HK40000286A - Gene products differentially expressed in tumors and their uses - Google Patents

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 from tumors for tumor-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 specificities that could result in 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 disclosure, a strategy for identifying and providing tumor-associated expressed antigens and the nucleic acids coding for them was pursued, based on the fact that certain genes expressed organ-specifically, e.g. exclusively in colon, lung or kidney tissue, are also ectopically and unlawfully reactivated in tumor cells in the relevant organs and in other tissues. Data mining first generates a complete list of all known organ-specific genes and then assesses their expression by means of a special RT-PCR but in different actors. A valid method for tumor identification is the use of the DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA DNA

The apparent approach, however, which has proved much more successful, is based on using data mining to electronically extract all organ-specific genes and then evaluate them for expression in tumors.

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A combined strategy based on two different bioinformatics scripts enabled the identification of new tumor genes, which have been classified as purely organ-specific, and the recognition that these genes are aberrantly activated in tumor cells allows them to be assigned a substantially new quality with functional implications.

The identified tumour-associated antigens 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, 119 and 138, 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 degenerates with respect to the nucleic acid under (a) or (b), and (d) a nucleic acid which is a complement to the nucleic acid under (a), (b) or (c) NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO: NO

This disclosure is generally concerned with the use of tumour-associated antigens or parts or derivatives thereof, of nucleic acids coding for them or of nucleic acids directed against the nucleic acids coding for them, or of antibodies directed against the tumour-associated antigens or parts or derivatives thereof, identified by disclosure, for therapy and diagnosis, which may be individual or combinations of several of these antigens, functional fragments, nucleic acids, antibodies, etc., in one embodiment or in combination with other tumour-associated genes and antigens for diagnosis, therapy and control.

Diseases of preference 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 by the exposure.

The disclosure also concerns nucleic acids and gene products expressed in tumour cells.

In addition, the present technical theory concerns gene products, i.e. nucleic acids and proteins or peptides, which are produced by modified splicing (splice variants) of known genes or by modified translation using alternative open reading frames. In this aspect, nucleic acids are revealed which contain a nucleic acid sequence selected from the group consisting of the sequences according to SEQ ID NO: 3-5 of the sequence protocol. In addition, the theory presented in this aspect concerns proteins or peptides which contain an amino acid sequence selected from the group consisting of the sequences according to SEQ ID NO: 10 and 12-14 of the sequence protocol. The revealed splice variants are revealed as targeted for use in the diagnosis and treatment of tumours.

In particular, this theory concerns the amino acid sequence according to SEQ ID NO: 10 of the sequence protocol, which is encoded by an alternative open reading rod 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 are, as it appears, suitable, in particular oligonucleotides that have or contain a sequence selected from SEQ ID NO: 34-36, 39, 40 and 107-110 of the sequence protocol. All sequence-dependent detection systems are suitable for detection. In addition to PCR, these include e.g. gene-chip/microarray systems, Northern blot, RNAse protection assays (RDA) and others.In this respect, the disclosure concerns in particular peptides that have 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 them. The detection of tumour cells can also be carried out by antibodies that detect tumour-specific altered glycosylation variants. Peptide regions that differ in terms of glycosylation in tumour cells and healthy cells can be used to generate such antibodies. In this respect, the disclosure concerns in particular peptides that contain or contain a sequence selected from SEQ ID NO: 17-19, 111-115, 120, 1372-145, and specific antibodies directed against and containing sequence specific antibodies of the sequence protocol.Asparagine is transformed into aspartic acid by endogenous deglycosylation of N-coupled sugar residues. It is therefore apparent that the proteins described here may be tumor-specific sequentially altered and thus exhibit other biochemical and antibody binding properties. In this respect, the disclosed theory concerns particularly peptides that have or contain a sequence selected from SEQ ID NO: 146-150 of the sequence protocol and specific antibodies directed against it. Amino acids that exhibit significant epitopunters to the product variant (s) which are subsequently detected in healthy target cells are particularly suitable for immunisation. The detection of antibodies against tumours can be done by means of an antibody or by means of an antibody injected into a patient.In addition to diagnostic usefulness, splice variants that have new or altered epitopes are attractive targets for immunotherapy. The epitopes can be used to target therapeutically effective monoclonal antibodies or T lymphocytes. Passive immunotherapy involves adoptive transfer of antibodies or T lymphocytes that detect splice variant-specific epitopes. The generation of antibodies as with other antigens can also be done using standard technologies (immunization of polyps, panning strategies for isolation of recombinant antibodies) using polypeptides that contain these epitopes.For example, Kessler JH, et al. 2001, Sahin et al., 1997) and are also based on the use of oligo- or polypeptides containing the splice-variant-specific epitopes or nucleic acids encoding them.

Proteins are also described which differ in the type and amount of their secondary modifications in normal and tumour tissues (e.g. Durand & Seta, 2000; Cl. 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, Roonder Diagnostics). A Western blot is then performed. When the size of a target protein is reduced, a specific glycosidase pathway can be detected after incubation with a glycosidase. However, the specific glycosidase pathways and tumor types have been shown to be of interest.

Gastrointestinal carcinomas, pancreatic carcinomas, esophageal tumors, prostate tumors, and lung tumors all show a less glycosylated form of claudin-18. Glycosylation in healthy tissues masks claudin-18 protein pitopes, which are exposed on tumor cells due to lack of glycosylation. Accordingly, ligands and antibodies can be selected according to the revelation 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 aspect, the disclosure concerns a pharmaceutical composition comprising a substance that detects the tumour-associated antigen identified by the disclosure and is preferably selective for cells that exhibit expression or abnormal expression of a tumour-associated antigen identified by the disclosure. The substance may induce cell death, reduce cell growth, damage the cell membrane or secrete cytokines in certain embodiments and preferably exhibit tumour-inhibiting activity. In one embodiment, the substance is a selective antigen-isomeric acid that selectively binds to the nucleic acid that codes for the tumour-associated antigen. The substance is a selective antigen that binds to the tumour-associated antigen.In particular, a complement-activated or toxin-conjugated antibody that selectively binds to the tumor-associated antigen. In another embodiment, the agent includes several agents that selectively detect different tumor-associated antigens, each with at least one tumor-associated antigen being a tumor-associated antigen identified by the exposure. The detection need not be directly accompanied by an inhibition of antigen activity or expression. In this aspect of the revealed technical theory, selectively binding to tumor-confined antigens is preferably used as a marker of recruitment mechanisms at that specific site. In an advanced embodiment, the agent is a cytotoxic lymphocyte antigen that detects and acts on a ZLA-molecule and the lytic effect of the HLA.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 aspect, the disclosed doctrine concerns a pharmaceutical composition comprising a drug that inhibits the expression or activity of a tumor-associated antigen identified in accordance with the invention; in a preferred embodiment, the drug is an antisense nucleic acid that selectively hybridizes with the nucleic acid that encodes for the tumor-associated antigen; in another embodiment, the drug is an antibody that selectively binds to the tumor-associated antigen; in another embodiment, the drug includes several drugs, each selectively inhibiting the expression or activity of several tumor-associated antigens, at least one of the tumor-associated drugs being an obvious tumor-associated antigen.

The disclosed doctrine also concerns a pharmaceutical composition comprising a product which, when administered, selectively increases the amount of complexes between an HLA molecule and a peptide pitop from the tumour-associated antigen identified by the disclosure, comprising in one embodiment 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 virus cell expressing the tumour-associated antigen or part thereof and (iv) isolated antigenic complexes between a tumour-associated peptide and a tumour-associated antigen. The selectively increasing amount of MHC and MHC is the most important factor in determining the amount of an antigen.

In addition, the disclosed doctrine concerns a pharmaceutical composition comprising one or more components selected from the group consisting of (i) a tumor-associated antigen or part thereof identified by disclosure, (ii) a nucleic acid encoding a tumor-associated antigen or part thereof identified by disclosure, (iii) an antibody binding to a tumor-associated antigen or part thereof identified by disclosure, (iv) an antisense nucleic acid specifically encoded with a hybrid nucleic acid that is apparently encoded for a tumor-associated antigen or part thereof identified by disclosure, (v) an antigen or part thereof identified by disclosure, or a complex between a tumor-associated antigen or part thereof identified by disclosure and an antigen or part thereof identified by disclosure.

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

A host cell in a pharmaceutical formulation 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-producing cell, specifically a dendritic cell, a monocyte, or a macrophage.

An antibody contained in a pharmaceutical composition may be a monoclonal antibody. In other embodiments, the antibody is a chimeric or humanized antibody, a fragment of a natural antibody, or a synthetic antibody, all of which can be made 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 may comprise a sequence of 6-50, in particular 10-30, 15-30 or 20-30 nucleotides from the nucleic acid that encodes the tumour-associated antigen identified by the disclosure.

In other embodiments, a tumour-associated antigen, provided by a pharmaceutical formula complying with disclosure, either directly or by nucleic acid expression, or a part thereof, binds to MHC molecules on the surface of cells, preferably by inducing a cytolytic response and/or cytokine release.

A pharmaceutical composition 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 is preferentially used to treat a disease characterized by the selective expression or abnormal expression of a tumor-associated antigen.

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In one aspect, the disclosure concerns a method for the diagnosis of a disease characterised by the expression or abnormal expression of a tumour-associated antigen identified by the disclosure, which includes the detection of (i) a nucleic acid encoding 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 which are pro-active for the tumour-associated antigen or part thereof in a biological patient isolated from a specific biological agent.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 aspect, the disclosed doctrine concerns a method for determining the regression, course or outbreak of a disease characterised by the expression or abnormal expression of an apparently identified tumour-associated antigen, involving the monitoring of a sample from a patient who has the disease 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 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 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 an initial time in a first sample and at a second time in a second sample, and the disease transmission is determined by comparing the two samples. In certain embodiments, the disease course is characterised by the expression or abnormal expression of several different tumour-associated antigens and monitoring includes monitoring of (i) the amount of several nucleic acids,which code 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 performed 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 aspect, the disclosed doctrine concerns a procedure for the treatment, diagnosis or monitoring of a disease characterised by the expression or abnormal expression of a tumour-associated antigen identified by the disclosure, including the administration of an antibody that binds to the tumour-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 is a chimeric or humanized antibody or a fragment of a natural antibody.

In some embodiments, the methods described for the diagnosis or monitoring of a disease characterised by the expression or abnormal expression of a tumour-associated antigen identified by the exposure are by means of or by detection of disseminating tumour cells or tumour metastases, for example in blood, serum, bone marrow, sputum, bronchial aspirate and/or bronchial effusion.

The disclosed doctrine also concerns a procedure for the treatment of a patient with a disease characterised by the expression or abnormal expression of an apparently identified 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 T cells against the tumour-associated antigen or part thereof, and (iii) the introduction of the cytolytic T cells into the patient in a quantity suitable to lyse the cells which excrete 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, this disclosure concerns a procedure for the treatment of a patient with a disease characterized by the expression or abnormal expression of a tumor-associated antigen, comprising (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 the transfection rate is high), and (iv) the introduction of a host cell or extract thereof into the patient in a quantity,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 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 the tumour-associated antigen or part thereof or specific to the tumour of the patient or part thereof.

The disclosed doctrine also concerns a method for the treatment of a disease characterised by the expression or abnormal expression of an apparently identified tumour-associated antigen, involving (i) the identification of cells from the patient expressing abnormal levels 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 disclosure are non-proliferative or are made non-proliferative.

Furthermore, this disclosure concerns 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 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 comparable to the nucleic acid under (a), (b) or (c), and (c) a nucleic acid which encodes a protein or polypeptide containing an amino acid, including a nucleic acid selected from group NO: 10, 12-150, and a derivative thereof, including a nucleic acid containing a protein or a polypeptide containing an amino acid, SEQ ID: 146-150, and (c).

In another aspect, the disclosed theory concerns promoter sequences of revealed nucleic acids which can be functionally linked to another gene, preferably in an expression vector, and thus ensure the selective expression of this gene in the corresponding cells.

In another respect, the invention relates to a recombinant nucleic acid molecule, in particular a DNA or RNA molecule containing a nucleic acid as described above.

This doctrine also applies to host cells containing a manifested nucleic acid or a recombinant nucleic acid molecule containing a manifested nucleic acid.

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 expressed nucleic acid or part thereof recombinantly. Preferably, the host cell is non-proliferative. In a preferred embodiment, the host cell is an antigen-presenting cell, particularly a monocyt, dendritic cell, or macrophage.

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In another aspect, the disclosure concerns 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 hybridizes with the nucleic acid under strict conditions (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 aspect, the disclosed doctrine concerns an immunogenic fragment of a revealed tumor-associated antigen, 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 disclosure concerns in particular a peptide that 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, 135-137, 139 and 142-150, or a derivative thereof.

In another aspect, the disclosure doctrine concerns an antibody that selectively binds to a complex of (i) a disclosure-identified tumor-associated antigen or part thereof; in a preferred embodiment, the agent is an antibody; in other embodiments, the antibody is a chimeric, humanized or combinatorially produced antibody or a fragment of an antibody; and in a disclosure, the disclosure concerns an antibody that selectively binds to a complex of (i) a disclosure-identified tumor-associated antigen or part thereof; and (ii) an MHC molecule to which the disclosure-identified tumor-associated antigen or part thereof binds, whereby all (i) the antibodies may be a chimeric or non-chimeric form of an antibody or a monomer of the disclosure-identified tumor-associated antigen or part thereof.

In particular, this disclosure concerns such a product, in particular an antibody that specifically binds to a peptide that 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, 135-137, 139 and 142-150, or a derivative thereof.

As regards claudin-18, the invention also concerns agents, in particular antibodies, which bind specifically to a variant of claudin-18. In one embodiment, the agent, in particular an antibody, binds specifically to the variant claudin-18A1 (SEQ ID NO: 118). In another embodiment, the agent, in particular an antibody, binds specifically to the variant claudin-18A2 (SEQ ID NO: 16). Such specific antibodies may be obtained, for example, by immunization with the peptides described in example 4.

In addition, the disclosure concerns claudin-18 agents, in particular antibodies, which bind specifically to a form of claudin-18A2 which has a specific glycosylation pattern; in one embodiment, the agent, in particular an antibody, binds specifically to a form of claudin-18A2 which is non-glycosylated at one or more possible glycosylation sites; in another embodiment, the agent, in particular an antibody, binds specifically to a form of claudin-18A2 which is glycosylated at one or more possible glycosylation sites; preferably, one of the embodiments concerns one or more possible glycosylation sites which are further removed from the group consisting of positions 37, 38, 45, 116, 146 and 205A.

A substance specific to a variant or form of claudin-18 and in particular a variant or form of claudin-18 specific antibody means in this context that the substance or antibody binds more strongly to the variant or form to which it is specific than to another variant or form. A substance, in particular an antibody, binds more strongly to a first variant or form or to a first epitope than to a second variant or form or to a second epitope when it binds to the first variant or form or to the first epitope with a dissociation constant (KD) that is less than the dissociation constant for the second variant or form.Form or second epitope. Preferably, the dissociation constant (KD) for the variant or form or epitope to which the agent, in particular an antibody, specifically binds is more than 10 times, preferably more than 20 times, more preferably more than 50 times, more preferably more than 100 times and in particular more than 200 times, 500 times or 1000 times lower than the dissociation constant (KD) for the variant or form or epitope to which the agent, in particular an antibody, does not specifically bind. Preferably, an agent, in particular an antibody, does not or does not substantially bind to the variant or form or epitope for the agent,The antibody is not specific.

The agents described above, in particular antibodies and derivatives thereof as described herein, which bind specifically to a variant or form of claudin-18 are also intended for use in the formulations and procedures described.

Furthermore, the disclosure concerns a conjugate between a disclosure-compliant agent binding to a disclosure-identified tumour-associated antigen or part thereof, or a disclosure-compliant antibody and a therapeutic or diagnostic agent or substance.

In another respect, the disclosure concerns 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 assembly of 10 to 30 nucleic acid or parts thereof, in particular, 20-30 to 50 nucleic acid sequences, which are composed of a selective amplitude of 15 to 30 nucleic acid sequences.

Detailed description of the invention

The scope of the invention is covered by the scope of the claims attached, which describes genes that are selectively expressed or aberrantly expressed in tumor cells and represent tumor-associated antigens.

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

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Complementary nucleic acids are shown to 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.

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So on the one hand, the tumor-associated antigens shown here can be combined with any expression control sequence and promoters, and on the other hand, the promoters of the tumor-associated gene products shown here can be combined with any other genes, which allows us to use the selective activity of these promoters.

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The nucleic acids that code for a tumor-associated antigen that has been identified 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 by stabilizing sequences, capping and polyadenylation before application.

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

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

In cases of exposure 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 may be cotransmitted into a cell. If one cell does not express the antigen or part thereof, the HLA vector can be expressed on both nucleic acids, or in the case of the tumor, only the part already expressed in the HLA vector can be expressed on the different expression vectors.

Such kits include, for example, a pair of amplification primers that hybridise to the nucleic acid that encodes for the tumor-associated antigen. The primers preferably comprise a sequence of 6-50, especially 10-30, 15-30 or 20-30 related 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 that encodes for the hybridiser-associated antigen, and the other primer is 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 consists of ribonucleotides, deoxyribonucleotides or a combination thereof, with the 5' end of one nucleotide and the 3' end of another nucleotide linked by a phosphodiester bond. These oligonucleotides can be synthesized or recombinantly produced in a conventional manner.

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

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

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Such proteins and polypeptides are used, for example, to produce 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)

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A part or fragment of a tumor-associated antigen has 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, an apparent part or fragment of a tumor-associated antigen includes a sequence of at least 6, especially at least 8, at least 10, 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 that encodes for a tumor-associated antigen is revealed to be the part of the nucleic acid that encodes at least for the tumor-associated antigen and/or for 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 theory also allows the isolation of proteins that bind to the tumour-associated antigens described herein, including antibodies and cellular binding partners of tumour-associated antigens.

Err1:Expecting ',' delimiter: line 1 column 98 (char 97)

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

The disclosed theory also includes substances such as polypeptides that bind to tumour-associated antigens, which can be used, for example, in screening assays to detect tumour-associated antigens and tumour-associated antigen complexes with their binding partners and in clearance of tumour-associated antigens and tumour-associated antigen complexes with their binding partners, and can also be used to inhibit tumour-associated antigen activity, for example by binding to tumour-associated antigens.

Therefore, binders such as antibodies or antibody fragments, which have the ability to bind selectively to tumour-associated antigens, are apparently included.

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.

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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 CDR3 regions have been replaced by homologous human or non-human sequences, chimeric fragment 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 fragment antibodies in which the polycyclic and/or CDR1 and/or CDR2 and/or light chain CDR3 regions have been replaced by homologous human or non-human sequences, chimeric fragment antibodies in which the CDR1 and/or CDR2 antibodies are homologous to specific human or non-human pepper-like antibodies, or homologous antibodies in which the CDR1 and/R2 antibodies are not homologous to human or non-human, and can also be produced in a combination with a CDR or CDR2 or CDR2 antibodies, and are not easily produced in a homologous or homologous or homologous or homologous cell-like form, and are not synthetic.

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A biological sample may be a tissue and/or cellular sample by revelation and may be obtained in the conventional way 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 body fluids.

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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 isolated in vitro in the presence of β2-microglobulin and a peptide antigen bound to the class I molecule. After purification, the MHC class I molecule/peptide complexes are biomarked. These tetramers are then sorted by the biomarked LHC class T-Lymphocyte mix. These cytotoxic tetramenes can be detected in the blood by means of a 4:1 ratio of isototoxicity to the cytotoxic T-Lymphocyte.

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 to a host organism that has not previously had contact with a specific receptor; cells presenting the desired complex (e.g. dendritic cells) are also combined 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 a host organism from a human host organism by a specific T-cell receptor; these T-cells can then be transferred to a host organism from a human host organism (e.g. T-cell receptor V.S.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.T.

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 limited to the treatment of cancer in the first two stages of the disease, but is also used to treat cancer in the second stage.

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Adjuvants can enhance the immune response by providing a reservoir of antigen (extracellular or inrophagenic), activating a specific type of vitamin D, and/ or stimulating certain lymphocytes. Adjuvants are known and include, but are not limited to, monophosphoryl QL-LMPL (QLQL), QLQL (QLQL), QLQL (QLQL), QLQL (QLQL), and QLQL (QLQL), which are typically produced in a range of approximately 1:10 to 1 μg, and are produced in a range of doses from approximately 1 to 10 mg/ ml (SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL, SQL

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 T cell proliferation and effectiveness. B7 also interacts with BLACDL4 (ZL1511) and TLC4 (ZL1511) and interacts with the CD28 molecule expressed on the T cells. Studies have shown that BLACDL4 and CLACDL4 (CTL98) can enhance the antigenic and immunological function of the T cells.

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 Th1 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.

In one embodiment, nucleic acid administration is done by ex vivo procedures, i.e. by removing cells from a patient, genetically modifying the cells to insert a tumor-associated antigen target, and reinserting the altered cells into the patients. This generally involves introducing a functional copy of a gene into a patient's cells in vitro and returning the genetically altered cells back into the patient. The functional copy sectors of the gene are under the control of regulatory functional sectors that allow gene expression 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 techniques can be used to induce nucleic acid in cells in vitro or in vivo, according to the findings. Such techniques include transfection of nucleic acid CaPO4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the leading viruses carrying the nucleic acids of interest, liposome-mediated transfection, and the like. In certain embodiments, control of nucleic acid binding to specific cell acids is preferred. In such embodiments, a vehicle 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 a lipid molecule.

The therapeutic formulations described in the disclosure 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 may be administered by any conventional route, including injection or infusion. For example, oral, intravenous, intraperitoneal, intramuscular, subcutaneous or transdermal. Therapeutic antibodies are preferably given 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 are preferably sterile and contain an effective amount of the therapeutically active substance to produce the desired response or effect.

The doses of the compounds to be administered may depend on various parameters such as the route of administration, the patient's condition, the desired duration of administration, etc. If 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.

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The pharmaceutical compositions 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.

Pharmaceutical formulations are usually presented in a uniform dosage form and may be prepared in a way that is known to the consumer.

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 to be understood in a restrictive manner.

The following images:

Claudin-18A2.1 expression in stomach and esophagus and stomach and pancreatic tumors Other 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 of the stomach tumour biopsies and 3/6 of the pancreatic tumour biopsies. Other According to the revelation, the claudin-18A2-polypeptide can be present in two conformations on the cell. In conformation 1, the protein is present as a membrane molecule with 4 transmembrane domains (TM) and has two separate, extracellular domains. In conformation 2, the two middle hyrophobic regions (h-phob) do not perform a transmembrane domain function.This results in extracellular peptide regions in this conformation compared to conformation 1. Furthermore, this conformation results in an additional N-glycosylation site at position 116 (thicker arrow). All predicted glycosylation domains are listed at the bottom of the figure. Ex1: extracellular domain 1, Ex2: extracellular domain 2, TM: transmembrane domains, H-phobic: extracellular hydrophobic region.Figure 3. Quantitative expression of Claudin-18, variant A1 Other Claudin-18A1 is not detectable in any normal tissue other than lung and stomach tissue. Claudin-18A1 is highly expressed in a wide variety of tumour tissues. Particularly strong expression is found in gastric tumours, lung tumours, pancreatic tumours and oesophageal tumours.Figure 4. Other Claudin-18A2 is not detectable in any normal tissue other than gastric tissue.Claudin-18A2 is expressed in a wide variety of tumour tissues, particularly in stomach, lung, pancreatic and oesophageal tumors.Figure 5.Use of Claudin-18A2 specific antibodies (extracellular domain)A: staining of Claudin-18A2-positive magento tumor cells (SNU-16, methanol-fixed) with an antibody produced by immunization with a peptide (SEQ ID NO:17).A domain staining is particularly strong at cell/cell interaction sites.The protein is aggravated in focal membrane sites.B, C, D: Detection of the specificity of the antibody by collec-tion in Claudin-18A2-transfused cells.Typical use: 29A-FGF2A;CFP-A-FGF2A-specific antibodies. Other Membrane staining of Claudin-18A2-positive stomach tumour cells (SNU-16) with an antibody produced by immunisation with a peptide (SEQ ID NO: 113, N-terminal extracellular domain).A monoclonal antibody directed against E-cadherin was used for the anti-staining. A: Claudin-18A2 antibody; B: anti-E-cadherin anti-staining; C: overlapping.Figure 7. Other Left: Membrane staining of Claudin-18A2-positive stomach tumour cells (SNU-16) with an antibody produced by immunisation with a peptide (SEQ ID NO: 116, C-terminal extracellular domain). A monoclonal antibody directed against E-cadherin was used for the counterstaining (right) (Figure 8). Use of Claudin-18A1-specific antibodies Other Upper: weak to no staining of stomach tumour 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: overlapping.Other Other Below: Detection of antibody specificity by collocation analysis in 293T cells translocated with Claudin-18A1 GFP. A: GFP fluorescence; B: anti-Claudin-18A1; C: superposition.Fig. 9 Detection of Claudin-18A2 in the western blot. Other Western blot with lysates from various healthy tissues with a claudin-18A2-specific antibody directed against the epitope with SEQ ID NO: 17.1: stomach; 2: testicles; 3: skin; 4: breast; 5: liver; 6: colon; 7: lung; 8: kidney; 9: lymph node normal tissue.Fig. 10. Other Lysates from stomach and stomach tumours (A, B) and tumour cell lines (C, D) were bloated and tested with a claudin-18A2-specific antibody against the epitope with SEQ ID NO:17.A PNGase F treatment of gastric lysates results in the formation of the low-glycosylated form. A: 1: stomach normal tissue #A; 2: stomach tumour #A; 3: stomach normal tissue #B; 4: stomach tumour #BB: 1: stomach normal tissue #A; 2: stomach normal tissue #B; 3: stomach normal tissue #B + PNGase F; 4: stomach tumour #C; 5: stomach tumour #D; 6: stomach tumour #D + PNGase FC: 1: stomach normal tissue; 2: MDA-MB-231; 3: SK-MEL-37; 4: AGS; 5: SNU-1; S-16; 7: EFO-27; 8: OV-112D; 9: Claudin 9: Note that the antibody cell lines express the deglycosylated strains of claudin-18A. Other As shown in Figure 1.30 Low-glycosylated claudin-18A2 variants were detected in lung tumours. 1: normal stomach tissue; 2: stomach tumour; 3-9: lung tumours.Fig. 12 Immunohistochemical analysis of claudin-18 with claudin-18A2-specific antibodies in normal tissues Other In the gastric mucosa, only differentiated epithelial cells at the opening and bottom of the glands are stained. Claudin-18A2 is not detectable in stomach stem cells. All other normal tissues we studied also do not express this gene, as shown in the kidney, lung and colon.Figure 13. Results of immunohistology with Claudin-18A2-specific polyclonal antiserumA: Examples of specific staining of lung tumor tissues.B: Examples of specific tumor staining of esophageal tumors. It should be noted that healthy cells in the environment are not stained. C: Examples of specific tumor staining of gastric tumor epithelia. Again, healthy cells in the environment are not stained. D: Exemplary tabular summary of immunohistochemical staining data with claudin-18A2-specific antibodies. AdenoCa: adenocarcinoma; SCC: squamous cell carcinoma; RCC: renal carcinoma.Fig. 14 sequence. Other The sequences referred to herein are shown.Figure 15.Detection of extracellular regions of claudin-18A2 Other Three constructs were produced, each carrying a marker sequence (myc or HA tag) in one of the domains EX1 (= extracellular domain 1), EX2 (= extracellular domain 2), or D3 (= domain 3) (above).whether an antibody directed against these marker sequences binds to non-permeabilised cells. This requires that the corresponding region of the protein is topologically extracellular. Flow cytometry showed that all three regions of the molecule are accessible to the antibodies (below).Figure 16. Other According to our data, claudin-18A2 can be present in conformation 2, where the inner two hydrophobic domains do not pass through the cell membrane in its entirety. This makes larger areas of this molecule extracellular. This also contains glycosylation domains, which according to our data are glycosylated in normal gastric tissue but not in tumors. Thus, purely tumor tissue-specific epitopes are produced.Figure 17. Other The figure shows flow cytometric analyses on non-permeabilised cells.The mAB1 and mAB2 antibodies are specific to Claudin-18A2 (left column) and the extracellular domain 2 (Ex2, column 3) on the cell surface, while Claudin-18A1 (second column) and the negative control (last column) are negative. mAB1 also specifically binds to the extracellular domain 1 (Exl, column 4) unlike mAB2.

Examples: Materials and methods

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All other terms and terms are used as understood by the practitioner unless explicitly defined otherwise. The techniques and methods mentioned are in a way that is familiar to the practitioner and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. All procedures involving the use of kits and reagents are performed according to the manufacturer's instructions.

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

Err1:Expecting ',' delimiter: line 1 column 502 (char 501)Err1:Expecting ',' delimiter: line 1 column 266 (char 265)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 capacity > 1000 were approved. Pool B was digitally subtracted from Pool A using the BUT NOT operator. The set of GOIs found in this way was also subjected to eNorthern studies and backed up by a literature search. Other 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). Other 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). Other 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). Other 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).20 μl of this reaction was separated and analysed on an agarose seal stained with ethidium bromide.

The following primers were used for the expression analysis of the corresponding antigens at the specified hybridisation temperature.Claudin-18A1 (64°C)

Sense: 5'-GAGGCAGAGTTCAGGCTTCACCGA-3'	(SEQ ID NO: 109)
Antisense: 5'-TGTTGGCTTTGGCAGAGTCC-3'	(SEQ ID NO: 110)

The following shall be added to the list of substances:

Sense1: 5'-GGTTCGTGGTTTCACTGATTGGGATTGC-3'	(SEQ ID NO: 39)
Antisense1: 5'-CGGCTTTGTAGTTGGTTTCTTCTGGTG-3'	(SEQ ID NO: 40)
Sense2: 5'-TGTTTTCAACTACCAGGGGC-3'	(SEQ ID NO: 107)
Antisense2: 5'-TGTTGGCTTTGGCAGAGTCC-3'	(SEQ ID NO: 108)

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.

To detect specific O- and N-glycosidic bonds, 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). A Western protein assay is then described. When reducing the size of a target protein after incubation with a specific glycosidase, a tumor can be predicted and the position of the glycosidase can be predicted by this method.

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-stained staining are confirmed in addition to the target protein by staining the coupled amino acid markers or other marker proteins, the localization of which has already been described in the literature.

Immunohistochemistry

The IHC is specifically used to (1) estimate the amount of target protein in tumour and normal tissues, (2) analyse how many cells in tumour and healthy tissues synthesize the target gene, and/or (3) define the cell type in a tissue (tumor, healthy cells) in which the target protein is detectable.

Err1:Expecting ',' delimiter: line 1 column 138 (char 137)

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 active substance in the active substance is not recommended for the treatment of patients with severe allergic reactions. Other 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. Other 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 191 (char 190)

In the subsequent Westem 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 specific membrane (e.g. nitrodulose, silica & shell) by one of several blotting procedures (e.g. semi-dry electroblot; biospira) and the antibodies are then detected on this membrane. The antibodies are then first incubated with the target protein, which should be detectable by the antigen (e.g. ECP, or peroxide) in a specific membrane (e.g. a target protein, which can be incubated with a protein of approximately 60 micrometres per second).

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 Westem-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 precipitation) are well established (e.g. Lemoine et al., Molinosols. Biol. 751-7, 1997).

Err1:Expecting ',' delimiter: line 1 column 96 (char 95)

The analysis usually allows for the safe localization of the target protein, whereby, in addition to the target protein, the coupled amino acid markers or other marker proteins whose localization has already been described in the literature are also stained to confirm the antibody quality and the target protein in double staining. A special case is GFP and its derivatives, which can be directly stimulated and self-fluoresce.

Alternatively, extracellular domains can be detected by flow cytometry, which uses cells fixed under non-permeabilising conditions (e.g. PBS/Na-acid/2% FCS/ 5 mM EDTA) and analysed in a flow cytometer as specified by the manufacturer. Only extracellular epitopes can be detected by the antibody to be analysed in this method. Unlike immunofluorescence, the use of e.g. trypanid or propanidine can distinguish between dead and living cells, thus avoiding 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 further washing 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 GFP transfectors

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

Flow cytometry

Flow cytometry measurements were performed according to known methods (e.g. Robinson (Editor) Handbook of flow cytometry methods, Wiley-Liss, New York, 1993).

Example 1: 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 hydrophobic regions. According to the prediction programs (TMHMM, TMPred) and the topology described for many other members of this family, claudin-18 has four transmembrane and thus two extracellular domains EX1 and EX2, whose extracellular localization (conformation 1) is shown in Figure 2. The D3 domain, located between the two extracellular epithelial regions, is described as intracellular in the literature for claudin-18 and other members of this family and is also predicted as such by conventional prediction programs. The N-terminus and C-terminus are intrinsically exogenous and differentiated (M. Niol. Biol.

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 was investigated, a quantitative PCR was established to distinguish between the two variants by selecting A1-specific (SEQ ID NO: 109, 110) and A2-specific (SEQ ID NO: 107, 108) primer pairs, respectively, and the splice variant A2 was additionally tested with a second primer pair in a conventional PCR (SEQ ID NO: 39, 40).

The A1 variant is described as only active in healthy lung tissue, but surprisingly we found that A1 was also active in the gastric mucosa (Fig. 3). The stomach and lungs are the only normal tissues to show significant activation. All other normal tissues are negative for claudin-A1. An examination of tumors found that claudin-A1 is highly active in a wide variety of tumor tissues. Particularly strong expression is found in magenta, lung, pancreatic, esophageal, HNOT, and claustral tumors (Fig. 3).

The study of the claudin A2 splice variant used oligonucleotides that specifically amplify this transcript (SEQ ID NO: 39, 40 and 107, 108, respectively). The study showed that the A2 splice variant is not expressed in any of the more than 20 normal tissues studied except in gastric mucosa and to a lesser extent in test tissues (Fig. 4). We found that, like the A1 variant, the A2 variant is activated in many tumors (Fig. 4).

These include stomach, pancreatic, esophageal and liver tumours, although no activation of claudin-18A2 has been found in healthy lungs, surprisingly a number of lung tumours have been found to express the A2 splice variant. Other Tabelle 1A. Expression von Claudin-18A2 in Normal- und Tumor-Geweben

Gehirn	-
Cerebellum	-
Myokard	-
Skelettmuskel	-
Endometrium	-
Magen	+++
Kolon	-
Pankreas	-
Niere	-
Leber	-
Testis (Hoden)	+
Thymus	-
Brust	-
Ovar	-
Uterus	-
Haut	-
Lunge	-
Schilddrüse	-
Lymphknoten	-
Milz	-
PBMC	-
Ösophagus	-

Tabelle 1A. Expression von Claudin-18A2 in Normal- und Tumor-Geweben

Kolon	-
Pankreas	++
Ösophagus	++
Magen	+++
Lunge	++
Brust	-
Ovar	-
Endometrium	n.u.
HNO	++
Niere	-
Prostata	-

Tabelle 1B. Expression von Claudin-18A1 in Normal- und Tumor-Geweben

Gehirn	-
Cerebellum	-
Herzmuskel	-
Muskel	-
Endometrium	-
Magen	+++
Kolon	-
Pankreas	-
Niere	-
Leber	-
Testis	+
Thymus	-
Brust	-
Ovar	-
Uterus	-
Haut	-
Lunge	+++
Schilddrüse	-
Lymphknoten	-
Milz	-
PBMC	-
Ösophagus	-

Tabelle 1B. Expression von Claudin-18A1 in Normal- und Tumor-Geweben

Kolon	-
Pankreas	++
Ösophagus	++
Magen	+++
Lunge	++
Brust	+
Ovar	n.u
Endometrium	n.u.
HNO	++
Niere	-
Prostata	++

Conventional PCR as an independent control also confirmed the results of quantitative PCR. In particular, oligonucleotides (SEQ ID NO: 39, 40) were used to allow specific amplification of the A2 splice variant. Most stomach tumors and half of the pancreatic tumors tested were shown to have strong expression of this splice variant (Figure 1). However, expression in other tissues is not detectable with conventional PCR. In particular, no expression is found in important normal tissues such as the lungs, liver, blood, lymph nodes, upper chest and kidney (Tables 1).

These gene products are attractive therapeutic targets because their absence in most toxicity-relevant organs does not lead to side effects on these organs, while the high activation in cells of the mentioned cancers leads to a good binding to these and the mediation of corresponding cell damage effects.

To confirm these protein-level data, claudin-specific antibodies or immunosers were generated by immunisation of animals. Other The N-terminal extracellular domain EX1 is different in sequence in the two splice variants A1 and A2 (SEQ ID NO: 111 for A1 and SEQ ID NO: 112 for A2). The C-terminal extracellular domain EX2 is identical in both variants (SEQ ID NO: 137). No antibodies binding to the extracellular domains of claudin-18 have been described to date. No antibodies specifically distinguishing between A1 and A2 variants have been described. Varicella-specific peptides and antibodies specific to A1 or A2 or to both of these proteins have been selected for immunization to generate antibodies. Seq ID NO: 17: DQWSTQDLYN (N-terminal extracellular domain, A2-specific, binding independent of glycosylation) SEQ ID NO: 18: NNPVTAVFNYQ (N-terminal extracellular domain, A2-specific, binding mainly to the unglycosylated form, N37) SEQ ID NO: 113: STQDLYNNPVTAVF (N-terminal extracellular domain, A2-specific, binding only to the unglycosylated form, N37) SEQ ID NO: 114: DMNPWSTQYD (N-terminal extracellular domain, A1-specific) NOQ: 115 CRYPY (N-terminal extracellular domain, A2-specific, also known as A-terminal extracellular domain, A-specific, A-specific, A-specific)

Among other things, we were able to generate antibodies that selectively recognize the N-terminal domain of the claudin-18-A1 splice variant but not the A2 variant (Figure 8). By using epitopes for immunizations that are in the C-terminal extracellular domain, which is identical in both splice variants, we were able to generate antibodies that recognize both variants (Figure 7).

The specific antibody can be used under different fixation conditions for immunofluorescence studies. In comparative staining of RT-PCR positive and negative cell lines, the corresponding protein is specific in a detectable amount, including in the positively typed gastric, esophageal and pancreatic tumour cell lines (Fig. 5). The endogenous protein is membrane localized and forms larger focal aggregates on the membrane (A. 5). Several immunoassays have been performed on human tissue with this antibody. We have confirmed the selective distribution of this protein in almost all types of proteins that are not normally studied.The A2 variant of claudin-18 is found in the differentiated cells of the gastric mucosa, but not in the stem cells. Differentiated gastric mucosa cells are subject to constant renewal. Physiologically, the entire gastric epithelium is continuously replaced starting from the stomach stem cells. This supports the usefulness of the A2 variant as a targeted therapeutic structure, as we show that stem cells of the A2 variant, which are indigenously susceptible to the A2 receptor, cannot be specifically targeted by the A2 receptor as all other organs of the stomach, and therefore cannot be targeted by the A2 receptor as specifically as the other A2 receptor.We detected the A2 variant of claudin-18 in a number of human tumors using this antibody (Figure 13), particularly in tumors of the stomach, esophagus and lungs, which we had already detected in RT-PCR studies.

The antibody described above was also used for protein detection in the Western blot. As expected, the protein is detected only in the stomach and not in any other normal tissue, including the lungs, where only the A1 variant is activated (Fig. 9). When comparing the staining of stomach tumors and adjacent normal stomach tissue from patients, it was surprising that in all stomach tumors where Claudin-18A2 is detected, this protein has only a smaller mass (Fig. 10 left). In a series of experiments, it was found that a band of this size is produced by treating lysosyl magnesiam with the deglycoside agent FPC (Fig. 10). While Magnesium is found in all normal tissues, it is also found in the right side of the lungs. Although it is only found in the RT-A2 form, the quantitatively detectable form of A2 is also found in the right side of the lung, whereas the glycoside is found in the normal form (Fig. 10).

Claudin-18 itself is a highly selective differentiation antigen of the stomach (A2 variant) and of the lungs and stomach (A1 variant).Our data indicate that it is obviously affected by tumour-associated changes in the glycosylation machinery and that a special form of the A2 variant is produced in tumours which is deglycosylated.Results from PNGaseF treatment show that claudin-18A2 differs in its N-glycosylation in tumour and normal tissues.

The glycosylation of an epitope can prevent the binding of an epitope-specific antibody and in this case helps to ensure that such an antibody cannot bind to claudin-18A2 in normal tissue but exclusively to the non-glycosylated form in cancer cells. To produce antibodies that bind exclusively to non-glycosylated epitopes, this was taken into account in the selection of immunogens. We have identified several regions of claudin-18A2 that may be present in tumor tissue and normal tissue in different glycosylations. As potential glycosylation sites for claudin-18A2, we have identified, among others, the regions that include the amino acids 37, 45, 38, 116, 146, 205 and 182, which are different from those in normal tissue (see below and one or more of these glycosylation sites).Most of these domains are not classical glycosylation sites but contain asparagine, serine and threonine, which can be glycosylated in rare cases (prediction Figure 2 below). Both variants of claudin-18 have a single classical glycosylation motif in the D3 domain, which is considered intracellular in the literature and accordingly common prediction algorithms. However, in a tetraspanin structurally similar to claudin-18, PMP 22, the hydrophobic membranes 2 and 3 of PMP 22 were shown not to cross the cell membrane entirely but to intercalate only partially in the plasma membrane (TMP et al., J. Neosclorus Res. 62:15-27, 2000).

We hypothesized and verified the possibility of such a topology for claudin-18A2. To this end, we constructed 3 constructs, each carrying a marker sequence (his or HA tag) in one of the EX1, EX2, or D3 domains (Fig. 15 above). These were translated into cell lines and tested for whether an antibody directed against these marker sequences binds to non-permeabilising cells, requiring that the corresponding region of the protein be topologically extracellular. Since all regions of the molecule were topometrically included as extracellular by three flow cytometrically (Fig. 15 below), we were able to confirm that claudin-18A2 is present in a conformation with two transmembrane and large biochemically-bound antibody domains (A, B, B, C, D, E, F, F, G, F, G, H, I, O, F, G, H, I, O, F, G, H, I, O, F, G, H, I, F, G, G, H, I, G, H, I, G, H, I, G, G, H, H, I, G, G, H, H, G, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H, H,

This allows the production of antibodies that distinguish between glycosylated and non-glycosylated variants of claudin-18A2. These have a particularly high specificity for tumour cells. In the production of glycosylation specific antibodies, we have taken into account these different conformational options in addition to the glycosylation domains.

Preferably, but not exclusively, proteins from the D3 region of claudin-18A2 are suitable for immunization of animals. This is illustrated for two antibodies mAB1 and mAB2 (Fig. 17). We investigated the binding properties of these antibodies to cell lines expressing either the A1 or A2 variant of claudin-18. We were able to show that claudin-18A2 is accessible to antibodies on the cell surface. Such antibodies are specific to the A2 variant and do not bind to the A1 variant (Fig. 17). We introduced short myc-tags (myc-tags) into the regions of the extellalomas Ex1 and Ex2 (Fig. 431 mAB).The generated antibodies can be used for both diagnostic and therapeutic purposes. Immunizers such as the one described here (against peptide SEQ ID NO: 17) can be used for diagnostic purposes, e.g. in the Western blot. Immunization with peptides containing at least one of these regions (e.g. peptide SEQ ID NO: 113 (Figure 6), peptide SEQ ID NO: 142-145) can produce antibodies that cannot bind to the glycosylated EOP at all. Such antibodies specifically bind to the glycosylated EOPs on tumor cells. The absence of glycosylation at any of these positions compared to normal tissue may also be a secondary endogenous condition caused by deglycosylation in tumors.Therefore, claudin-18A2-derived peptides can be used to produce antibodies against such modified tumour-associated variants, in which the amino acid Asn (N) is replaced by an Asp (D) at at least one of the positions 37, 38, 45, 116, 141, 146, 205 of the claudin-18A2-peptide (e.g. SEQ ID NO: 146-150). In particular, such antibodies can be used therapeutically, as they are highly selective for tumour cells. The antibodies produced can also be directly converted to produce chimeric or humanised recombinant antibodies. This can also be done directly with antibodies obtained from canine chemicals (see J. Biol. 2000 5; May Ritter, May Ritter, 2000:2751:1686-78).Err1:Expecting ',' delimiter: line 1 column 125 (char 124)

The following shall be added:

< 110 > Ganymed Pharmaceuticals GmbH < 120 > Gene products expressed differently in tumours and their use < 130 > 342-23 EPT6 < 150 > DE 10 2004 024 617.3 Other The Commission has decided to grant a Community patent on a new type of motor vehicle.

Claims (8)

1. A pharmaceutical composition for use in treating an adenocarcinoma of the stomach characterised by the expression of a tumor-associated antigen, wherein the pharmaceutical composition comprises an antibody which specifically binds to the tumor-associated antigen, wherein the tumor-associated antigen has an amino acid sequence encoded by a nucleic acid sequence according to SEQ ID NO: 7 or 8, and wherein the antibody is a single-chain antibody, or wherein the antibody is coupled to a therapeutic agent, wherein the therapeutic agent is selected from the group consisting of toxin, cytostatic or cytolytic drugs, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and vincristine sulfate, pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, Pseudomonas exotoxin, and cobalt-60.
2. The pharmaceutical composition for the use of claim 1, wherein the antibody is a monoclonal, chimeric or humanized antibody or an antigen-binding fragment of an antibody.
3. The pharmaceutical composition for the use of claim 1 or 2, wherein the tumor-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19, 112, 113, 116, 137 and 142-145.
4. The pharmaceutical composition for the use of any one of claims 1-3, wherein the antibody binds to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19, 112 and 113.
5. Use of an antibody which specifically binds to a tumor-associated antigen and is coupled to a therapeutic agent for the preparation of a pharmaceutical composition for treating an adenocarcinoma of the stomach characterised by the expression of the tumor-associated antigen, wherein the tumor-associated antigen has an amino acid sequence encoded by a nucleic acid sequence according to SEQ ID NO: 7 or 8, wherein the therapeutic agent is selected from the group consisting of toxin, cytostatic or cytolytic drugs, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, cytarabidine, dacarbazine, dactinomycin, daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon-α, lomustine, mercaptopurine, methotrexate, mitotane, procarbazine HCl, thioguanine, vinblastine sulfate and vincristine sulfate, pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin, Pseudomonas exotoxin, and cobalt-60.
6. The use of claim 5, wherein the antibody is a monoclonal, chimeric or humanized antibody or an antigen-binding fragment of an antibody.
7. The use of claim 5 or 6, wherein the tumor-associated antigen comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19, 112, 113, 116, 137 and 142-145.
8. The use of any one of claims 5 to 7, wherein the antibody binds to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16-19, 112 and 113.