CD86) expressed on dendritic cells (DC) and interact with the CD28 molecule expressed on the T cells. This interaction provides a co-stimulation (Signal 2) for an antigen/MHC/TCR-stimulated (Signal 1) T cell, which enhances the proliferation of T cells and the effectiveness of the immune system. B7 also interacts with BLACDL4 (ZL1511) and TLC4 (ZL14-L11), and studies on CLACDL4 and CLACDL4 (CTL98, 954-CLACDL94) have shown that the antigen and its function can be enhanced.

B7 is typically not expressed on tumor cells, so these are not effective antigen presenting cells (APCs) for T cells. Induction of B7 expression would allow tumor cells to more effectively stimulate cytotoxic T cell proliferation and effector function. Co-stimulation by a combination of B7/IL-6/IL-12 showed induction of IFN gamma and Thl cytokine profiles in a T cell population, resulting in further enhanced T cell activity (Gajewski et al., J. Immunol. 154:5637-5648 (1995)).

Complete cytotoxic T-lymphocyte activation and full effector function require the involvement of T helper cells through the interaction between the CD40 ligand on the T helper cells and the CD40 molecule expressed by dendritic cells (Ridge et al., Nature 393:474 (1998), Bennett et al., Nature 393:478 (1998), Schönberger et al., Nature 393:480 (1998)). The mechanism of this co-stimulatory signal is likely to involve the increase of B7 and associated IL-6/IL-12 production by the dendritic cells (antigen-presenting cells). The CD40-CD40 interaction thus complements the interactions of the signal 1 (HCL/MTCL) and the signal 2 (CDL/MTCL) of the dendritic cells (CDL/MTCL-27-B28).

The use of anti-CD40 antibodies for dendritic cell stimulation would be expected to directly enhance a response to tumor antigens, which are usually outside the range of an inflammatory response or are presented by non-professional antigen-presenting cells (tumor cells). In these situations, T helper and B7 co-stimulating signals are not provided. This mechanism could be used in conjunction with therapies based on dendritic cell antigen stimulation.

The present invention also includes the administration of nucleic acids, polypeptides or peptides. The administration of polypeptides and peptides may be done in a known manner. In one embodiment, the administration of nucleic acids is done by ex vivo procedures, i.e. by removing cells from a patient, genetically modifying the cells to incorporate a tumor-associated target antigen, and reintroducing the altered cells into the patient. This generally involves the introduction of a functional copy of a gene into a patient's cells in vitro and the return of the genetically modified cells back into the patient. The functional copy sectors of the virus genome are subject to regulatory functional factors that allow the expression of the gene in the cells.

In a preferred embodiment, a viral vector for the administration of a nucleic acid coding for a tumor-associated antigen is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses including vaccinaviruses and attenuated poxviruses, semliki forest virus, retroviruses, sindbis virus and typhoid-like particles. Particularly preferred are adenoviruses and retroviruses. The retroviruses are usually replication deficient (i.e. they are unable to produce infectious particles).

Various methods can be used to introduce nucleic acids into cells in vitro or in vivo according to the invention. Such methods include transfection of nucleic acid CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the above viruses carrying the nucleic acids of interest, liposome-mediated transfection, and the like. In certain embodiments, a control of nucleic acid binding to specific cell acids is preferred. In such embodiments, a carrier used to deliver an upper nucleic acid to a cell (e.g., a lipid or lipid molecule) may be an antibody. For example, an antibody may be a lipid or lipid molecule for specific tumours. An antibody may be a specific target for specific targeted binding, such as a protein or a ligand, which may be built into a target cell or a protein, and may be used to target specific proteins or proteins, which may be specifically designed to bind to a specific target cell or to target a specific protein, and which may be used to target and recombine the target cell.

The therapeutic formulations of the invention may be administered in pharmaceutically compatible preparations, which may normally contain pharmaceutically compatible concentrations of salts, buffers, preservatives, carriers, complementary immunosupporters such as adjuvants, CpG and cytokines and, where appropriate, other therapeutic agents.

The therapeutic agents of the invention may be administered by any conventional route, including injection or infusion. For example, oral, intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal. Antibodies may be administered therapeutically preferably by a pulmonary spray.

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An effective dose of a composition of the invention will depend on the condition to be treated, the severity of the disease, the individual parameters of the patient, including age, physiological condition, height and weight, duration of treatment, type of concomitant therapy (if any), specific route of administration and similar factors.

The pharmaceutical formulations of the invention are preferably sterile and contain an effective amount of the therapeutically active substance to produce the desired reaction or effect.

The doses of the compositions of the present invention to be administered may depend on various parameters such as the mode of administration, the patient's condition, the desired duration of administration, etc. In the event that a response in a patient is insufficient at an initial dose, higher doses (or effectively higher doses obtained by another, more localized route of administration) may be used.

Generally, doses of 1 ng to 1 mg, preferably 10 ng to 100 μg, of the tumour-associated antigen are formulated and administered for treatment or to produce or enhance an immune response If the administration of nucleic acids (DNA and RNA) coding for tumour-associated antigens is desired, doses of 1 ng to 0.1 mg are formulated and administered.

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The pharmaceutical formulations of the invention may contain suitable buffers such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.

The pharmaceutical formulation may also contain, where appropriate, suitable preservatives such as benzalkonium chloride, chlorbutanol, parabens and thimerosal.

The pharmaceutical formulations are usually presented in a uniform dosage form and may be prepared in a way that is known in itself.

Compounds suitable for parenteral administration usually include a sterile aqueous or non-aqueous preparation of the active substance, preferably isotonic with the recipient's blood. Compatible carriers and solvents include Ringer's solution and isotonic sodium chloride solution. In addition, sterile fixed oils are usually used as a solvent or suspension medium.

The present invention is described in detail by the following illustrations and examples, which are intended for illustrative purposes only and are not intended to be comprehensible in a restrictive manner.

The following images:

GPR35 mRNA expression in colon cancer biopsies is shown in Figure 1. RT-PCR tests with DNA-free RNA show GPR35 expression in the majority of colon cancer biopsies, but no expression is detectable in normal tissue (1 breast, 2 lungs, 3 lymph nodes, 4 thymus, 5 colon, 6-15 colon cancer, 16 negative control).Figure 2. Real-time PCR with GUCY2C-specific primers (SEQ ID NO: 22-23) shows selective mRNA expression in the normal ileum, colon, and in all colon cancer biopsies. Significant amounts of GUCY2C transcript were also detected in a colon cancer metastasis in the liver.Figure 3. Identification of tumour-specific GUCY2C splice variants PCR products from normal colon tissues and colon carcinomas were cloned and clones from both groups were examined and sequenced by EcoRI.Figure 4. The following substances are to be used in the manufacture of the test chemical:38) shows cDNA amplification only in normal lung (track 8, 14-15) and in lung cancer biopsies (track 16-24). (1-liver-N, 2-PBMC-N, 3-lympho node-N, 4-stomach-N, 5-test-N, 6-mamm-N, 7-kidney-N, 8-lung-N, 9-thymus-N, 10-ovary-N, 11-adrenal-N, 12-spleen-N, 14-15-lung-N, 16-24 lung cancer, 25-negative controls).Figure 5. Claudine-18A2.1 expression in the stomach, esophagus, stomach and pancreas RT-PCR analysis with claudin-18A2.1 specific primers (SEQ ID NO: 39, 40) showed, according to the invention, a pronounced expression of claudin-18A2.1 in 8/10 gastric carcinoma biopsies and 3/6 pancreatic carcinoma biopsies. RT-PCR analysis with SLC13A1-specific primers (SEQ ID NO: 49, 50) showed expression in 7/8 of the renal cell carcinoma samples.Otherwise, transcripts within normal tissue were detected only in the kidney (1-2 kidney, 3-10 renal cell carcinoma, 11 breast, 12 lung, 13 liver, 14 colon, 15 lymph nodes, 16 spleen, 17 esophagus, 18 thymus, 19 thyroid, 20 PBMCs, 21 ovary, 22 testicles).Figure 7. RT-PCR studies with CLCA1-specific primers (SEQ ID NO: 67, 68) confirmed selective expression in the colon and showed high expression in (3/7) colon and (1/3) gastric cancer samples examined. RT-PCR studies with FLJ21477 specific primers (SEQ ID NO: 69, 70) showed selective expression in the colon and, in addition, varying levels of expression in colon cancer samples examined (7/12).The remaining normal tissues (NG) showed no expression.Figure 9.FLJ20694 expression in the colon and colon cancer RT-PCR studies with FLJ20694 specific primers (SEQ ID NO: 71, 72) showed selective expression in the colon, and in addition, varyingly pronounced expression in (5/9) colon cancer samples examined. RT-PCR studies with von Ebner-specific primers (SEQ ID NO: 73, 74) showed selective expression in the stomach, lung and (5/10) samples of lung cancer examined. RT-PCR studies with Plunc-specific primers (SEQ ID NO: 75, 76) showed selective expression in the thymus,The remaining normal tissues showed no expression.Figure 12 SLC26A9 expression in lung, lung and thyroid cancer RT-PCR studies with SLC26A9 specific primers (SEQ ID NO: 77, 78) showed selective expression in the lung and in all (13/13) lung cancer samples examined. The remaining normal tissues (NG) except the thyroid did not show expression.Fig.13 THC1005163 expression in stomach, ovarian, lung and lung cancer RT-PCR studies with a THC1005163 specific primer (SEQ ID NO: 79) and a non-specific Oligo dT tag primer showed expression in stomach, ovary, lung and (5/9) lung cancer biopsies; the remaining normal tissues (NG) showed no expression.Fig. 14. The following substances are to be used in the test:81) showed selective expression in the kidney and in (5/8) renal cell carcinoma biopsies examined.Figure 15. RT-PCR studies with THC943866 specific primers (SEQ ID NO: 82, 83) showed selective expression in the kidney and in (4/8) renal cell carcinoma biopsies examined.Fig.16 FLJ21458 expression in colon and colon cancer RT-PCR studies with FLJ21458 specific primers (SEQ ID NO: 86, 87) showed selective expression in the colon and in (7/10) colon cancer biopsies examined (1-2-colon, 3-liver, 4-PBMCs, 5-spleen, 6-prostate, 7-kidney, 8-ovary, 9-skin, 10-ileum, 11-lung, 12-testis, 13-22 colon cancer, 23-negative control).Figure 17. Immunofluorescence to detect the cellular localization of GPR35 after transfection of a plasmid that expresses a GPR35-GFP fusion protein.The arrows characterized the membrane-bound fluorescence of the fluorescent GFP.Figure 18 Quantitative expression of GPR35A. Quantitative RT-PCR with GPR35-specific primers (SEQ ID NO: 88, 89) show selective expression in the gut, in colon tumor samples and in metastases from colon tumors.The following normal tissues were analysed: liver, lung, lymph nodes, stomach, spleen, secondary tissues, kidney, esophagus, ovary, testis, thymus, skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate, thyroid, ovaries, endothelium, small brain.GPR35A is expressed in both metastases and metastases of the metastases in which the expression is greater than 90% in both the prostate, thyroid, ovaries, endothelium, small brain. The following information shall be provided in the form of a summary of the results of the analysis:GUCY2C is detectable in 11/12 colon cancers and 7/10 gastric cancers.Figure 20.Quantitative expression of SCGB3A2 RT-PCR with SCGB3A2 specific primers (SEQ ID NO: 103.104) show selective expression in lung and lung tumour samples. 19/20 lung tumour samples are SCGB3A2-positive, SCGB3A2 is overexpressed by at least a factor of 10 in more than 50% of the samples. The following normal tissues were analysed: liver, lungs, lymph nodes, stomach, spleen, adrenal glands, kidney, esophagus, ovary, testis, thymus, SC, skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate, thyroid, ovary, endometrium, small brain, brain.Absorption of 21.AGB3A2-specific antibodies. COS7 cells were transplanted with a plasmid,A. Detection of the transfected fusion protein with a SCGB3A2-specific rabbit antiserum (immunisation with SEQ ID NO: 105). B. Detection of the transfected fusion protein by GFP fluorescence. C. Overlapping of the two fluorescences from A and B. The yellow colouration occurs at the points where the two fluoresces overlap and thus indicates the specificity of the SCGB3A2-antiserum.Figure 22. Schematic representation of claudin-18 splice variant The two claudin-18 splice variants A1 and A2 differ in the N-terminus and have different potential glycosylation sites.Figure 23.Quantitative expression of clandin-18, variant A1 Claudin-Al is highly activated in a wide variety of tumor tissues, with a particularly strong expression in stomach tumors,The following table shows the most commonly used methods for the determination of the concentration of claudin-18. Like the A1 variant, the A2 variant is activated in many tumours.Figure 25.Use of claudin-18A2-specific antibodies (extracellular domain) (above) staining of claudin-18A2-positive gastric cancer cells (SNU-16) with an antibody produced by immunisation with a peptide (SEQ ID NO:17). Membrane staining is particularly strong in the cell/cell interaction areas. A-primmun., MeOH; B-immuniserum MeOH, 5 μg/ml (below) Detection of antibody specificity by collocation analysis in 293T cells transferred to Claudin-18A2-GFP. A-claudin-18A2 GFP; B-anti-claudin-A2; C-superposition.Figure 26. Use of claudin-18A2-specific antibodies (extra-neural) Membrane staining of claudin-18A2-positive gastric cancer cells (SNU-16) with an antibody produced by immunisation with a peptide (SEQ ID NO.:113, N-terminal extracellular domain).Anti-staining was carried out with a monoclonal antibody directed against E-cadherin.A antibody;B anti-staining;C overlay.Figure 27.Use of antibodies against the C-terminal extracellular domain of claudin-18 (left, top and bottom) Membrane staining of Claudin-18A2-positive gastric cancer cells (SNU-16) with an antibody produced by immunization with a peptide (SEQ ID NO.:116, C-terminal extracellular domain). A monoclonal antibody directed against E-cadherin was used for the staining (top right, bottom).Figure 28.Use of Claudin-18A1-specific antibodies (above) weak to no staining of gastric cancer cells (SNU-16; Claudin18A2 positive) with an antibody produced by immunisation with a claudin-18A1-specific peptide (SEQ ID NO:115) A-anti-E-cadherin; B-anti-Claudin-18A1; C-overlay. (bottom) Demonstration of specificity of the antibody by collocation analysis in Claudin-18A1-GFP-transferred 293T cells.A-GFP-claudin-18A1; B-anti-claudin-18A1; C-overloading.Figure 29. Detection of claudin-18A2 in the western blot. Western blot with lysates from various healthy tissues with a claudin-18A2 specific antibody, directed against the epitope with SEQ ID NO:17. Magenic tumors have a low glycosylated form of claudin-18A2. PNGase F treatment of gastric lysates results in the formation of the low glycosylated form. Left: 1-stomach No #A; 2-stomach Tu #A; 3-stomach No #B; 4-stomach Tu #B The following are the main features of the newly-created system:Expression of claudin-18 in lung tumours In accordance with Figure 30, low-glycosylated Claudin-18A2 variants were detected in lung tumours. 1-stomach No; 2-stomach Tu; 3-9 lung Tu.Figure 32. A. staining with a pre-immuniserum generated prior to immunisation; B. staining with the claudin-18-specific serumFig. 34 Quantitative expression of SLC13A1 Quantitative RT-PCR with SLC13A1-specific primers (SEQ ID NO: 121, 122) show high and selective expression in normal renal tissue (A) and SLC13A1-specific expression in renal cell carcinomas (B). SLC13A1 transcription is detectable in 5/8 renal cell carcinomas.Figure 35. Immunofluorescence to detect the cellular localization of SLC13A1 following plasmid transfectionThe membrane-bound fluorescence (as a ring around the transfected cell) of the SLC13A1 fusion protein is clearly visible.Figure 36.Quantitative expression of CLCA1 Quantitative RT-PCR with CLCA1-specific primers (SEQ ID NO: 125, 126) show high and selective expression in normal colon and gastric tissue (A) and CLCA1-specific expression in colon and gastric tumour samples (B). CLCA1 is detectable in 6/12 colon cancers and 7/10 gastric cancers.Figure 37. Quantitative RT-PCR with FLJ21477 specific primers (SEQ ID NO: 127, 128) show high and selective expression in normal colon and gastric tissues and weak expression in thymus, esophagus and brain (A) and FLJ21477 specific expression in colon tumour samples (B).FLJ21477 is detectable in 11/12 colon cancers.Figure 38.Quantitative expression of FLJ20694 Quantitative RT-PCR with FLJ20694 specific primers (SEQ ID NO: 129, 130) show high and selective expression in normal colon and gastric tissue (A) and FLJ20694 specific overexpression in colon and gastric tumour samples (B). FLJ20694 is detectable in 11/12 colon cancers and 7/10 gastric cancersFigure 39. Quantitative RT-PCR with FLJ21458 specific primers (SEQ ID NO: 133, 134) show selective expression in testis, gastric and intestinal tissues. In addition, FLJ21458 specific transcripts were detected in 20/20 colon tumors and 7/11 colon metastases. The following normal tissues were analysed: liver, lung, lymph nodes, spleen, adrenal glands, kidney, esophagus, ovary, testis,Thymus, skin, breast, pancreas, lymphocytes, activated lymphocytes, prostate, thyroid, fallopian tube, endometrium, cerebrum, brain.Figure 40 Immunofluorescence with FLJ21458 specific antibodies (above) 293 cells were translocated with a plasmid encoding a FLJ21458 GFP fusion protein A: Detection of the translocated fusion protein with a FLJ21458 specific rabbit antiserum (immunisation with SEQ ID NO: 136) B: Detection of the translocated fusion protein by GFP fluorescence C: Overlapping of the fluorescence from A and B. The yellow colouration occurs at the two sites where both fluorescences cross, indicating the specificity of the FLJ21458 anti-serum. A: Protein detection with a FLJ21458 specific rabbit antiserum (immunisation with SEQ ID NO: 136).B: Detection of membrane protein E-Cadherin. C: Overlapping of the two fluorescences from A and B. The yellow colouration is produced at the points where the two fluorescences overlap and indicates the membrane localization of FLJ21458. The sequences referred to herein are shown.

Examples: Materials and methods

Err1:Expecting ',' delimiter: line 1 column 56 (char 55)

Data mining strategy for the identification of new tumour-associated genes

Err1:Expecting ',' delimiter: line 1 column 497 (char 496)Err1:Expecting ',' delimiter: line 1 column 261 (char 260)This assessment also took into account the existence of mis-annotated cDNA banks in the public domain (Scheurle et al., Cancer Res. 60: 4037-4043, 2000) (www.fau.edu/cmbb/publications/cancergenes6.htm). As a second data mining method, the cDNA xProfiler of the NCBI Cancer Genome Anatomy Project (http://cgap.nci.nih.gov/Tissues/xProfiler) was used (Hillier et al., Genome Research 6:807-828, 1996; Penn Normal Science, 27:10623-1024, 1997). This allowed pools of transcripts stored in databases to be related by logical operators. A pool was defined, for example, where all the colon expressions produced by the Library were assigned to the cDNA pool, with the exception of those produced by the Library of Genome Expressions.In general, all cDNA banks were used regardless of the underlying manufacturing process, but only those with a potency > 1000 were approved. Using the BUT NOT operator, Pool B was digitally subtracted from Pool A. The set of GOIs found in this way was also subjected to eNorthern studies, as well as backed up by a literature search. This combined data mining includes all of the approximately 13,000 full-length genes in the public domain and predicts from these genes with potential organ-specific expression.

All other genes were first evaluated by specific RT-PCR in normal tissues; all GOIs that were found to be expressed in non-organ-specific normal tissues were considered false-positive and excluded from further testing; the remainder were examined in a large panel of various tumour tissues; the antigens shown below were found to be activated in tumour cells.

RNA extraction, production of poly-d (((T) primed cDNA and conventional RT-PCR analysis

Total RNA from native tissue material was extracted using guanidium isothiocyanate as a chaotrophemic agent (Chomczynski & Sacchi, Anal. Biochem. 162:156-9, 1987). From 2-4 μg total RNA, a first-strand cDNA synthesis was performed in a 20 μl reaction approach using Superscript II (invitrogen) according to the manufacturer's specifications. A dT(18) oligonucleotide was used as a primer. The integrity and quality of the cDNA were verified by amplification of p53 in a 30 cycles PCR (sense CGTGAGCGCTTCGAGATTTCCG, antisense CCTAACCAGCTGCCCAACTTTAG, hybridisation temperature 67°C). An archive of primary cDNAs from a range of normal tissues and tumor entities was produced.The test chemical was then amplified with a 30 μl reaction approach using GOI-specific primers (see below) and 1 U HotStarTaq DNA polymerase (Qiagen). The primers were selected to be in 2 different exons and the elimination of interference by contaminating genomic DNA as the reason for false positive results was confirmed by testing non-reversed transcribed DNA as a matrix. After 15 minutes at 95°C to activate the HotStarTaq DNA polymerase, 35 cycles of PCR were performed (1 min 94°C, 1 min respective hybridization temperature, 2 min 72°C and final elongation at 72°C for 6 min).Other 20 μl of this reaction was separated and analysed on an agarose seal stained with ethidium bromide.

The following primers were used for the analysis of expression of the corresponding antigens at the specified hybridisation temperature. The following is a list of the main types of waste which are to be disposed of in the waste water treatment plants: waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water treatment plants, waste water, waste water, waste, waste, waste, waste, etc.

Manufacture of random hexamer-primed cDNA and quantitative real-time PCR

The increase in SYBR-Green fluorescence as a result of specific amplification by GOI-specific primers after each PCR cycle is used for control quantification. The total expression quantification of the target DNA is performed or the total expression of a tested DNA with constant expression in the samples is suppressed and the dye is active only after binding to double stranded DNA fragments. The expression was expressed as 18 μ μ μ by means of a Z-DNA clearance system. The test was conducted by the US Bioclimate Testing Kit (CQC) (A) 30C. The test was conducted in the USA with the high-capacity DNA clearance test set at 30°C. The test was conducted in the USA with the manufacturer of the C-DNA test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test results were presented in the USA with the test kit (CQC) 30°C. The test kit (C) 30°C) 30°C. The test was performed in the USA with the test kit (C) 30°C) 30°C. The test kit (C) 30°C) 30°C. The test was performed in the USA with the test kit (C) 30°C) 30°C. The test kit (C) 30°C) 30°C. The test was performed in the test kit (C) 30°C) 30°C. The test kit (C) 30°C) was performed in the test kit (C) 30°C) 30°C. The test kit (C) 30°C) was performed in the test kit (C) 30°C) 30°C) 30°C. The test kit (C) was performed in the test kit (C) 30°C) 30°C) 30°C. The test kit (C) was performed in the test kit (C

Cloning and sequence analysis

The cloning of full lengths or gene fragments was performed using standard methods. To determine the sequence, corresponding antigens were amplified by means of the proofreading polymerase pfu (stratagen). After the PCR was completed, adenosine was ligated to the ends of the amplicon by HotStarTaq DNA polymerase to clone the fragments into the TOPO-TA vector as specified by the manufacturer. The sequencing was performed by a commercial service. The sequences were analyzed using standard prediction programs and algorithms.

Western blot

The lysates of an experimental approach are separated into 8-15 percent of the size of the saturating polyacrylamide glycogen (containing 1% SDS) by electrophoresis (SDS-Polyacrylamide-resultant alkalinity membrane reaction, SDS-PAGE). The resulting antibodies are then detected by the semi-dry membrane (Biorad) antigen called anti-cellulose membrane (AMP) which is translocated to the target protein (ECP) after 60 minutes. The enzyme is then incubated on a single membrane (a second membrane) using a 1:20-percent (B) or a second membrane (B) enzyme. The enzyme is then incubated on a single membrane (a second membrane) and then incubated on a single membrane (a second membrane) using a specialised enzyme called an enzyme called an enzyme called an enzyme called an enzyme called an enzyme called an enzyme called an enzyme.

Glycosylations, which are usually several kDa in size, result in a larger total mass of the target protein, which can be separated in the SDS PAGE To detect specific O- and N-glycosidic bindings, protein lysates from tissues or cells are incubated with O- or N-glycosides prior to denaturation by SDS (according to the manufacturer, e.g. PNGase, endoglycosidase F, endoglycosidase H, Rolier Diagnostics).

Immunofluorescence

Err1:Expecting ',' delimiter: line 1 column 728 (char 727)The cells are then permeabilised by incubation with detergents (e.g. 0.2% Triton X-100) if necessary. After fixation/permeabilisation, the cells are incubated with a primary antibody directed against the target protein or one of the coupled markers. After a wash, the approach is followed by a second antibody coupled with a fluorescent marker (e.g. Fluorescin, Texas, Red Dako), which is then incubated with the first antibody. The cells thus marked are then incubated with a Glycerin fluorescent cell and analyzed using a microscope according to the manufacturer's instructions.Specific fluorescence emissions are achieved by specific stimulation, depending on the substances used. The analysis usually allows for the safe localization of the target protein, whereby the antibody quality and the target protein in double staining are confirmed by staining the coupled amino acid markers or other marker proteins, the localization of which has already been described in the literature, in addition to the target protein.

Immunohistochemistry

Err1:Expecting ',' delimiter: line 1 column 472 (char 471)

Err1:Expecting ',' delimiter: line 1 column 895 (char 894)

Typically histologically defined tumour tissue and comparable healthy tissue are used as reference in the IHC. Positive and negative controls may also be cell lines where the presence of the target gene is known by RT-PCR analysis.

The samples are washed with TBS-T and blocked in serum. Then incubation with the first antibody (diluting: 1:2 to 1:2000) for 1-18 hours, usually with affinity-cleaned antibodies, is followed by a 30-60 minute incubation with a second antibody, the alkyphosphate enzyme (E.P. 742-701, et al., 1991; et al., 1993; et al., 1993; et al., 1993; et al., 1993; and may be followed by a reaction with the first antibody, the antibody being used in the laboratory.

Immunisation

The use of the test chemical in the manufacture of the test chemical is recommended for the control of the presence of antibodies in the test chemical. The following is a brief description of the antibody production process, details of which can be found in the cited publications. First, animals (e.g. rabbits) are immunized by a first injection of the desired target protein. A second or third immunization within a defined period (about 2-4 weeks after the previous immunization) can enhance the animal's immune response to the immunogen.Blood is drawn from the animals and an immunoserum is obtained. Immunisation of animals is usually carried out by one of four well-established methods, although other methods are available, which may include peptides specific to the target protein, the entire protein or extracellular sub-sequences of a protein which can be identified experimentally or by prediction programmes. (1) In the first case, keyhole limpet hemocyanin (KLH) conjugated peptides (length 8-12 amino acids) are synthesized in a standardized in vitro process and these peptides are used for immunization.Immunization can also be provided as a service by service providers. (2) Alternatively, immunization can be performed by recombinant proteins. To this end, the cloned DNA of the target gene is cloned into an expression vector and the target protein is cloned in accordance with the conditions of the respective manufacturer (e.g. Roche Diagnostics, Invitrogen, Clontech, Qiagen), e.g. cell-free in vitro, in bacteria (e.g. E. coli), in yeast (e.g. S. pombe), in insect cells or in mammalian cells. After synthesis in a system, the target protein is purified, which can be done in standardized chromatographic methods.Err1:Expecting ',' delimiter: line 1 column 168 (char 167)The transferred DNA is taken up by the animal's cells, the target gene is expressed and the animal eventually develops an immune response to the target gene (Jung et al., Mol Cells 12:41-49,2001; Kasinrerk et al., Hybrid Hybridomics 21:287-293, 2002).

Quality control of the polychlorinated serum or antibody

Err1:Expecting ',' delimiter: line 1 column 192 (char 191)

In the subsequent Western blot, cells from cell cultures or tissue samples that may contain the target protein are lysed in a 1% SDS solution and the proteins denatured. The lysates are separated electrophoresis on 8-15% denaturing polyacrylamide gels (containing 1% SDS) of size (SDS polyacrylamide gel electrophoresis, SDS-PAGE). The proteins are then transferred to a special membrane (e.g. nitrosulphate, silica & silica shell) by one of several blotting procedures (e.g. semi-dry electroblot; biorad) and the membrane itself can be detected by an antibody of 8-15% denaturing polyacrylamide gel (containing 1% SDS). An antibody of this membrane can be detected by an antibody of approximately 60 minutes.

A number of methods are used to confirm the membrane localization of the target protein identified in the silico approach. An important and well-established method using the antibodies described above is immunofluorescence (IF). This uses cells from established cell lines that either synthesize the target protein (detection of RNA in RT-PCR or protein in Western blot) or have been translocated with plasmid DNA. For transfection of cell lines with DNA, a variety of methods (e.g. electroporation, liposome-based transfection, calcium phosphate pre-prepitation) are well established (e.g. Linosemoine et al., Molodols. Biol. 751-7, 1997).Err1:Expecting ',' delimiter: line 1 column 97 (char 96)The specific fluorescence emission is achieved by specific stimulation, depending on the substances used. The analysis usually allows the target protein to be securely localized, confirming the antibody and target protein in double-stained form, by presenting the target protein in addition to the paired amino acid markers or other marker proteins, the localization of which has already been described in the literature.Alternatively, extracellular domains can be detected by flow cytometry. For this purpose, cells are fixed under non-permeabilising conditions (e.g. with PBS/Na-acid/2% FCS/ 5 mMTA) and analyzed in the flow cytometer as specified by the manufacturer. Only extracellular epitopes can be detected by this method to distinguish from antibodies.B. Propidium iodide or trypanosulphate to distinguish between dead and living cells and thus avoid false positives.

Affinity cleaning

The purification of the polyclonal serums was carried out by the companies in charge of the cleaning of the peptide antibodies in full or, in the case of the antibodies to recombinant proteins, in part as a service. In both cases, the corresponding peptide or recombinant protein was covalently bound to a matrix, which was then balanced after coupling with a native buffer (PBS) and then incubated with the raw serum. After a further wash with PBS, the antibody was eluted with 100 mM glycine, pH 2.7, and the eluate was immediately neutralized in 2 M TRIS, pH 8.

Manufacture of EGFP transfectors

For immunofluorescence microscopy of heterologously expressed tumour-associated antigens, the complete ORF of the antigens in pEGFP-C1 and pEGFP-N3 vectors (Clontech) was cloned, CHO and NIH3T3 cells cultured on-demand were translocated with the appropriate plasmid constructs using a Fugene Transfection Reagent (Roche) as specified by the manufacturer and analysed after 12-24 h by immunofluorescence microscopy.

Example 1: Identification of GPR35 as a diagnostic and therapeutic target for cancer

GPR35 (SEQ ID NO:1) and its translation product (SEQ ID NO: 9) have been described as putative G-protein coupled receptors. The sequence is published in the gene bank under access number AF089087. This transcript encodes for a 309-amino acid protein with a molecular weight of 34 kDa. GPR35 has been predicted to belong to the G-protein coupled receptor superfamily with 7 transmembrane domains (O'Dowd et al., Genomics 47:310-13, 1998). To confirm the predicted localization of GPR35 in the cell, the eGFP protein was fused with a reporter as a heterogeneous molecule and transfection of the corresponding plasmid was confirmed in 293 plasma cells.The results of the previous studies on human GPR35 (including Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Mashima H, Schwarz PE, del Bosque-Plata L, Horikawa Y, Oda Y, Yoshiuchi I, Colilla S, Polonsky KS, Wei S, Concannon P, Iwasaki N, Schulze J, Baier LJ, Bogardus C, Groop L, Boerwinkle E, Hanis CL, Bell GI Genet 2000 Oct;262) NO:163-75) suggest that GPR35 is activated in many healthy tissues. The reading of the GPR35 gene contains a single gene.In contrast, significant expression in other normal tissues is not detectable. Due to the peculiarity of GPR35 being composed of a single exon, genomic DNA contamination cannot be detected with intron-spanning primers. Therefore, to rule out genomic contamination of the RNA samples, all RNAs were treated with DNAse. According to the invention, DNA-free RNA GPR35 transcripts were detected only in colon, rectum, testes and colon cancers.

Gehirn	-
Cerebellum (Kleinhirn)	-
Herzmuskel	-
Skelettmuskel	-
Rektum	++
Magen	-
Kolon	++
Pankreas	-
Niere	-
Hoden	-
Thymus	-
Brustdrüse	-
Ovar	-
Uterus	n.d.
Haut	-
Lunge	-
Schilddrüse	-
Lymphknoten	-
Milz	-
PBMC	-
Nebenniere	-
Ösophagus	-
Dünndarm	+
Prostata	-

The selective and high expression of GPR35 transcripts in normal colon tissue, as well as in colon cancer biopsies (Fig. 1) was not previously known and can be used according to the invention for molecular diagnostic techniques such as RT-PCR to detect tumor cells spreading in serum and bone marrow and to detect metastases in other tissues. Quantitative RT-PCR with specific primers (SEQ ID NO:88 and 89) also confirm that GPR35 is a highly selective intestinal specific and also a differentiated antigen in intestinal tumours and intestinal metastases. In some intestines it is even a log-preserved antigen in comparison to normal intestinal antigen (Fig. 18).The following peptides were used to propagate these antibodies: The following information is provided for the purpose of the calculation of the amount of the premium: All 4 extracellular domains of GPR35 (position of predicted extracellular domains in the sequence of SEQ ID NO:9: AS 1-22 (SEQ ID NO: 94); AS 81-94 (SEQ ID NO: 95); AS 156-176 (SEQ ID NO: 96); AS 280-309 (SEQ ID NO: 97)) can be used as target structures of monoclonal antibodies according to the invention. These antibodies bind specifically to the cell surface of tumor cells and can be used for both diagnostic and therapeutic procedures.The overexpression of GPR35 in tumors supports such use furthermore, the protein coding sequences of the invention can be used as vaccines (RNA, DNA, peptide, protein) to induce tumor-specific immune responses (T-cell and B-cell-mediated immune responses) and surprisingly, 5' before the common start dodec exists another start dodec expressing an N-terminally elongated protein.

Previous research on human GPR35 has shown that a protein previously described as ubiquitously expressed, GPR35, is selectively overexpressed in gastrointestinal tumours, particularly tumours of the colon, and is therefore particularly suitable as a molecular target structure for the diagnosis and treatment of these tumours. Previous research on human GPR35, e.g. Horikawa Y, Oda N, Cox NJ, Li X, Orho-Melander M, Hara M, Hinokio Y, Lindner TH, Himaima, Black PE, del Bosque-Plata L, Horikawa Mash, Oda Y, Mashaki Y, Yucchi I, Killer S, Polonsky S, Pannon, Concannon, NPR, NPR, NPR, Jeneriser, Jeneriser, Boggess, LJ, Hanseye, C.E.E., has shown that it is activated by the gene, and a significant alternative to the G35 gene has been found in most studies, including the newly discovered G35 gene, E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E.E

Err1:Expecting ',' delimiter: line 1 column 599 (char 598)

Example 2: Identification of GUCY2C in liver and ovarian tumours and new GUCY2C splice variants as diagnostic and therapeutic cancer targets

Guanylate cyclase 2C (SEQ ID NO:2; translation product: SEQ ID NO: 11) - a type I transmembrane protein - belongs to the natriuretic peptide receptor family. The sequence is published in the gene bank under the access number NM_004963. Recent studies suggest that GUCY2C expression extends to extraintestinal areas such as primary and metastatic adenocarcinomas of the stomach and esophagus (Park et al., Cancer Epidemiol Biomarkers Prev. 11: 739-44, 2002). A splice variant of GUCYC found in both normal and transformed intestinal tissue involves a 142 bp deletion in exon 1, which prevents translation of a GUCY2C-like product (Pearlman et al., Dig. Sci. 45:298-05, 2000).

One of the goals of the invention was to identify tumour-associated splice variants for GUCY2C that are both diagnostic and therapeutically useful. RT-PCR studies with a GUCY2C-specific primer pair (SEQ ID NO: 22, 23, 98, 99) show a pronounced expression of GUCY2C transcripts in the normal colon and stomach, and weak expression in liver, testis, ovary, thymus, spleen, brain and lung (Table 2, Fig. 19). Expression in the colon and stomach was at least 50 times higher than in all other normal tissues. Pronounced GUCY2C transcript levels were detected in many colon and gastric cancers (Table 2). These results were further described by quantitative PCR analysis and revealed a pronounced expression of GUCY2C-activated OxyBlastoma Ovarium in almost all colon, ovary, brain and lung tumors we have examined (Table 2, Fig. 19), and a massive expression of the gene in collagen, ileum, carboxyl carboxyl, and other tumors (Table 2, Fig. 19), which was not found in the previously tested OxyBlastoma Ovarium (Table 2, Fig. 10).

The following pairs of primers were used for the detection of splice variants in colon and colon cancer tissues: GUCY2C-118s/GUCY2C-498as (SEQ ID NO:24, 29); GUCY2C-621s/GUCY2C-1140as (SEQ ID NO:25, 30); GUCY2C-1450s/GUCY2C-1790as (SEQ ID NO:26, 31); GUCY2C-1993s/GUCY2C-2366as (SEQ ID NO:27, 32); GUCY2C-2717s/GUCY2C-3200as (SEQ ID NO:28, 33); GUCY2C-118s/GUCY2C-1140as (SEQ ID NO:24, 30); GUCY2C-621s/GUCY2C-1790as (SEQ ID:25, 31); GUCY2C-236as (SEQ ID:26, 32); GUCY2C-336/GUCY2C-336 (SEQ ID:26, 32); GUCY2C-360/GUCY2C-336 (SEQ ID:26, 32); GUCY2C-336/GUCY2C-336 (SEQ ID:26, 32).

In the study of splice variants in colon cancer tissues, three previously unknown forms of the invention were identified. (a) A deletion of exon 3 (SEQ ID NO: 3) resulting in a variant of GUCY2C only 111 amino acids long, wherein asparagine is replaced by a proline at position 111. (b) A deletion of exon 6 (SEQ ID NO: 4) resulting in a 258-amino acid long expression product, resulting in a C-terminal neoepitope comprising 13 amino acids. (c) A variant in which the nucleotides at positions 1606-1614 and the corresponding amino acids L536, L537, and Q538 are deleted (SEQ ID NO: 5). The splice variants of the invention with deletions in exon 3,The main characteristic of the product (SEQ ID NO: 12, 13) is that the translation products (SEQ ID NO: 12, 13) do not have transmembrane domains. In the case of exon 6 deletion, the C-terminal forms a neoepitope of 13 amino acids, which has no homology to previously known proteins. This makes this neoepitope a target structure for immunotherapy. The splice variant of the invention with base deletions at positions 1606-1614 (SEQ ID: 5) and its translation product (SEQ ID NO: 14) also contain a neoepitope. The following information shall be provided in the form of a summary of the results of the evaluation: Such antibodies can in principle be used for both diagnostic and therapeutic purposes.

In particular, the extracellular domain of GUCY2C (position of the predicted extracellular domain from the sequence of SEQ ID NO:11: AS 454-1073 (SEQ ID NO. 102)) can be used as a target structure of monoclonal antibodies according to the invention. However, the prediction of the structure is not entirely clear and has not yet been experimentally proven, so an alternative membrane orientation is also conceivable. In this case, the amino acids 1-431 DNA would be extracellular and would be suitable as a starting point for monoclonal DNA. These antibodies specifically bind to the cell surface of tumor cells and can be used for both diagnostic and therapeutic purposes. The GUCY2 gene, in particular, can be widely used in immunosuppression therapy, in addition to the use of a protein such as a peptidase (T) protein, which can be used to induce immune responses. Furthermore, the cellular function of the GUCY2C molecule allows the development of substances, in particular small molecules, which modulate the enzyme's function on tumour cells, according to the invention.

Example 3: Identification of SCGB3A2 as a diagnostic and therapeutic target for cancer

SCGB3A2 (SEQ ID NO: 6) (translation product: SEQ ID NO: 15) belongs to the secretoglobin gene family. The sequence is published in the GenBank under access number NM_054023. SCGB3A2 (UGRP1) is a homodimeric secretory protein of 17 kDa size, expressed exclusively in the lung and trachea (Niimi et al., Am J Hum Genet 70:718-25, 2002). RT PCR studies with a primer pair (SEQ ID NO: SC 37, 38) confirmed selective expression in normal lung tissue. Lung and lung-specific genes, such as surfactant proteins, are highly active in the context of tumor regulatory and tumor-transferase pathways and have been shown to be highly active in primary and secondary metastases.The investigations according to the invention showed that SCGB3A2 is highly and frequently expressed in bronchial carcinomas (Fig. 4). All other 23 normal tissues tested have no expression except in the lungs and trachea (see Fig. 20). This was further confirmed by a specific quantitative RT-PCR (SEQ ID NO:103, 104) (Fig. 20), which additionally showed overexpression of at least one log in more than 50% of bronchial carcinomas. The selective and high expression of SCGB3A2 in normal lung tissue, as well as in lung cancer biopsies, is suitable for molecular diagnostic methods such as RT-PCR for detection of spreading tumour cells in blood and bone marrow, sputum, bronchial aspirate or lavage and for detection of metastases in other tissues, e.g.In healthy lungs, SCGB3A2 is excreted by specialized cells exclusively into the bronchi. Accordingly, SCGB3A2 protein is not expected to be detectable in body fluids outside the respiratory tract in healthy individuals. In contrast, especially metastatic tumor cells secrete their protein products directly into the bloodstream. One aspect of the invention therefore concerns the detection of SCGB3A2 products in serum or plasma of patients via a specific antibody test as a diagnostic finding for lung tumors.

Antibodies to the SCGB3A2 protein were produced by immunizing rabbits and the following peptides were used to propagate these antibodies: The following shall be indicated in the table: A SCGB3A2 specific response was detected in immunofluorescence (Fig. 21). As expected for a secreted protein, a distribution of SCGB3A2 was found in the cell, which could be attributed to the endoplasmic reticulum and secretion granule (Fig. 21A). To control specificity, the cells were translocated in parallel with a plasmid that synthesized a SCGB3A2 GFP fusion protein. The protein detection was done here via the autofluorescent GFP (green fluorescent protein) (Fig. 21B). An overlap of both fluorescent images clearly shows that the immune system recognizes the specific SCGB3A2-protein (Fig. 21 C). Such antibodies may be used, for example, in the form of immunoassays for diagnostic and therapeutic purposes.

Example 4: Identification of claudin-18A1 and claudin-18A2 splice variants as diagnostic and therapeutic targets for cancer

The claudin-18 gene encodes a surface membrane molecule with 4 transmembrane domains and intracellular N- and C-terminus. Niimi and colleagues (Mol. Cell. Biol. 21:7380-90, 2001) described two splice variants of mouse and human claudin-18 that were described as selectively expressed in lung tissue (Claudin-18A1) and gastric tissue (Claudin-18A2) respectively. These variants differ in the N-terminus (Fig. 22). The extent to which the splice variants Claudin-18A2 (SEQ ID NO:7) and Claudin-18A1 (SEQ ID NO: 117) and their respective translation products (SEQ ID NO:16 and 118) can be used as markers or therapeutic target structures for tumours has been investigated in accordance with the invention.The A2 splice variant was additionally tested with a second primer pair in a conventional PCR (SEQ ID NO: 39 & 40). For the A1 variant, it is described as active only in normal lung However, surprisingly, the invention has shown that the A1 variant is also active in gastric mucosa. The stomach and lungs are the only normal tissues that show significant activation. All other normal tissues are negative for claudin-A1.Expression levels of claudin-A1 in tumours of the lung, prostate, pancreas and esophagus are 100-10000 higher than levels in corrosponent normal tissue. The study of the claudin-A2 splice variant used oligonucleotides that specifically enable amplification of this transcript (SEQ ID NO: 39 & 40 and 107 & 108, respectively). The study showed that the splice variant A2 does not express itself in any of the more than 20 normal tissues studied except in the stomach lining and to a lesser extent.24) These include stomach tumours (8/10), pancreatic tumours (6/6), esophageal cancer (5/10), and liver cancer. Although no activation of claudin-18A2 has been found in healthy lungs, a surprising number of lung tumours were found to express the A2.1 splice variant. Tabelle 3B. Expression von Claudin-18A1 in Normal- und Tumor-Geweben

Gehirn	-		Colonkarzinom	-
Cerebellum	-		Pankreaskarzinom	++
Myokard	-		Ösophaguskarzinom	++
Skelettmuskel	-		Magenkarzinom	+++
Endometrium	-		Bronchialkarzinom	++
Magen	+++		Mammakarzinom	+
Colon (Dickdarm)	-		Ovarialkarzinom	n.u
Pankreas	-		Endometriumkarzino	n.u.
Niere	-		HNO-Tumoren	++
Leber	-		Nierenzellkarzinom	-
Testis (Hoden)	+		Prostatakarzinom	++
Thymus	-
Mamma (Brust)	-
Ovar	-
Uterus	-
Haut	-
Lunge	+++
Thyroid	-
Lymphknoten	-
Milz	-
PBMC	-
Ösophagus	-

Conventional PCR as an independent control also confirmed the results of quantitative PCR. Oligonucleotides (SEQ ID NO: 39, 40) were used to specifically amplify the A2 splice variant. According to the invention, 8/10 of gastric carcinomas and half of pancreatic carcinomas tested showed strong expression of this splice variant (Fig. 5). However, expression in other tissues is not detectable with conventional PCR. In particular, no expression is found in the lungs, liver, blood, lymph nodes, upper chest and kidney tissues (NO. 3).The detection of tumours can be carried out according to the invention with the oligonucleotides mentioned (SEQ ID No 39, 40, 107-110), in particular, primary pairs, at least one of which binds under strict conditions to a 180 base pair long section of the transcript specific to one (SEQ ID No 8) or another spleiB variant (SEQ ID No 119). To confirm these protein-level data, claudin-specific antibodies and/ or immunosers were generated by immunization of animals.The plasma membrane localization and protein topology of claudin-18 was confirmed by analysis of the transmembrane domains with bioinformatics tools (TMHMM, TMPRED) and immunofluorescence studies of cells expressing enhanced GFP-tagged claudin-18 fusion proteins.Claudin 18 has two extracellular domains. The N-terminal extracellular domain is different in sequence in the two splice variants (SEQ ID NO: 111 for A1 and SEQ ID NO: 112 for A2). The C-terminal extracellular domain is identical in both variants (SEQ ID NO: 137). No antibodies have yet been described to bind to the extracellular domains of claudin-18; according to the invention, extracellular peptide peptides, specific to the A1 or A2 variants, were selected for immunization. Both variants of claudin-18 therefore have no classical glycosylated protein and a glycosylated protein was not to be expected.However, the selection of epitopes has taken into account that epitopes containing asparagine, serine, threonine are in rare cases potentially glycosylated even without classical glycosylation sites. The glycosylation of an epitope may prevent the binding of an epitope-specific antibody. According to the invention, epitopes were selected in such a way that the antibodies generated by this method allow a distinction to be made between the glycosylation status of the antigen. Among other things, the following antibody-making peptides were selected for immunization: The following substances are to be classified in the active substance as the active substance:The test chemical is a chemical that is used to test the concentration of a substance in a test medium. The specific antibody can be used under various fixation conditions for immunofluorescence studies. In comparative staining of RT-PCR positive and negative cell lines, the corresponding protein is detectable in a well-detectable amount specifically in the positive-typed gastric cancer cell lines (Fig. 25). The endogenous protein is membrane localized and forms larger focal aggregates on the membrane.This antibody was also used for protein detection in the Western blot. As expected, protein is detected only in the stomach and no other normal tissue, including the lung (Fig. 29). When comparing the staining of stomach tumours and adjacent normal stomach tissue from patients, it was surprising that in all stomach tumours in which claudin-18 A2 is detected, this protein has a smaller mass (Fig. 30 on the left). In a series of experiments, it was found that a band on this surface also occurs when normal stomach tissue is treated with the PNG deglycosylating agent glycans F (Fig. 30).A2 as such is detectable in over 60% of the gastric carcinomas studied, and only in the deglycosylated form. Although the A2 variant of claudin-18 is not detected at the protein level in normal lungs, it is still present in quantitative RT-PCR in bronchial carcinomas. Again, only the deglycosylated variant is present (Fig. 31). Antibodies have been produced according to the invention that recognize the extracellular domain of the spleen variant Claudin-18-A2.Claudin-18 itself is a highly selective differentiation antigen of gastric tissue (A2) and of the lungs and stomach (A1). Since it is obviously affected by changes in the glycosylation machinery in tumors, a special variant of A2 is produced in tumors that is glycosylated. This can be used both diagnostically and deglycidally. The immuno-antibodies described here (against NOQ: IDB 17) can be used in Western diagnostic tests, such as the E-Pepperidine, which is not even a glycosylated antigen.Err1:Expecting ',' delimiter: line 1 column 658 (char 657)For this purpose, lymphocytes were assayed from the immunized animals. Amino acids 1-47 (SEQ ID NO: 19 and 120) are also particularly good epitopes for immunotherapeutic procedures such as vaccines and the adoptive transfer of antigen-specific T-lymphocytes.

Example 5: Identification of SLC13A1 as a diagnostic and therapeutic target for cancer

SLC13A1 belongs to the family of sodium sulfate cotransporters. The human gene is selectively expressed in the kidney, unlike its mouse homolog (Lee et al., Genomics 70:354-63). SLC13A1 encodes for a 595-amino acid protein and contains 13 putative transmembrane domains. Alternative splicing produces 4 different transcripts (SEQ ID NO: 41-44) and its corresponding translation products (SEQ ID NO: 45-48). It was investigated whether SLC13A1 can be used as a marker for kidney tumors.

RT-PCR studies with a SLC13A1 specific primer pair (SEQ ID NO: 49, 50) confirmed near-selective expression in the kidney and showed high expression in almost all (7/8) renal cell carcinoma biopsies examined (Table 4, Figure 6). Quantitative RT-PCR studies with specific primers (SEQ ID NO: 121, 122) also confirm these data (Figure 34). Weak signals were detected in the following normal tissues: colon, stomach, testis, breast, liver and brain. Expression in renal carcinomas was however at least 100 times higher than in all other normal tissues. The subcellular localization of SFP13ALC1 in the plasma was analysed to fuse with the corresponding trans transgene and expressed in 293 eGZ as a heterogeneous protein.The localization was then analysed in fluorescence microscopy, and our data strongly confirm that SLC13A1 is an integral transmembrane molecule (Figure 35). To detect the SLC13A1 protein, antibodies were produced by immunizing rabbits, and the peptide sequence of SEQ ID NO: 123 and 124 was used to propagate these antibodies. The SLC13A1 protein has 13 transmembrane domains and 7 extracellular regions. In particular, these extracellular domains of SLC13A1 can be used as target structures of monoclonal antibodies according to the invention. SLC13A1 is involved in ion transport as a channel protein.However, high molecular monoclonal antibodies used in therapy are not excreted into the urinary tract, so that no binding to SLC13A1 occurs in the healthy kidney. However, the polarity of SLC13A1 in tumor cells is removed and the protein is directly accessible via the bloodstream for antibody targeting. The pronounced expression and high incidence of SLCl3A1 in renal cell carcinomas make this protein a highly interesting diagnostic and therapeutic marker according to the invention. This includes the detection of disseminated tumor cells in the serum, bone, and urine, as well as the detection of other organs in the immune system by means of antibodies. Therefore, the SLCl3A1 can be used as a target and extra-immunological diagnostic tool according to the invention.Err1:Expecting ',' delimiter: line 1 column 301 (char 300)

Example 6: Identification of CLCA1 as a diagnostic and therapeutic target for cancer

CLCA1 (SEQ ID NO: 51; translation product: SEQ ID NO: 60) belongs to the family of Ca++ activated Cl channels. The sequence is published in the gene bank under the access number NM_001285. CLCA1 is expressed exclusively in the intestinal cryptenepithelium and cup cells (Gruber et al., Genomics 54:200-14, 1998). It was investigated whether CLCA1 can be used as a marker for colon and gastric cancer. Oligonucleotides (SEQ ID NO: 67, 68) were used to allow a very broad specification of CLCA1. RT-PCR studies with this primer confirmed that it shows a selective expression, and that it shows a high expression in the colon or stomach.This was further confirmed by specific quantitative RT-PCR (SEQ ID NO: 125, 126) with no expression detected in the normal tissues analysed (Fig. 36). In the tumour samples examined in this experiment, 6/12 colon cancer samples and 5/10 stomach cancer samples were positive for CLCA1. Overall, gene expression in tumours appears to be dysregulated. In addition to very highly expressive samples, CLCA1 was significantly downregulated in other samples. The protein is predicted to have 4 transmembrane domains with a total of 2 extracellular regions, and in particular these extracellular domains of CLCA1 can be used as target structures of monoclonal antibodies according to the invention. The high expression and incidence of CLCA1 for gastric and colon cancers make this protein an interesting diagnostic and therapeutic marker according to the invention.Err1:Expecting ',' delimiter: line 1 column 643 (char 642)

Example 7: Identification of FLJ21477 as a diagnostic and therapeutic target for cancer

FLJ21477 (SEQ ID NO: 52) and its predicted translation product (SEQ ID NO: 69, 71) were published as a hypothetical protein in the gene bank under access number NM_025153. It is an integral membrane protein with ATPase activity and 4 transmembrane domains, which is suitable for specific antibody therapy. RT-PCR studies with FLJ21477 specific primers (SEQ ID NO: 69, 70) showed selective expression in the colon and also varying expression in (7/12) colonic carcinoma monomers (Fig. 8). The remaining 10 are non-transposable. This was not confirmed by specific expression in colonic carcinoma (Fig. 12), although it was also confirmed by a quantitatively normal expression in FLJ127 (A12A12A127) (RTQ 377A128).In addition to expression in colon tissue, expression in gastric tissue was detected, and under quantitative RT-PCR conditions, significantly weaker expression in brain, thymus and esophagus was detected compared to colon and stomach (Fig. 37A). The protein is predicted to have 4 transmembrane domains with a total of 2 extracellular regions, and in particular these extracellular domains of FLJ21477 can be used as target structures of monoclonal antibodies according to the invention. The expression and high incidence of FLJ21477 for gastric and colon cancers make this protein a valuable diagnostic and therapeutic marker of the invention.This includes the detection of disseminated tumor cells in serum, bone marrow, urine, and the detection of metastases in other organs by RT-PCR. Furthermore, the extracellular domains of FLJ21477 can be used as a target structure for immunological diagnosis and therapy by monoclonal antibodies, according to the invention. Furthermore, FLJ21477 can be used as a vaccine (RNA, DNA, protein, peptide) to induce tumor-specific immune responses (T- and B-cell-mediated immune responses).

Example 8: Identification of FLJ20694 as a diagnostic and therapeutic target for cancer

Err1:Expecting ',' delimiter: line 1 column 1407 (char 1406)

Example 9: Identification of the von Ebner protein (c20orf114) as a diagnostic and therapeutic target for cancer

The Ebner protein (SEQ ID NO: 54) and its translation product (SEQ ID NO: 63) were published as a Plunc-related protein of the upper respiratory tract and nasopharyngeal epithelium in the gene bank under access number AF364078. According to the invention, the study investigated whether the Ebner protein could be used as a marker of lung cancer. Oligonucleotides (SEQ ID NO: 73, 74) were used to allow specific amplification of the Ebner protein. RT-PCR tests with this primer showed selective expression in the lungs and in (5/10) samples of lung cancer examined (A. 10).

Example 10: Identification of Plunc as a diagnostic and therapeutic target for cancer

Plunc (SEQ ID NO: 55) and its translation product (SEQ ID NO: 64) have been published in the gene bank under the access number NM_016583.The human Plunc encodes for a protein of 256 amino acids and has a 72% homology to the murine Plunc protein (Bingle and Bingle, Biochim Biophys Acta 1493:363-7, 2000).Expression of Plunc is limited to the trachea, upper airways, nasopharyngeal epithelium and salivary gland. The purpose of the study was to investigate the use of Plunc as a marker of lung cancer by using oligonucleotides (SEQ ID NO: 75, 76) that allow specific amplification of Plunc. RT-PCR studies with this primer set showed selective expression in the thymus, lung and (6/10) lung cancer samples examined (Fig. 11).

Example 11: Identification of SLC26A9 as a diagnostic and therapeutic target for cancer

SLC26A9 (SEQ ID NO: 56) and its translation product (SEQ ID NO: 65) have been published in the gene bank under the access number NM_134325 SLC26A9 belongs to the family of anion exchangers SLC26A9 is expressed only in the bronchiolar and alveolar epithelium of the lungs (Lohi et al., J Biol Chem 277:14246-54, 2002). The use of SLC26A9 as a marker of lung cancer was investigated by using oligonucleotides (SEQ ID NO: 77, 78) that allow specific amplification of SLC26A9. RT-PCR studies using SLC26A9 specific primers (SEQ ID NO: 77, 78) showed selective expression in the lung and in all (13/13) lung cancer samples examined (Figure 12).The remaining normal tissues, except the thyroid, showed no expression. Quantitative RT-PCR experiments with the primaries SEQ ID NO. 131 and 132 confirmed these results and provided additional insights. In pooled samples of 4-5 tumor tissues, high expression levels for SLC26A9 specific RNA were detected in lung, colon, pancreatic and stomach tumors. SLC26A9 is a member of a family of transmembrane anion transporters. In the healthy lung, the protein is directed in the direction of the airway and thus not directly accessible to IgG antibodies from the blood.The high expression and high incidence of SLC26A9 for lung, stomach, pancreatic and esophageal carcinomas make this protein an excellent diagnostic and therapeutic marker according to the invention. This includes the detection of disseminated tumor cells in serum, bone marrow and urine, and the detection of metastases in other organs by RT-PCR. Furthermore, the extracellular domain of SLC26A9 can be used as a target for immunotherapy and immunotherapy by means of monospecific antibodies. This includes the detection of disseminated tumor cells in serum, bone marrow and urine, and the detection of metastases in other organs by RT-PCR. Furthermore, the extracellular domain of SLC26A9 according to the invention can be used as a target for immunotherapy and immunotherapy by means of monospecific antibodies.Err1:Expecting ',' delimiter: line 1 column 110 (char 109)

Example 12: Identification of THC1005163 as a diagnostic and therapeutic target for cancer

THC5163 (SEQ ID NO: 57) is a gene fragment from the TIGR gene index. The gene is defined only in the 3' range, while an ORF is missing. RT-PCR tests were performed with a THC1005163 specific primer (SEQ ID NO: 79) and an Oligo dT18 primer, which had a specific tag of 21 specific bases at the 5' end. This tag was checked with database search engines for homology with known sequences. This special primer was initially used in cDNA synthesis to exclude genomic DNA contamination. RT-PCR tests with this primer showed a variation in expression in O, L100, and L/A carcinoma (59) in the 5' end.

Example 13: Identification of LOC134288 as a diagnostic and therapeutic target for cancer

The following information is provided for the purpose of the analysis of the data: The use of LOC134288 as a marker of renal cell carcinoma was investigated using oligonucleotides (SEQ ID NO: 80, 81) that allow specific amplification of LOC134288 RT-PCR studies showed selective expression in the kidney and in renal cell carcinoma biopsies (5/8) (Figure 14).

Example 14: Identification of THC943866 as a diagnostic and therapeutic target for cancer

THC943866 (SEQ ID NO: 59) is a gene fragment from the TIGR gene index. It was investigated whether THC943866 can be used as a marker of renal cell carcinoma. RT-PCR studies with THC943866 specific primers (SEQ ID NO: 82, 83) showed selective expression in the kidney and in (4/8) examined renal cell carcinoma biopsies (Figure 15).

Example 15: Identification of FLJ21458 as a diagnostic and therapeutic target for cancer

FLJ21458 (SEQ ID NO: 84) and its predicted translation product (SEQ ID NO: 85) were published in the gene bank under access number NM_034850.Sequence analyses indicated that the protein represents a new member of the butyrophillin family.Structural analyses indicated that it is a type 1 transmembrane protein with extracellular immunoglobulinamines.Expression testing used oligonucleotides (SEQ ID NO: 86, 87) that allow specific amplification of FLJ21458 RT-PCR studies with FLJ21458 specific primes (SEQ: 86, 87) show a selective expression in 39 colons (AQAQ, 16, 137) and a quantitative expression in this colony (AQAQ, 13, 134) (AQAQAQ, 13, 5).In addition, in the FLJ21458 experiment, gastrointestinal specificity was detected in the colon, as well as in the stomach, rectum and appendix in the testes. 7/11 colonic metastasis samples were also positive in quantitative PCR. FLJ21458 specific expression was extended to other tumors and protein specific expression was detected in stomach, pancreatic and liver tumors (Table 5). The following information shall be provided in the form of a summary of the results of the evaluation: A FLJ21458 specific response was detected in immunofluorescence (Fig. 40).293 cells were translocated with a plasmid encoding a FLJ21458 GFP fusion protein to control the specificity of the antibodies.The evidence of specificity was obtained by both localization tests with the FLJ21458 specific antibody and autofluorescent GFP. An overlay of both fluorescence images clearly showed that the immune serum specifically recognizes FLJ21458 protein (Fig. 40A). Due to the over-expression of the protein, diffuse cell staining resulted, which did not allow for clear protein localization. From this, a further immunofluorescence experiment was performed with the STL cell line S16, which is endogenously expressed in FLJ21458, and at least one other immunofluorescence experiment (Fig. 41B). The cells were further stained with the FLJ2145 specific antibody and a new antibody, the FLJ21458C2, which is localized in the FLJ2145 membrane and is detected in the cell membrane.

Err1:Expecting ',' delimiter: line 1 column 1248 (char 1247)

Normalgewebe	Expression		Tumortyp	Expression
Gehirn	-		Kolonkarzinom	7/10
Cerebellum (Kleinhirn)	-		Pankreaskarzinom	5/6
Myokard	nd		Ösophaguskarzinom	nd
Skelettmuskel	-		Magenkarzinom	8l10
Herzmuskel	-		Bronchialkarzinom	nd
Magen	++		Mammakarzinom	nd
Colon (Dickdarm)	+++		Ovarialkarzinom	nd
Pankreas	-		Endometriumkarzinom	nd
Niere	-		HNO-Tumoren	nd
leber	-		Nierenzellkarzinom	nd
Testis (Hoden)	++		Prostatakarzinom	nd
Thymus	nd		Kolonmetastasen	7/11
Mamma (Brust)	nd		Leberkarzinom	5/8
Ovar	-
Uterus	-
Haut	-
Lunge	-
Thyroid (Schilddrüse)	nd
Lymphknoten	-
Milz	-
PBMC	-
Nebenniere	nd
Ösophagus	-
Dünndarm	-
Prostata	-

Claims (16)

1. Pharmaceutical formulation consisting of one or more constituents selected from the group consisting of: Other

   (i) a tumor-associated antigen or part thereof,

   (ii) a nucleic acid that codes for a tumor-associated antigen or part thereof,

   (iii) an antibody binding to a tumour-associated antigen or part thereof,

   (iv) an antisense nucleic acid specifically hybridised with a nucleic acid coding for a tumour-associated antigen,

   (v) a host cell expressing a tumour-associated antigen or part thereof; and

   (vi) isolated complexes between a tumour-associated antigen or part thereof and an HLA molecule,

   Other where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: Other

   (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

   (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

   (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

   (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
2. Pharmaceutical composition according to claim 1, where the host cell secretes the tumour-associated antigen or part thereof, expresses on the surface or expresses an HLA molecule binding to the tumour-associated antigen or part thereof.
3. Pharmaceutical formulation according to claim 1 or 2 for the treatment of a disease characterised by the expression or abnormal expression of a tumour-associated antigen.
4. procedures for the diagnosis of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including

   (i) the detection of a nucleic acid coding for the tumour-associated antigen or part thereof, and/or

   (ii) the detection of the tumour-associated antigen or part thereof and/or

   (iii) the detection of an antibody against the tumour-associated antigen or part thereof and/or

   (iv) the detection of cytotoxic or helper T lymphocytes specific to the tumour-associated antigen or part thereof;

   Other in a biological sample isolated from a patient, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: Other

   (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

   (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

   (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

   (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
5. procedures to determine the regression, course or outbreak of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including monitoring of a sample from a patient who has or is suspected of having the disease, with respect to one or more parameters selected from the group consisting of: Other

   (i) the amount of nucleic acid encoding the tumour-associated antigen or part thereof,

   (ii) the amount of tumor-associated antigen or part thereof,

   (iii) the amount of antibodies binding to the tumour-associated antigen or part thereof; and

   (iv) the number of cytolytic or cyclin-expressing T cells specific for a complex between the tumour-associated antigen or part thereof and an MHC molecule,

   Other where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: Other

   (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

   (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

   (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

   (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
6. An antibody that specifically binds to a protein or polypeptide or part thereof, where the protein or polypeptide is encoded by a nucleic acid selected from the group consisting of: Other

   (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

   (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

   (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

   (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
7. Pharmaceutical composition as claimed 1 or antibody as claimed 6 where the antibody is coupled with a therapeutic or diagnostic agent.
8. Antibodies as claimed by claim 7 for use in a procedure for the treatment, diagnosis or monitoring of a disease characterised by the expression or abnormal expression of a tumour-associated antigen, where the procedure involves the administration of the antibody and the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of:

   (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

   (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

   (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

   (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
9. Pharmaceutical composition according to claim 1 or 7 or antibody according to any of claims 6 to 8, where the antibody is a monoclonal, chimeric or humanized antibody or a fragment of an antibody.
10. Cytolytic or cytokine-expressing T cells for use in a procedure to treat a patient with a disease characterised by the expression or abnormal expression of a tumour-associated antigen, including: Other

    (i) the removal of a sample of immune-reactive cells from the patient;

    (ii) contact of the sample with a host cell expressing the tumour-associated antigen or part thereof under conditions which favour the production of cytolytic or cytokine-expressing T cells against the tumour-associated antigen or part thereof; and

    (iii) the introduction of cytolytic or cytokine-expressing T cells into the patient in a quantity suitable for lycing cells expressing the tumour-associated antigen or part thereof, where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: Other

    (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

    (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

    (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

    (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).
11. Cytolytic or cytokine-expressing T cells as claimed 10, where the host cell expresses an HLA molecule recombinantly or endogenously that binds to the tumour-associated antigen or part thereof.
12. Pharmaceutical composition according to claim 3, procedure according to claim 4 or 5, antibodies according to claim 8 or 9, or cytolytic or cytokine-expressing T cells according to claim 10 or 11, where the disease is cancer.
13. Pharmaceutical composition according to claim 3, procedure according to claim 4 or 5. Antibodies according to claim 8 or 9 or cytolytic or cytokine-expressing T cells according to claim 10 or 11 where the disease is a lung tumour, breast tumour, prostate tumour, melanoma, colon tumour, stomach tumour, pancreatic tumour, tumour of the ENT, renal cell carcinoma or cervical cancer, colon cancer or breast cancer.
14. Pharmaceutical composition according to one of claims 1 to 3, 7, 9, 12 and 13, procedure according to one of claims 4, 5, 12 and 13, antibody according to one of claims 6 to 9, 12 and 13, or cytolytic or cytokine-expressing T-cells according to one of claims 10 to 13, where the protein or polypeptide or tumour-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 105, 106, 9-14, 16-19, 45-48, 60-66, 85, 90-97, 100-102, 111-116, 118, 120, 123, 124, and 135-137, a part or derivative thereof.
15. Protein or polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 105, 106, 10, 12-14, 17-19, 90-97, 100-102, 111-116, 120, 123, 124, and 135-137, a part or derivative thereof, or an immunogenic fragment of the protein or polypeptide.
16. Kit for detecting the expression or abnormal expression of a tumour-associated antigen, including means of detection Other

    (i) nucleic acid, or part thereof, which codes for the tumour-associated antigen,

    (ii) the tumour-associated antigen or part thereof,

    (iii) antibodies binding to the tumour-associated antigen or part thereof, and/or

    (iv) T cells specific for a complex between the tumour-associated antigen or part thereof and an MHC molecule,

    Other where the tumour-associated antigen has a sequence encoded by a nucleic acid selected from the group consisting of: Other

    (a) a nucleic acid containing a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6, 1-5, 7, 8, 41-44, 51-59, 84, 117 and 119, a part or derivative thereof,

    (b) a nucleic acid which hybridizes under strict conditions with the nucleic acid referred to in (a);

    (c) a nucleic acid which is degenerated with respect to the nucleic acid referred to in (a) or (b); and

    (d) a nucleic acid which is complementary to the nucleic acid referred to in (a), (b) or (c).