EP1664103A2 - Produits geniques exprimes de maniere differentielle dans des tumeurs et leur utilisation - Google Patents

Produits geniques exprimes de maniere differentielle dans des tumeurs et leur utilisation

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Publication number
EP1664103A2
EP1664103A2 EP04765088A EP04765088A EP1664103A2 EP 1664103 A2 EP1664103 A2 EP 1664103A2 EP 04765088 A EP04765088 A EP 04765088A EP 04765088 A EP04765088 A EP 04765088A EP 1664103 A2 EP1664103 A2 EP 1664103A2
Authority
EP
European Patent Office
Prior art keywords
nucleic acid
tumor
associated antigen
antibody
expression
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP04765088A
Other languages
German (de)
English (en)
Inventor
Özlem TÜRECI
Ugur Sahin
Michael Koslowski
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ganymed Pharmaceuticals GmbH
Original Assignee
Ganymed Pharmaceuticals GmbH
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Ganymed Pharmaceuticals GmbH filed Critical Ganymed Pharmaceuticals GmbH
Priority to EP10011193.9A priority Critical patent/EP2327721B1/fr
Priority to EP10011190A priority patent/EP2336156A3/fr
Priority to EP10011191A priority patent/EP2336157A3/fr
Priority to EP10011194A priority patent/EP2322544A3/fr
Priority to EP10011192A priority patent/EP2314613A3/fr
Priority to EP16176546.6A priority patent/EP3095791B1/fr
Publication of EP1664103A2 publication Critical patent/EP1664103A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4748—Tumour specific antigens; Tumour rejection antigen precursors [TRAP], e.g. MAGE
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/30—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants from tumour cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/112—Disease subtyping, staging or classification
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/154—Methylation markers

Definitions

  • cancer continues to be a leading cause of death.
  • More recent therapeutic concepts aim to include the patient's own immune system in the overall therapeutic concept by using recombinant tumor vaccines and other specific measures such as antibody therapy.
  • the 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, the effector functions of which are to be enhanced by intervention.
  • Tumor cells differ biologically from their non-malignant cells of origin. These differences are due to genetic changes acquired during tumor development and also lead to the formation of qualitatively or quantitatively changed molecular structures in the cancer cells.
  • tumor-associated antigens are recognized by the specific immune system of the tumor-bearing host, one speaks of tumor-associated antigens.
  • tumor-associated antigens arises from the fact that the recognition of antigens on neoplastic cells by the immune system leads to the initiation of cytotoxic effector mechanisms and, in the presence of T helper cells, can cause the cancer cells to be eliminated (Pardoll, Nat. Med. 4: 525-31, 1998). Accordingly, it is a central objective of tumor immunology to define these structures on a molecular basis. The molecular nature of these antigens has long remained enigmatic.
  • CTA cancer / testis antigens
  • CTA and genes encoding it are defined by their characteristic expression pattern [Tureci et al, Mol Med Today. 3: 342-9, 1997]. They are not found in normal tissues except for testis or germ cells, but are expressed in a number of human malignancies, and not specifically for the tumor type, but with different frequencies in tumor entities of very different origins (Chen & Old, Cancer J Sei. Am. 5:16 -7, 1999). Serum reactivities against CTA are also not found in healthy controls, but only in tumor patients.
  • a strategy for identifying and providing tumor-associated expressed antigens and the nucleic acids coding therefor was pursued.
  • This strategy is based on the fact that it is actually testis-specific and therefore germ-cell-specific Genes that are normally silent in adult tissues are reactivated ectopically and illegally in tumor cells.
  • data mining a complete list of all known Testis-specific genes is first drawn up and then evaluated by expression analysis using specific RT-PCR for their aberrant activation in tumors. Datamining is a well-known method for the identification of tumor-associated genes.
  • transcriptomes from normal tissue banks are usually electronically subtracted from tumor tissue banks on the assumption that the remaining genes are tumor-specific (Schmitt et al., Nucleic Acids Res. 27: 4251-60, 1999; Vasmatzis et al, Proc. Natl. Acad. Sci. USA 95: 300-4, 1998. Scheurle et al, Cancer Res. 60: 4037-43, 2000).
  • the invention relates to a strategy for identifying genes differentially expressed in tumors. This combines data mining from public sequence banks ("in silico") with subsequent evaluating laboratory experiments ("wet bench”).
  • CT Cancer / Testis
  • gene products were produced independently of an immunogenic effect.
  • the tumor-associated antigens identified according to the invention have an amino acid sequence which is encoded by a nucleic acid which is selected from the group consisting of (a) a nucleic acid which comprises a nucleic acid sequence which is selected from the group consisting of SEQ ID NOs: 1 -5, 19-21, 29, 31-33, 37, 39, 40, 54-57, 62, 63, 70, 74, 85-88 a part or derivative thereof is selected, (b) a nucleic acid which under stringent conditions with the nucleic acid hybridized under (a), (c) a nucleic acid which is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).
  • a tumor-associated antigen identified according to the invention has an amino acid sequence which is encoded by a nucleic acid which is selected from the group consisting of SEQ ID NOs: 1-5, 19-21, 29, 31-33, 37, 39 , 40, 54-57, 62, 63, 70, 74, 85-88 is selected.
  • a tumor-associated antigen identified according to the invention comprises an amino acid sequence which is selected from the group consisting of SEQ ID NOs: 6-13, 14-18, 22-24, 30, 34-36, 38, 41, 58- 61, 64, 65, 71, 75, 80-84, 89-100, 101-117 a part or derivative thereof is selected.
  • the present invention relates generally to the use of tumor-associated antigens identified according to the invention or of parts thereof, of nucleic acids coding therefor or of nucleic acids which are directed against the coding nucleic acids or of antibodies which are directed against the tumor-associated antigens or parts thereof identified according to the invention are directed for therapy and diagnosis.
  • This use can relate to individual, but also combinations of several of these antigens, functional fragments, nucleic acids, antibodies, etc., in one embodiment also in combination with other tumor-associated genes and antigens for diagnosis, therapy and follow-up.
  • Preferred diseases for therapy and / or diagnosis are those in which there is selective expression or abnormal expression of one or more of the tumor-associated antigens identified according to the invention.
  • ectopic activation of genes in tumors can also be traced back to an altered gene methylation pattern of their nucleotide sequence. For example, that alterations of methylation on cytosine contribute to this (De Smet et al, 1996 and 1999).
  • the invention also relates to nucleic acids and gene products which are expressed in association with tumor cells and which result from altered splicing (splice variants) of known genes or from altered translation using alternative open reading frames.
  • These nucleic acids comprise the sequences according to (SEQ ID NO: 2-5, 20, 21, 31-33, 54-57, 85-88) of the sequence listing.
  • the gene products include sequences according to (SEQ ID NO: 7-13, 23, 24, 34-36, 58-61, 89-100) of the sequence listing.
  • splice variants can be used as targets for the diagnosis and therapy of tumor diseases.
  • a wide variety of mechanisms can be responsible for the formation of splice variants, for example the use of variable transcription initiation sites, the use of additional exons - complete or incomplete splice of single or several exons, mutation-modified splice regulator sequences (deletion or creation of new donor / acceptor sequences), incomplete elimination of intron sequences.
  • the changed splicing of a gene leads to a changed transcript sequence (splicing variant). If a splice variant is translated in the area of its changed sequence, a modified protein results, which can differ significantly in structure and function from the original one. With tumor-associated splice variants, tumor-associated transcripts and tumor-associated proteins / antigens can arise. These can be used as molecular markers both for the detection of tumor cells and for the therapeutic targeting of tumors.
  • the detection of tumor cells can be carried out in accordance with the invention, for example after extraction of nucleic acids by PCR amplification with splice variant-specific oligonucleotides.
  • all sequence-dependent detection systems are suitable for detection.
  • these are, for example, gene chip / microarray systems, Northern blot, RNAse protection assays (RDA) and others. All detection systems have in common that the detection is based on a specific hybridization with at least one splicing variant-specific nucleic acid sequence.
  • tumor cells can also be detected by antibodies which recognize a specific epitope coded by the splice variant.
  • Peptides for immunization that are specific for this splice variant can be used for the production of the antibodies.
  • Particularly suitable for immunization are the amino acids which have clear epitope differences from the variant (s) of the gene product, which is (are) preferably formed in healthy cells.
  • the detection of the tumor cells with antibodies can be carried out on a sample isolated from the patient or as imaging with intravenously applied antibodies.
  • splice variants which have new or modified epitopes, attractive targets for immunotherapy.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent which recognizes the tumor-associated antigen identified according to the invention and is preferably selective for cells which have expression or abnormal expression of a tumor-associated antigen identified according to the invention.
  • the agent can induce cell death, reduce cell growth, damage the cell membrane or secrete cytokines and preferably has a tumor-inhibiting activity.
  • the agent is an antisense nucleic acid that selectively hybridizes to the nucleic acid encoding the tumor associated antigen.
  • the agent is an antibody which selectively binds to the tumor-associated antigen, in particular a complement-activated antibody which selectively binds to the tumor-associated antigen.
  • the agent comprises several agents, each of which selectively recognizes different tumor-associated antigens, at least one of the tumor-associated antigens being a tumor-associated antigen identified according to the invention.
  • the detection does not have to be directly associated with an inhibition of activity or expression of the antigen.
  • this is selectively limited to tumors Antigen preferably as a marker for recruiting effector mechanisms at this specific location.
  • the agent is a cytotoxic T lymphocyte which recognizes the antigen on an HLA molecule and lyses the cell labeled in this way.
  • the agent is an antibody which selectively binds to the tumor-associated antigen and thus recruits natural or artificial effector mechanisms for this cell.
  • the agent is a T helper lymphocyte that enhances effector functions of other cells that specifically recognize this antigen.
  • the invention relates to a pharmaceutical composition
  • a pharmaceutical composition comprising an agent which inhibits the expression or activity of a tumor-associated antigen identified according to the invention.
  • the agent is an antisense nucleic acid that selectively hybridizes with the nucleic acid that codes for the tumor-associated antigen.
  • the agent is an antibody that selectively binds to the tumor-associated antigen.
  • the agent comprises several agents, each of which selectively inhibit the expression or activity of different tumor-associated antigens, at least one of the tumor-associated antigens being a tumor-associated antigen identified according to the invention.
  • the activity of a tumor-associated antigen identified according to the invention can be any activity of a protein or peptide.
  • the activity of an enzymatic activity is like the lactate dehydrogenase activity in the case of sequences which relate to LDHC (cf. Example 1).
  • this activity relates to involvement in cellular migration and / or metastatization as in the case of the sequences relating to TPTE (cf. Example 2).
  • the therapy and diagnostic methods according to the invention can thus also aim to inhibit or reduce this activity or to test this activity.
  • the agent comprises several agents, each of which selectively increases the amount of complexes between MHC molecules and peptide epitopes of different tumor-associated antigens, at least one of the tumor-associated antigens being a tumor-associated antigen identified according to the invention.
  • a nucleic acid which codes for a tumor-associated antigen identified according to the invention or a part thereof can be present in the pharmaceutical composition in an expression vector and can be functionally linked to a promoter.
  • An antisense nucleic acid contained in a pharmaceutical composition according to the invention can comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides from the nucleic acid which codes for the tumor-associated antigen identified according to the invention.
  • a pharmaceutical composition according to the invention can comprise a pharmaceutically acceptable carrier and / or an adjuvant.
  • the adjuvant can be selected from saponin, GM-CSF, CpG nucleotides, RNA, a cytokine or a chemokine.
  • a pharmaceutical composition according to the invention is preferably used for the treatment of a disease which is characterized by the selective expression or abnormal expression of a tumor-associated antigen. In a preferred embodiment, the disease is cancer.
  • the invention relates to a method for diagnosing a disease which is characterized by the expression or abnormal expression of a tumor-associated antigen identified according to the invention.
  • the procedure includes the proof (i) of one Nucleic acid coding for the tumor-associated antigen or a part thereof and / or (ii) the detection of the tumor-associated antigen or a part thereof and / or (iii) the detection of an antibody against the tumor-associated antigen or a portion thereof and / or (iv) the detection of cytotoxic or helper T lymphocytes which are specific for the tumor-associated antigen or a portion thereof in a biological sample isolated from a patient.
  • the disease is characterized by the expression or abnormal expression of several different tumor-associated antigens
  • the detection includes a detection of several nucleic acids coding for the several different tumor-associated antigens, or parts thereof, the detection of the several different tumor associated antigens or parts thereof, the detection of several antibodies which bind to the several different tumor-associated antigens or parts thereof or the detection of several cytotoxic or helper T lymphocytes which are specific for the several different tumor-associated antigens.
  • the isolated biological sample from the patient is compared with a comparable normal biological sample.
  • the diagnostic methods according to the invention can also relate to the use of the tumor-associated antigens identified according to the invention as prognostic markers in order to predict metastasis, for example by testing the migration behavior of cells and therefore a worsened course of the disease, which among other things enables the planning of a more aggressive therapy.
  • the sequences relating to TPTE are suitable for this purpose. They are also suitable for differentiating benign changes, such as hyperplasia, from tumor precursors that are already to be assessed as unfavorable, and thus to predict a tendency to cancer even before an invasive tumor has formed.
  • an increased migration behavior indicates a metastasis and / or formation of lymph node and / or distant metastases or a potential therefor.
  • the tumor-associated antigen identified according to the invention preferably has a sequence encoded by a nucleic acid selected from the group consisting of: (a) a nucleic acid comprising a nucleic acid sequence consisting of the group consisting of SEQ ID NOs: 19-21, and 54-57, a part or derivative thereof, is selected (b) a nucleic acid which hybridizes under stringent conditions with the nucleic acid under (a), (c) a nucleic acid which is related to the nucleic acid under (a) or (b) is degenerate and (d) a nucleic acid which is complementary to the nucleic acid under (a), (b) or (c).
  • the tumor associated antigen preferably comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 22-24, 58-61, 81, 82,
  • methods of treatment according to the invention are preferably aimed at
  • the invention relates to a method for diagnosing a disease which is characterized by the expression or abnormal expression of a tumor-associated antigen identified according to the invention, the method determining the methylation pattern and / or the degree of methylation within a nucleic acid which comprises a nucleic acid sequence which codes for the tumor-associated antigen, in particular within the non-coding regions thereof, preferably within of the promoter region thereof.
  • the tumor-associated antigen to be detected or the part thereof is present in a complex with an MHC molecule, in particular an HLA molecule.
  • Detection of an antibody or monitoring the amount of antibodies can be carried out according to the invention with a protein or peptide that binds specifically to the antibody.
  • Detection of cytolytic T cells or helper T cells or monitoring the amount of cytolytic T cells or helper T cells which are specific for complexes between an antigen or a part thereof and MHC molecules can be carried out according to the invention a cell that presents the complex between the antigen or part thereof and an MHC molecule.
  • Cell receptor can identify the specific T lymphocytes.
  • the invention relates to a method for the treatment, diagnosis or monitoring of a disease which is characterized by the expression or abnormal expression of a tumor-associated antigen identified according to the invention, comprising the administration of an antibody to the tumor-associated antigen or a Part of it binds and is coupled with a therapeutic or diagnostic agent.
  • the antibody can be a monoclonal antibody.
  • the antibody is a chimeric or humanized antibody or a fragment of a natural antibody.
  • the invention also relates to a method for treating a patient with a disease which is characterized by the expression or abnormal expression of a tumor-associated antigen identified according to the invention, comprising (i) the removal of a Sample with immunoreactive cells from the patient, (ii) contacting the sample with a host cell that expresses the tumor-associated antigen or part thereof, under conditions that produce cytolytic T cells against the tumor-associated antigen or part of this favor, and (iii) introducing the cytolytic T cells into the patient in an amount suitable for lysing cells expressing the tumor associated antigen or a portion thereof.
  • the invention also relates to the cloning of the T cell receptor of cytolytic T cells against the tumor-associated antigen. This can be transferred to other T cells, which thus obtain the desired specificity and can be introduced into the patient as in (iii).
  • the host cell endogenously expresses an HLA molecule.
  • the host cell expresses an HLA molecule and / or the tumor-associated antigen or the part thereof recombinantly.
  • the host cell is preferably non-proliferative.
  • the host cell is an antigen-presenting cell, in particular a dendritic cell, a monocyte or a macrophage.
  • the method may further include identifying an MHC molecule that presents the tumor-associated antigen or a portion thereof, the host cell expressing the identified MHC molecule and presenting the tumor-associated antigen or a portion thereof.
  • the immune response can include a B cell response or a T cell response.
  • a T cell response may include the production of cytolytic T cells and / or helper T cells that are specific for the host cells that the tumor associated Present antigen or part of it or are specific to the patient's cells expressing the tumor associated antigen or part thereof.
  • the host cells used according to the invention are preferably non-proliferative or are made non-proliferative.
  • a disease that is characterized by the expression or abnormal expression of a tumor-associated antigen is cancer in particular.
  • the present invention further relates to a nucleic acid selected from the group consisting of (a) a nucleic acid comprising a nucleic acid sequence consisting of the group consisting of SEQ ID NOs: 2-5, 20-21, 31-33 , 39, 54-57, 62, 63, 85-88 a part or derivative thereof is selected, (b) a nucleic acid which hybridizes with the nucleic acid under (a) under stringent conditions, (c) a nucleic acid which is related is degenerate 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).
  • the invention relates to a recombinant nucleic acid molecule, in particular a DNA or RNA molecule, which comprises a nucleic acid according to the invention.
  • the invention also relates to host cells which contain a nucleic acid according to the invention or a recombinant nucleic acid molecule which comprises a nucleic acid according to the invention.
  • the invention relates to oligonucleotides that hybridize with a nucleic acid identified according to the invention and can be used as genetic probes or as "antisense" molecules.
  • Nucleic acid molecules in the form of oligonucleotide primers or competent samples which hybridize with a nucleic acid or parts thereof identified according to the invention can be used to find nucleic acids which are homologous to the nucleic acid identified according to the invention.
  • PCR amplification, Southern and Northern hybridization can be used to find homologous nucleic acids.
  • the hybridization can take place under low, better under medium and best under high-stringent conditions.
  • the term “stringent conditions” relates to conditions which allow specific hybridization between polynucleotides.
  • the invention relates to a protein or polypeptide that is encoded by a nucleic acid that is selected from the group consisting of (a) a nucleic acid that comprises a nucleic acid sequence that consists of the group consisting of SEQ ID NOs: 2 -5, 20-21, 31-33, 39, 54-57, 62, 63, 85-88 a part or derivative thereof is selected, (b) a nucleic acid that hybridizes under stringent conditions with the nucleic acid under (a) , (c) a nucleic acid that is degenerate with respect to the nucleic acid under (a) or (b) and (d) a nucleic acid that is complementary to the nucleic acid under (a), (b) or (c).
  • a nucleic acid that is selected from the group consisting of (a) a nucleic acid that comprises a nucleic acid sequence that consists of the group consisting of SEQ ID NOs: 2 -5, 20-21, 31-33, 39, 54
  • the invention relates to a protein or polypeptide which comprises an amino acid sequence selected from the Group consisting of SEQ ID NOs: 7-13, 14-18, 23-24, 34-36, 58-61, 64, 65, 89-100, 101-107 a part or derivative thereof.
  • the invention relates to an immunogenic fragment of a tumor-associated antigen identified according to the invention.
  • the fragment preferably binds to a human HLA receptor or human antibody.
  • a fragment according to the invention preferably comprises 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.
  • the invention relates to an agent that binds to a tumor-associated antigen identified according to the invention or to a part thereof.
  • the agent is an antibody.
  • the antibody is a chimeric, a humanized or combinatorial-made antibody or a fragment of an antibody.
  • the invention further relates to an antibody which binds selectively to a complex of (i) a tumor-associated antigen identified according to the invention or a part thereof and (ii) an MHC molecule to which the tumor-associated antigen or part identified according to the invention binds thereof, the antibody not binding to (i) or (ii) alone.
  • An antibody according to the invention can be a monoclonal antibody.
  • the antibody is a chimeric or humanized antibody or a fragment of a natural antibody.
  • the invention relates to a conjugate between an agent according to the invention which binds to a tumor-associated antigen identified according to the invention or to a part thereof, or an antibody according to the invention and a therapeutic or diagnostic agent.
  • the therapeutic or diagnostic agent is a toxin.
  • the invention relates to a kit for detecting the expression or abnormal expression of a tumor-associated antigen identified according to the invention, comprising means for detecting (i) the nucleic acid which codes for the tumor-associated antigen, or a part thereof, (ii ) the tumor-associated antigen or a part thereof, (iii) antibodies which bind to the tumor-associated antigen or a part thereof, and / or (iv) T cells specific for a complex between the tumor-associated antigen or a part thereof and an MHC molecule.
  • the means for detecting the nucleic acid or part thereof are nucleic acid molecules for the selective amplification of the nucleic acid, which in particular comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides from the nucleic acid.
  • genes are described which are selectively expressed or aberrantly expressed in tumor cells and which represent tumor-associated antigens.
  • these genes or their derivatives are preferred target structures for therapeutic approaches.
  • the therapeutic approaches can aim to inhibit the activity of the selectively expressed tumor-associated gene product. This makes sense if the aberrant or selective expression is functionally of tumor-physiological importance and its suppression is accompanied by selective damage to the corresponding cells.
  • Other therapeutic concepts consider tumor-associated antigens as markers that selectively recruit effector mechanisms with cell-damaging potential to tumor cells. The function of the target molecule itself and its role in tumor development are completely irrelevant.
  • “derivative” of a nucleic acid means that single or multiple nucleotide substitution, deletion and / or addition are present in the nucleic acid. Furthermore, the term “derivative” also includes chemical derivatization of a nucleic acid on a nucleotide base, on sugar or on phosphate. The term “derivative” also includes nucleic acids that contain nucleotides and nucleotide analogues that do not occur in nature.
  • a nucleic acid is preferably deoxyribonucleic acid (DNA) or
  • nucleic acids include genomic DNA, cDNA, mRNA, recombinantly produced and chemically synthesized molecules.
  • a nucleic acid can be present as a single-stranded or double-stranded and linear or covalently circular molecule.
  • the nucleic acids described according to the invention are preferably isolated.
  • isolated nucleic acid means that the nucleic acid (i) was amplified in vitro, for example by polymerase chain reaction (PCR), (ii) was produced recombinantly by cloning, (iii) was purified, for example by cleavage and gel electrophoretic separation or (iv) was synthesized, for example by chemical synthesis.
  • An isolated nucleic acid is a nucleic acid that is available for manipulation by recombinant DNA techniques.
  • Hybridization is preferably carried out under conditions that allow specific hybridization between polynucleotides (stringent conditions). Stringent conditions are described, for example, in Molecular Cloning: A Laboratory Manual, J. Sambrook et al., Ed., 2.
  • New York described and concern, for example, hybridization at 65 ° C in
  • the membrane to which the DNA has been transferred is, for example, in 2 ⁇ SSC at room temperature and then in
  • Complementary nucleic acids according to the invention 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% identity of the nucleotides.
  • nucleic acids coding for tumor-associated antigens can be present alone or in combination with other nucleic acids, in particular heterologous nucleic acids.
  • a nucleic acid is functionally associated with expression control sequences or regulatory sequences, which can be homologous or heterologous with respect to the nucleic acid.
  • a coding sequence and a regulatory sequence are "functionally 11 connected to each other, if they are covalently linked to one another, that the expression or transcription of the coding sequence is under the control or under the influence of the regulatory sequence.
  • the coding sequence is to be translated into a functional protein
  • a regulatory sequence when a regulatory sequence is functionally linked to the coding sequence, induction of the regulatory sequence leads to transcription of the coding sequence without any shifting of the reading frame in the coding sequence or to inability the coding sequence comes to be translated into the desired protein or peptide.
  • control sequence or “regulatory sequence” encompasses promoters, enhancers and other control elements which control the expression of a gene.
  • regulatory sequence encompasses promoters, enhancers and other control elements which control the expression of a gene.
  • Sequences can vary depending on species or cell type, but generally includes 5'-non-transcribed and 5'-non-translated sequences that are involved in the initiation of transcription or translation such as TATA box, capping sequence, CAAT-
  • 5'-non-transcribed regulatory sequences include a promoter region that includes a promoter sequence for transcriptional control of the functionally linked gene.
  • Regulatory sequences can also be enhancer
  • Sequences or upstream activator sequences include.
  • the tumor-associated antigens shown here can be combined with any expression control sequences and promoters.
  • the promoters of the tumor-associated gene products shown here can be combined with any other genes. This allows the selective activity of these promoters to be used.
  • a nucleic acid can be present in connection with another nucleic acid which codes for a polypeptide which controls secretion of the protein or polypeptide encoded by the nucleic acid from a host cell.
  • a nucleic acid can also be present in conjunction with another nucleic acid which codes for a polypeptide which anchors the coded protein or polypeptide on the cell membrane of the host cell or its compartmentalization in certain organelles of this cell.
  • a recombinant DNA molecule according to the invention is a vector, optionally with a promoter, which controls the expression of a nucleic acid, for example a nucleic acid which codes for a tumor-associated antigen according to the invention.
  • vector is used in its most general meaning and encompasses any intermediate vehicles for a nucleic acid which, for example, make it possible to introduce the nucleic acid into prokaryotic and / or into eukaryotic cells and, if appropriate, to integrate them into a genome. Such vectors are preferably replicated and / or expressed in the cell.
  • An intermediate vehicle can be adapted, for example, for use in electroporation, in microprojectile bombardment, in liposomal administration, in transfer with the aid of agrobacteria or in the insertion via DNA or RNA viruses.
  • Vectors include plasmids, phagemids, or virus genomes.
  • Mammalian cells such as cells from humans, mice, hamsters, pigs, goats and primates are particularly preferred.
  • the cells can be derived from a variety of tissue types and include primary cells and cell lines. Specific examples include keratinocytes, peripheral blood leukocytes, bone marrow stem cells and embryonic stem cells.
  • the host cell is an antigen-presenting cell, in particular a dendritic cell, monocyte or a macrophage.
  • a nucleic acid can be present in the host cell in a single or in multiple copies and, in one embodiment, is expressed in the host cell.
  • RNA or of RNA and protein are preferred expression systems in mammalian cells. It also includes partial expression of nucleic acids. Furthermore, the expression can be transient or stable.
  • Preferred expression systems in mammalian cells include pcDNA3.1 and pRc / CMV (Invitrogen, Carlsbad, CA), which contain a selectable marker such as a gene which confers resistance to G418 (and thus enables selection of stably transfected cell lines) and the enhancer-promoter sequences of cytomegalovirus (CMV).
  • CMV cytomegalovirus
  • an expression vector can also comprise a nucleic acid sequence which codes for the HLA molecule.
  • the nucleic acid sequence encoding the HLA molecule can be on the same expression vector as the nucleic acid encoding the tumor-associated antigen or part thereof, or both nucleic acids can be on different expression vectors. In the latter case, the two expression vectors can be cotransfected into one cell. If a host cell does not express the tumor-associated antigen or part of it or the HLA molecule, both nucleic acids encoding it are transfected into the cell either on the same expression vector or on different expression vectors. If the cell already expresses the HLA molecule, only the nucleic acid sequence that codes for the tumor-associated antigen or part thereof can be transfected into the cell.
  • kits for amplifying a nucleic acid which codes for a tumor-associated antigen include, for example, a pair of amplification primers that hybridize to the nucleic acid encoding the tumor associated antigen.
  • the primers preferably comprise a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides from the nucleic acid and are non-overlapping in order to avoid the formation of primer dimers.
  • One of the primers will hybridize to a strand of the nucleic acid encoding the tumor associated antigen and the other primer will hybridize to the complementary strand in an arrangement that allows amplification of the nucleic acid encoding the tumor associated antigen ,
  • an “antisense molecule” also comprises a construct which contains a nucleic acid or a part thereof in a reverse orientation with respect to its natural promoter.
  • An antisense transcript of a nucleic acid or a portion thereof can duplex with the naturally occurring mRNA that specifies the enzyme, thus preventing accumulation or translation of the mRNA into the active enzyme.
  • Another option is to use ribozymes to inactivate a nucleic acid.
  • Preferred antisense oligonucleotides according to the invention have a sequence of 6-50, in particular 10-30, 15-30 and 20-30 contiguous nucleotides from the target nucleic acid and are preferably completely complementary to the target nucleic acid or a part thereof.
  • the antisense oligonucleotide hybridizes to an N-terminal or 5 'upstream site such as a translation initiation, transcription initiation or promoter site. In other embodiments, the antisense oligonucleotide hybridizes to a 3 'untranslated region or mRNA splicing site.
  • an oligonucleotide according to the invention is a "modified" oligonucleotide.
  • the oligonucleotide can be modified in various ways in order to increase its stability or therapeutic effectiveness, for example, without impairing its ability to bind to its target.
  • modified oligonucleotide means an oligonucleotide in which (i) at least two of its nucleotides are linked to one another by a synthetic internucleoside bond (ie an internucleoside bond which is not a phosphodiester bond) and / or (ii) a chemical group is covalently linked to the oligonucleotide that does not normally occur with nucleic acids.
  • Preferred synthetic Internucleoside bonds are phosphorothioates, alkylphosphonates, phosphorodithioates, phosphate esters, alkylphosphonothioates, phosphoramidates, carbamates, carbonates, phosphate triesters, acetamidates, carboxymethyl esters and peptides.
  • isolated protein or isolated polypeptide
  • isolated polypeptide mean that the protein or polypeptide is separated from its natural environment.
  • An isolated protein or polypeptide can be in a substantially purified state.
  • substantially purified means that the protein or polypeptide is essentially free of other substances with which it is present in nature or in vivo.
  • proteins and polypeptides are used, for example, for the production of antibodies and can be used in an immunological or diagnostic assay or as therapeutic agents.
  • Proteins and polypeptides described according to the invention can be isolated from biological samples such as tissue or cell homogenates and can also be expressed recombinantly in a large number of pro- or eukaryotic expression systems.
  • Amino acid insertion variants include amino- and / or carboxy-terminal fusions, as well as insertions of single or several amino acids in a certain amino acid sequence.
  • amino acid sequence variants with an insertion one or more amino acid residues are introduced into a predetermined position in an amino acid sequence, although a random insertion with suitable screening of the resulting one Product is also possible.
  • Amino acid deletion variants are characterized by the removal of one or more amino acids from the sequence.
  • Amino acid substitution variants are characterized in that at least one residue in the sequence is removed and another residue is inserted in its place. The modifications are preferably at positions in the amino acid sequence that are not conserved between homologous proteins or polypeptides.
  • amino acids are replaced by others with similar properties, such as hydrophobicity, hydrophilicity, electronegativity, volume of the side chain and the like (conservative substitution).
  • Conservative substitutions relate, for example, to the replacement of an amino acid by another amino acid, listed below in the same group as the substituted amino acid:
  • amino acid variants described above can easily be obtained using known peptide synthesis techniques such as e.g. by “Solid Phase Synthesis” (Merrifield, 1964) and similar methods or by recombinant DNA manipulation. Techniques for introducing substitution mutations at predetermined locations in DNA that have a known or partially known sequence are well known and include, for example, M13 The manipulation of DNA sequences for the production of proteins with substitutions, insertions or deletions is described in detail, for example, in Sambrook et al. (1989).
  • “derivatives” of proteins or polypeptides also include single or multiple substitutions, deletions and / or additions of any molecules which are associated with the enzyme, such as carbohydrates, lipids and / or proteins or polypeptides. Furthermore the term “derivative” also extends to all functional chemical equivalents of the proteins or polypeptides.
  • Preferred parts or fragments of a tumor-associated antigen according to the invention preferably have one of the sequences listed below, which are derived from the tumor-associated antigens identified according to the invention, and are preferably peptide epitopes which, according to the invention, in particular also for stimulating cytotoxic T-lymphocytes in vivo are suitable for the production of expanded and stimulated T lymphocytes for therapeutic adoptive transfer ex vivo.
  • sequences are in particular peptide epitopes for the MHC class I alleles given below:
  • a part or a fragment of a nucleic acid which codes for a tumor-associated antigen relates to the part of the nucleic acid which codes at least for the tumor-associated antigen and / or codes for a part or a fragment of the tumor-associated antigen as defined above.
  • the isolation and identification of genes coding for tumor-associated antigens also enables the diagnosis of a disease which is characterized by the expression of one or more tumor-associated antigens.
  • These methods include determining one or more nucleic acids encoding a tumor associated antigen and / or determining the encoded tumor associated antigens and / or peptides derived therefrom.
  • the nucleic acid can be determined in a conventional manner, including by polymerase chain reaction or hybridization with a labeled probe.
  • a determination of tumor-associated antigens or peptides derived therefrom can be carried out by screening patient antisera with regard to recognition of the antigen and / or the peptides. It can also be done by screening the patient's T cells for specificity for the corresponding tumor-associated antigen.
  • the present invention also enables isolation of proteins that bind to tumor-associated antigens described herein, including antibodies and cellular binding partners of the tumor-associated antigens.
  • dominant negative polypeptides are also provided in certain embodiments, which are derived from tumor-associated antigens.
  • a dominant negative polypeptide is an inactive variant of a protein that, by interacting with the cellular machinery, displaces an active protein from its interaction with the cellular machinery or competes with the active protein, thereby reducing the activity of the active protein.
  • a dominant negative receptor that binds a ligand but does not produce a signal in response to ligand binding can reduce the biological activity of the ligand.
  • a dominant negative catalytically inactive kinase that normally interacts with target proteins but does not phosphorylate the target proteins can decrease the phosphorylation of the target proteins in response to a cellular signal.
  • a dominant negative transcription factor that binds to a promoter site in the control region of a gene can however, the transcription of the gene does not increase, reducing the effect of a normal transcription factor by occupying promoter binding sites without increasing transcription.
  • the result of the expression of a dominant negative polypeptide in a cell is a decrease in the function of active proteins.
  • the person skilled in the art can produce dominant negative variants of a protein, for example by conventional mutagenesis methods and evaluating the dominant negative effect of the variant polypeptide.
  • the invention also includes substances such as polypeptides that bind to tumor-associated antigens.
  • binders can e.g. in screening assays for the detection of tumor-associated antigens and complexes of tumor-associated antigens with their binding partners as well as in the purification of the tumor-associated antigens and of complexes thereof with their binding partners.
  • substances can also be used for inhibiting the activity of tumor-associated antigens, for example by binding to such antigens.
  • binding agents such as e.g. Antibodies or antibody fragments that have the ability to selectively bind to tumor-associated antigens.
  • Antibodies include polyclonal and monoclonal antibodies that are made in a conventional manner.
  • 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 Fc region was cleaved enzymatically or which was produced without the Fc region referred to as a Fab fragment
  • Fab fragments consist of a covalently bound light chain of an antibody and part of the heavy chain of Antibodies, referred to as Fd.
  • the Fd fragments are the main determinants of antibody specificity (a single Fd fragment can be associated with up to ten different light chains without changing the specificity of the antibody) and Fd fragments retain isolation the ability to bind to an epitope.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • Both the Fd fragment of the heavy chain and the light chain of IgG immunoglobulins contain four framework regions (FR1 to FR4), which are each separated by three complementarity-determining regions (CDR1 to CDR3).
  • CDRs and in particular the CDR3 regions and even more the CDR3 region of the heavy chain are largely responsible for the antibody specificity.
  • non-CDR regions of a mammalian antibody can be replaced by similar regions of antibodies with the same or a different specificity, while maintaining the specificity for the epitope of the original antibody.
  • WO 92/04381 describes the production and use of humanized RSV antibodies from mouse, in which at least some of the FR regions from mouse have been replaced by FR regions of human origin. Such antibodies, including fragments of intact antibodies with an antigen binding ability, are often referred to as "chimeric" antibodies.
  • An antibody used according to the invention is preferably directed against one of the sequences shown in SEQ ID NO: 14-18, 80-84 or 101-116 of the sequence listing and / or can be obtained by immunization with these peptides.
  • the "yeast two-hybrid system” can also be used to identify polypeptides that bind to a tumor-associated antigen.
  • Tumor-associated antigens or fragments thereof described in accordance with the invention can be used for screening peptide libraries, including phage display libraries, to identify and select peptide binding partners of the tumor-associated antigens.
  • Such molecules can be used, for example, for screening assays, purification protocols, for a Interference with the function of the tumor associated antigen and for other purposes known to those skilled in the art.
  • antibodies and other binding molecules can be used, for example, for the identification of tissue which expresses a tumor-associated antigen.
  • Antibodies can also be coupled to specific diagnostic agents for imaging cells and tissues that express tumor-associated antigens. They can also be coupled to therapeutically useful substances.
  • Diagnostic agents include, but are not limited to, barium sulfate, iocetamic acid, lopanoic acid, calcium ipodate, sodium diatrizoate, meglumine diatrizoate, metrizamide, sodium tyropanoate and radiodiagnostics, including positron emitters such as fluorine-18 and carbon-11, gamma emitters such as Iodine-123, Technitium-99m, Iodine-131 and Indium-111, nuclear magnetic resonance nuclides such as fluorine and gadolinium.
  • positron emitters such as fluorine-18 and carbon-11
  • gamma emitters such as Iodine-123, Technitium-99m, Iodine-131 and Indium-111
  • nuclear magnetic resonance nuclides such as fluorine and gadolinium.
  • therapeutically useful substance means any therapeutic molecule which, if desired, is selectively guided to a cell which expresses one or more tumor-associated antigens, including anti-cancer agents, radioactive iodinated compounds, toxins, cytostatic or cytolytic drugs, etc.
  • anti-cancer agents for example, aminoglutethimide, azathioprine, bleomycin sulfate, busulfan, carmustine, chlorambucil, cisplatin, cyclophosphamide, cyclosporine, Cytarabidin, dacarbazine, dactinomycin, Daunorubin, doxorubicin, taxol, etoposide, fluorouracil, interferon- ⁇ , lomustine, mercaptopurine, methotrexate, mitotane, Procarbazin- HCl, thioguanine, vinblastine sulfate and vincristine sulfate
  • Other anticancer agents are described, for example, in Goodman and Gilman, "The Pharmacological Basis ' of Therapeutics", 8th edition, 1990, McGraw-Hull, Inc., in particular chapter 52 (Antineoplastic Agents (Paul Calabresi and Bruce A.
  • Toxins k can be proteins such as pokeweed antiviral protein, cholera toxin, pertussis toxin, ricin, gelonin, abrin, diphtheria exotoxin or Pseudomonas exotoxin.
  • Toxin residues can also be high energy emitting radionuclides such as cobalt-60.
  • TPTE tumor-associated antigens according to the invention can themselves transport substances binding to them, in particular the therapeutic antibodies described above, from the membrane into the cytoplasm and thus serve an internalization, where these substances can preferably develop their effect like a cell-destroying effect.
  • the term “patient” means human, not human primacy or another animal, in particular mammal such as cow, horse, pig, sheep, goat, dog, cat or rodent such as mouse and rat.
  • the patient is a human.
  • the term “disease” relates to any pathological condition in which tumor-associated antigens are expressed or abnormally expressed.
  • abnormal expression means that the expression is changed, preferably increased, compared to the condition in a healthy individual.
  • An increase in expression relates to an increase of at least 10%, in particular at least 20%, at least 50% or at least 100%)
  • the tumor associated antigen is only expressed in tissue of a diseased individual while expression is repressed in a healthy individual, an example of such a disease is cancer, especially seminomas, melanomas, teratomas, gliomas, colorectal, breast, prostate -, uterine, ovarian, and lung cancer.
  • a biological sample may be a tissue and / or cellular sample in accordance with the invention and may be obtained for use in the various methods described herein in a conventional manner, such as by tissue biopsy, including punch biopsy, and by drawing blood, bronchial aspirate, urine, faeces or other body fluids.
  • the term “immunoreactive cell” means a cell which can mature into an immune cell (such as a B cell, T helper cell or cytolytic T cell) with suitable stimulation.
  • Immunoreactive cells include CD34 + hematopoietic stem cells, immature and mature T cells, and immature and mature B cells. If the production of cytolytic or helper T cells recognizing a tumor associated antigen is desired, the immunoreactive cell is contacted with a cell expressing a tumor associated antigen under conditions that allow for production, differentiation and / or selection of cytolytic as well as helper T cells favor.
  • the Differentiation of T cell precursors into a cytolytic T cell upon exposure to an antigen is similar to clonal selection of the immune system.
  • Some therapeutic methods rely on a patient's immune system response that leads to lysis of antigen-presenting cells, such as cancer cells that present one or more tumor-associated antigens.
  • antigen-presenting cells such as cancer cells that present one or more tumor-associated antigens.
  • autologous cytotoxic T lymphocytes which are specific for 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 method for differentiating T cells can be found in WO-A-9633265.
  • a sample with cells such as blood cells is taken from the patient and the cells are brought into contact with a cell which presents the complex and can trigger an increase in cytotoxic T-lymphocytes (eg dendritic cells).
  • Another method for selecting antigen-specific cytotoxic T lymphocytes uses fluorogenic tetramers of MHC class I molecule / peptide complexes for the detection of 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 folded in vitro in the presence of ⁇ 2 microglobulin and a peptide antigen that binds to the class I molecule. After the MHC / peptide complexes have been purified, they are labeled with biotin.
  • Tetramers are formed by mixing the biotinylated peptide-MHC complexes with labeled avidin (eg phycoerythrin) at a molar ratio of 4: 1. Tetramers are then contacted with cytotoxic T lymphocytes such as peripheral blood or lymph nodes. The tetramers bind to cytotoxic T lymphocytes that recognize the peptide-antigen / MHC class I complex. Cells that bind to the tetramers can be sorted by fluorescence-controlled cell sorting for isolation of reactive cytotoxic T lymphocytes. The isolated cytotoxic T lymphocytes can then be grown in vitro.
  • cytotoxic T lymphocytes such as peripheral blood or lymph nodes.
  • Cells that bind to the tetramers can be sorted by fluorescence-controlled cell sorting for isolation of reactive cytotoxic T lymphocytes. The isolated cytotoxic T lymphocytes can then be grown in vitro.
  • T cell receptor itself.
  • cells that present the desired complex e.g. dendritic cells
  • cytotoxic T lymphocytes from healthy individuals. This leads to an increase in high-affinity specific cytotoxic T lymphocytes if the donor has not had any contact with the specific complex.
  • the high affinity T cell receptor from these increased specific T lymphocytes is cloned and can be transferred by gene transfer e.g. with retroviral vectors can be transduced as desired in T cells from other patients.
  • the above therapeutic aspects assume that at least some of the patient's abnormal cells present a complex of a tumor-associated antigen and an HLA molecule. Such cells can be identified in a manner known per se. 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 presented.
  • Cytotoxic T lymphocytes can also be generated in vivo in a manner known per se become.
  • One method 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 that have been transfected with one or both of the genes necessary for presentation of the complex (ie, the antigen peptide and the presenting HLA -Molecule). Different cell types can be used.
  • Vectors can also be used which carry one or both of the genes of interest. Viral or bacterial vectors are particularly preferred.
  • nucleic acids encoding a portion of a tumor-associated antigen can be operably linked to promoter and enhancer sequences that direct expression of the tumor-associated antigen or a fragment thereof in certain tissues or cell types.
  • the nucleic acid can be incorporated into an expression vector.
  • Expression vectors can be unmodified extrachromosomal nucleic acids, plasmids or viral genomes into which an insertion of exogenous nucleic acids is possible.
  • Nucleic acids encoding a tumor associated antigen can also be inserted into a retroviral genome, thereby enabling the nucleic acid to be integrated into the genome of the target tissue or cell.
  • a microorganism such as vaccinia virus, pox virus, He ⁇ es simplex virus, retrovirus or adenovirus carries the gene of interest and "infects" de facto host cells.
  • Another preferred form is the introduction of the tumor-associated antigen in the form of recombinant RNA. This can be introduced into cells, for example, by liposomal transfer or by electroporation. The resulting cells present the complex of interest and are recognized by autologous cytotoxic T lymphocytes, which then multiply.
  • an effective amount of the tumor-associated antigen can be administered to a patient, for example, by intradermal injection.
  • the injection can also take place intranodally in a lymph node (Maloy et al, Proc Natl Acad Sei USA 98: 3299-303, 2001). It can also be used in combination with reagents that facilitate absorption into dendritic cells.
  • An in vivo preferred tumor associated antigens include those that react with allogeneic cancer antisera or with T cells from many cancer patients. Of particular interest are those against which there are no spontaneous immune responses.
  • Immune responses to these can be demonstrably induced, which can lyse tumors (Keogh et al, J Immunol 167: 787-96, 2001; Appella et al, Biomed Pept Proteins Nucleic Acids 1: 177-84, 1995; Wentworth et al, Mol Immunol 32: 603-12, 1995).
  • one or more tumor-associated antigens or stimulating fragments thereof with one or more adjuvants for inducing an immune response or increasing an immune response are administered.
  • An adjuvant is a substance that is incorporated into, or co-administered with, the antigen that enhances the immune response.
  • Adjuvants can enhance the immune response by providing an antigen reservoir (extracellular or in macrophages), activating macrophages and stimulating certain lymphocytes.
  • Adjuvants include, but are not limited to, monophosphoryl lipid-A (MPL, SmithKline Beecham), saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739), QS7, QS17, QS18 and QS-Ll (So et al, Mol. Cells 7: 178-186, 1997), incomplete Freund's adjuvant, complete Feundsch adjuvant, vitamin E.
  • MPL monophosphoryl lipid-A
  • saponins such as QS21 (SmithKline Beecham), DQS21 (SmithKline Beecham; WO 96/33739)
  • QS7, QS17, QS18 and QS-Ll So et al, Mol. Cells 7: 178-186, 1997)
  • incomplete Freund's adjuvant complete Feundsch adjuvant, vitamin E.
  • the peptides are preferably administered in a mixture with DQS21 / MPL.
  • the ratio of DQS21 to MPL is typically about 1:10 to 10: 1, preferably about 1: 5 to 5: 1 and in particular about 1: 1.
  • DQS21 and MPL are typically present in a vaccine formulation in a range from about 1 ⁇ g to about 100 ⁇ g.
  • cytokines can be used in vaccination due to their regulatory properties on lymphocytes.
  • cytokines include e.g. 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.
  • B7 is typically not expressed on tumor cells, so they are not effective antigen presenting cells (APCs) for T cells. Induction of B7 expression would allow tumor cells to more effectively stimulate cytotoxic T lymphocyte proliferation and effector function. Co-stimulation by a combination of B7 / IL-6 / IL-12 showed induction of IFN-gamma- and Thl-cytokine Profiles in a T cell population, which leads to a further increased T cell activity (Gajewski et al, J. Immunol 154: 5637-5648 (1995)).
  • anti-CD40 antibodies for stimulation of dendritic cells would, as expected, directly increase a response to tumor antigens that are normally 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 antigen-pulsed dendritic cells or in situations where T helper epitopes were not defined in known TRA precursors.
  • a viral vector for the administration of a nucleic acid encoding a tumor-associated antigen is selected from the group consisting of adenoviruses, adeno-associated viruses, poxviruses, including vaccinia virus and attenuated poxviruses, Semliki Forest virus, Retroviruses, Sindbis virus and Ty virus-like particles.
  • Adenoviruses and retroviruses are particularly preferred.
  • the retroviruses are usually replication-deficient (i.e. they are unable to produce infectious particles).
  • nucleic acids can be used to introduce nucleic acids into cells according to the invention in vitro or in vivo. Such methods include transfection of nucleic acid CaPO 4 precipitates, transfection of nucleic acids associated with DEAE, transfection or infection with the above viruses that carry the nucleic acids of interest, liposome-mediated transfection, and the like. In certain embodiments, control of the nucleic acid to certain cells is preferred.
  • a carrier used to deliver a nucleic acid to a cell e.g., a retrovirus or a liposome
  • the therapeutic agents of the invention can be administered by any conventional route, including by injection or infusion.
  • the administration can take place, for example, orally, intravenously, intraperitoneally, intramuscularly, subcutaneously or transdermally.
  • Therapeutic administration of antibodies is preferably carried out through a pulmonary aerosol.
  • Antisense nucleic acids are preferably administered by slow intravenous administration.
  • compositions according to 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, the duration of the
  • compositions according to the invention are preferably sterile and contain an effective amount of the therapeutically active substance for producing the desired reaction or the desired effect.
  • doses of the tumor-associated antigen from 1 ng to 1 mg, preferably from 10 ng to 100 ⁇ g, are formulated and administered for treatment or for generating or increasing an immune response. If the administration of nucleic acids (DNA and RNA) which code for tumor-associated antigens is desired, doses of 1 ng to 0.1 mg are formulated and administered.
  • nucleic acids DNA and RNA
  • compositions according to the invention are generally administered in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions.
  • pharmaceutically acceptable refers to a non-toxic material that does not interfere with the action of the active ingredient in the pharmaceutical composition.
  • Such preparations can usually contain salts, buffering agents, preservatives, carriers and optionally other therapeutic agents.
  • the salts should be pharmaceutically acceptable.
  • non-pharmaceutically acceptable salts can be used for the production of pharmaceutically acceptable salts thereof and are included according to the invention.
  • a pharmaceutical composition according to the invention can comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier relates to one or more compatible solid or liquid fillers, diluents or capsule substances which are suitable for administration to a human.
  • carrier refers to an organic or inorganic ingredient, natural or synthetic, in which the active ingredient is combined to facilitate application.
  • the components of the pharmaceutical according to the invention Compositions are usually such that there is no interaction that significantly affects the desired pharmaceutical efficacy.
  • compositions according to the invention can contain suitable buffer substances such as acetic acid in a salt, citric acid in a salt, boric acid in a salt and phosphoric acid in a salt.
  • compositions are usually presented in a unitary dosage form and can be prepared in a manner known per se.
  • Pharmaceutical compositions according to the invention can be present, for example, in the form of capsules, tablets, lozenges, suspensions, syrups, elixirs or as an emulsion.
  • compositions suitable for parenteral administration usually comprise a sterile aqueous or non-aqueous preparation of the active ingredient, which is preferably isotonic with the blood of the recipient.
  • Compatible carriers and solvents are, for example, Ringer's solution and isotonic sodium chloride solution.
  • sterile, fixed oils are usually used as the solution or suspension medium.
  • Fig. 1 Schematic representation of the cloning of eCT.
  • Fig. 2 Splicing of LDH C. Alternative splicing events lead to the absence of exon 3 (SEQ ID NO: 2), the two exons 3 and 4 (SEQ ID NO: 3), exons 3, 6 and 7 (SEQ ID NO : 4) or exon 7 (SEQ ID NO: 5).
  • Fig. 3 Alignment of possible LDH-C proteins.
  • SEQ ID NO: 8 and SEQ ID NO: 10 represent franked portions of the prototype protein (SEQ ID NO: 6).
  • the protein sequences of SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: II, SEQ ID NO: 12 and SEQ ID NO: 13 have also been changed and only contain tumor-specific epitopes (printed in bold).
  • the catalytic center is framed.
  • Fig. 4 Quantification of LDH C in different tissues by real-time PCR.
  • Fig. 5 Exon composition of TPTE variants.
  • splice variants were identified (SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57), which are expressed in testicular tissue and tumors and have reading frame shifts and thus changed sequence areas.
  • Fig. 6 Alignment of the possible TPTE proteins. Alternative splicing events result in changes in the encoded protein, whereby the reading frame is basically preserved. The putative transmembrane domains are in bold, the catalytic domain is framed.
  • Fig. 8 Alignment of TSBP variants at the protein level. Reading frame shifts in the proteins which are encoded by the TSBP variants found according to the invention (SEQ ID NO: 34, SEQ ID NO: 35, SEQ ID NO: 36) lead to substantial differences from the protein described above (SEQ ID NO: 30, NM_006781) and are marked in bold.
  • Fig. 9 RT-PCR for MS4A12.
  • expression could only be detected in testis, colon and colorectal carcinoma (colon ca's).
  • colon ca's colon and colorectal carcinoma
  • Fig. 10 RT-PCR for BRCO1.
  • BRCO1 is significantly overexpressed in breast tumors compared to expression in normal mammary gland tissue.
  • Fig. 11 RT-PCR for MORC, TPX1, LDHC, SGY-1.
  • Examination of various normal tissues shows expression only in testis (1 skin, 2 small intestine, 3 colon, 4 liver, 5 lungs, 6 stomach, 7 breast, 8 kidney, 9 ovary, 10 prostate, 11 thyroid, 12 leukocytes, 13 thymus, 14 negative Control, 15 testis).
  • the examination of tumors (1-17 lung tumors, 18-29 melanomas, 30 negative controls, 31 testis) shows ectopic expression in these at different frequencies for the individual eCT.
  • MCF-7 cells were transiently transfected with an LDHC expression plasmid.
  • the antigen was detected with LDHC-specific antibodies and showed a clear colocalization with the mitochondrial respiratory chain enzyme cytochrome C oxidase.
  • Fig. 13 Predicted topology of TPTE and subcellular localization on the cell surface of MCF-7 cells.
  • the left scheme shows the 4 putative trot membrane domains (arrows) of TPTE.
  • MCF-7 cells were transiently transfected with a TPTE expression plasmid. The antigen was detected with TPTE-specific antibodies and showed a clear colocalization with MHC I molecules located on the cell surface.
  • Fig. 14 MS4A12 localization on the cell membrane. Tumor cells were transiently transfected with a GFP-tagged MS4A12 construct and showed complete colocalization with plasma membrane markers in confocal immunofluorescence microscopy.
  • Fig. 15 Western blot detection of LDHC in normal tissues.
  • the expression of LDHC is only detectable in Testis, all other normal tissues tested are negative.
  • 1- testis normal tissue, 2-skin normal tissue, 3-breast normal tissue, 4- liver normal tissue, 5-spleen normal tissue, 6-column normal tissue, 7-lung normal tissue, 8-kidney normal tissue, 9 -lymph node normal tissue
  • Fig. 16 Expression of LDHC in the HCT116 DKO cell line. HCT116 P and HCT116 DKO were stained with an LDHC-specific antibody. Endogenous LDHC is only detectable in HCT116 DKO cells.
  • Fig. 17 Mitochondrial localization of LDHC in the MCF-7 breast cancer cell line.
  • Fig. 18 Membrane localization of heterologously and endogenously expressed TPTE in cell lines.
  • (Left) NIH3T3 cells were transiently transfected with a TPTE expression plasmid.
  • TPTE was detected with specific antibodies and showed a clear colocalization with MHC I molecules located on the cell surface.
  • (Right) Endogenous TPTE in SK-Mel 37 cells was detected with a specific antibody and shows clear membrane localization.
  • Fig. 19 Quantification of TPTE mRNA expression in different tissues by real-time PCR. Expression in normal tissues is only detectable in Testis. Significant expression levels can also be found in tumors and tumor cell lines.
  • Fig. 20 Evidence of antibody specificity. Sections of testis tissue were stained with TPTE-specific antibodies. The specific detection of TPTE is prevented by blocking (right) the antibody with the peptide used for immunization.
  • Fig. 21 Immunohistochemical detection of TPTE in bronchial carcinomas. Sections of bronchial carcinomas were stained with TPTE-specific antibodies. TPTE is expressed homogeneously throughout the tumor and is located on the cell membrane.
  • Fig. 22 Detection of TPTE localization in vital cells.
  • NIH3T3 cells were transiently transfected with a TPTE expression plasmid and analyzed using time-lapse microscopy. The localization of TPTE on the membrane of cell protrusions and pseudoposias results in the immediate refraction of the membrane areas concerned.
  • TPTE enhances cell migration in chemotactic gradients.
  • (Left) Schematic representation of the Boyden chamber assay. NIH3T3 / c-erbB-2 cells were transfected with TPTE-eGFP and the migration of the cells was determined in comparison with non-transfected cells and cells transfected with empty pEGFP vector.
  • the expression of TPTE leads to a 4 to 5-fold increase in cell migration when using 10% FCS as a chemotactic.
  • the expression of TPTE leads to a significant increase in migration even at very low concentrations of PDGF.
  • Fig. 27 Western blot detection of MS4A12 in colon and colon carcinoma.
  • the specific band is normal colon tissue and is detectable in some colon carcinomas.
  • M-MagicMark Invitrogen
  • 1 -colon normal tissue 2-6-colon tumor tissue
  • Fig. 30 Quantification of BRCO1 mRNA expression in different tissues by real-time PCR.
  • the expression of BRCOl in normal tissues is restricted to the breast and testis.
  • Significant expression levels of BRCO1 are detectable in all breast cancers examined. 50% of the tumors show an overexpression of BRCOl in comparison with expressing normal tissues.
  • Fig. 31 Overexpression of BRCOl in breast cancer.
  • Real-time PCR analysis of BRCOl in breast cancer and the adjacent normal tissue shows an overexpression of BRCOl in 50% of breast cancer.
  • Fig. 32 Quantification of PCSC mRNA expression in different tissues by real-time PCR. The expression of PCSC in normal tissues is restricted to the colon, rectum and terminal ileum. Expression of PCSC is detectable in all colon carcinomas. 85% of colon carcinoma metastases show significant overexpression of PCSC compared to primary tumors.
  • Fig. 33 Evidence of the inducibility of TPTE through genomic demethylation.
  • Fig. 34 Western blot detection of TPTE in normal tissues and tumor cell lines.
  • the expression of LDHC is only detectable in testis and tumor cell lines (SK-Mel-37, LCLC-107, PC-3), all other normal tissues tested are negative.
  • the specificity of the antibody is confirmed by the detection of TPTE in transfected NIH3T3 cells (TPTE-pcDNA3.1).
  • Fig. 36 Knock-down of TPTE expression in PC-3 cells by RNAi.
  • PC-3 cells were electroporated with l ⁇ M siRNA specifically for TPTE. After 24 hours, mRNA expression was quantified using real-time RT-PCR. Non-electroporated cells and cells that were electroporated with DsRed siRNA served as a control.
  • Fig. 38 Region from position 121 to 540 of the sequence shown in SEQ ID NO: 822 of the sequence listing. Examples:
  • Candidate genes were extracted from the databases using search queries with the keywords "testis-specif -c gene”, “sperm-specific gene”, “spermatogonia-specific gene”. The search was limited to part of the total information in these databases by using “homo sapiens” for the organism and "mRNA” for the type of molecule.
  • the list of GOI found was curated by determining different names for the same sequence and eliminating such redundancies. All candidate genes, which resulted from the keyword search, were in turn examined by the "electronic Northern” (eNorthern) method with regard to their tissue distribution.
  • the eNorthern is based on the fact that the sequence of a GOI is compared with an EST (expressed sequence tag) database (Adams et al, Science 252: 1651, 1991) (http://www.ncbi.nlm.nih.gov/BLAST ).
  • ESTs achieve a preliminary assessment of the tissue distribution of the GOI. Only those GOI who had no homology to EST from normal non-testicular tissues with the exception of placenta and fetal tissue were followed up. For this assessment, it was also taken into account that there are false-annotated cDNA banks in the public domain (Scheurle et al., Cancer Res. 60: 4037-4043, 2000) (www.fau.edu/cmbb/publications/cancergenes6.htm ).
  • the cDNA xProfiler of the Cancer Genome Anatomy Project of the NCBI (http://cgap.nci.nih.gov/Tissues/xProfiler) was used as the second data mining method (Hillier et al., Genome Research 6: 807-828, 1996; Pennisi, Science 276: 1023-1024, 1997).
  • This allows pools of transcriptomes stored in databases to be related to one another by logical operators.
  • RNA extraction preparation of poly-d (T) primed cDNA and RT-PCR analysis.
  • Total RNA from native tissue material was extracted using guanidium isothiocyanate as a chaotrophic agent (Chomczynski & Sacchi, Anal. Biochem. 162: 156-9 , 1987). After extraction with acidic phenol and precipitation with isopropanol, the RNA was dissolved in DEPC-treated water.
  • a first-strand cDNA synthesis was carried out from 2-4 ⁇ g of total RNA in a 20 ⁇ l reaction mixture using Superscript II (Invitrogen) according to the manufacturer's instructions.
  • a dT (18) oligonucleotide was used as the primer.
  • LDHC LDHC was quantified using real-time PCR.
  • the principle of quantitative real-time PCR with the ABI PRISM Sequence Detection System uses the 5'-3 'exonuclease activity of Taq DNA polymerase for the direct and specific detection of PCR products by the release of fluorescence reporter dyes.
  • a double fluorescence-labeled probe (TaqMan probe) is used in the PCR, which hybridizes to a sequence of the PCR product.
  • the probe is labeled 5 'with a reporter dye (e.g. FAM) and 3' with a quencher dye (e.g. TAMRA).
  • TPTE, MS4A12, PCSC and BRCOl The expression of TPTE, MS4A12, PCSC and BRCOl was also quantified using real-time PCR, but the PCR products were used with SYBR-Green as a reporter dye detected.
  • the reporter fluorescence of SYBR-Green is suppressed in solution and the dye is only active after binding to double-stranded DNA fragments.
  • the increase in SYBR-Green fluorescence as a result of the specific amplification using GOI-specific primers after each PCR cycle is used for the quantification.
  • the expression quantification of the target gene takes place absolutely or relative to the expression of a control gene with constant expression in the tissues to be examined.
  • LDHC Low Capacity cDNA Archive Kit
  • MS4A12 (65 °) sense CTGTGTCAGCATCCAAGGAGC, antisense TTCACCTTTGCCAGCATGTAG
  • PCSC (59 °) sense AGAATAGAATGTGGCCTCTAG, antisense TGCTCTTACTCCAAAAAGATG
  • the membrane is first blocked (e.g. with milk powder) and then incubated with the specific antibody at a dilution of 1: 20-1: 200 (depending on the specificity of the antibody) for 60 minutes.
  • the membrane is incubated with a second antibody coupled with a marker (e.g. enzymes such as peroxidase or alkaline phosphatase), which recognizes the first antibody.
  • a marker e.g. enzymes such as peroxidase or alkaline phosphatase
  • the target protein is then made visible on the membrane in a staining or chennuminescence reaction using an enzyme reaction (e.g. ECL, Amersham Bioscience). The result is documented by recording with a suitable camera.
  • Cells of established cell lines are used which either endogenously synthesize the target protein (detection of the RNA in RT-PCR or the protein in Western blot) or have been transfected with plasmid DNA before the IF.
  • Various methods eg electroporation, liposome-based transfection, calcium phosphate precipitation
  • the transfected plasmid can encode the unmodified protein or else can couple different amino acid markers to the target protein.
  • the most important markers are, for example, the fluorescent "green fluorescent protein” (GFP) in its differentially fluorescent forms, short peptide sequences of 6-12 amino acids, for which highly affine and specific antibodies are available.
  • GFP fluorescent "green fluorescent protein”
  • Cells that synthesize the target protein are included paraformaldehyde; Saponin or methanol fixed. If necessary, the cells can then be permeabilized by incubation with detergents (eg 0.2% Triton X-100). After fixation / permeabilization, the cells are incubated with a primary antibody which is directed against the target protein or against one of the coupled markers.
  • the mixture is incubated with a second antibody, which is coupled to a fluorescent marker (eg fluorescin, Texas Red, Dako) and which binds to the first antibody.
  • a fluorescent marker eg fluorescin, Texas Red, Dako
  • the cells marked in this way are then overlaid with glycerol and analyzed with the aid of a fluorescence microscope according to the manufacturer's instructions. Specific fluorescence emissions are achieved by specific excitation, depending on the substances used.
  • the analysis generally allows the target protein to be localized reliably, with the coupled amino acid markers or other brand proteins being stained in addition to the target protein in order to confirm the antibody quality and the target protein in double stains, the localization of which has already been described in the literature.
  • a special case is the GFP and its derivatives, which can be excited directly and fluoresce themselves, so that no antibodies are required for detection.
  • Tissue pieces with a thickness of approx. 4 ⁇ m fixed in formalin (other fixations: e.g. methanol) and embedded in paraffin are applied to a glass support and deparaffinized with xylene, for example.
  • the samples are washed with TBS-T and blocked in serum.
  • the incubation is then carried out with the first antibody (dilution: 1: 2 to 1: 2000) for 1-18 hours, with affinity-purified antibodies being used as a rule.
  • affinity-purified antibodies being used as a rule.
  • a second antibody coupled with alkaline phosphatase alternatively: eg peroxidase
  • a color reaction then takes place using the alkaline phosphatase (references: Shi et al. J Histochem Cytochem 1991 39: 741-748; Shin et al. Lab Invest. 1991 64: 693-702).
  • the reaction can be blocked by adding the immunogen beforehand.
  • the animals are usually immunized using one of four well-established methods, although other methods also exist. Immunization can be carried out with peptides that are specific for the target protein, the entire protein, with extracellular partial sequences of a protein that can be identified experimentally or via prediction programs.
  • peptides conjugated to KLH keyhole limpet hemocyanin
  • length: 8-12 amino acids are synthesized using a standardized in vitro method and these peptides are used for immunization.
  • 3 immunizations are carried out with a concentration of 5-1000 ⁇ g / immunization.
  • the immunization can also be carried out as a service by service providers.
  • Proteins can also be used for the immunization which have a molecular anchor as an aid for cleaning (for example His-Tag, Qiagen; FLAG day, Röche Diagnostics; Gst fusion proteins).
  • a molecular anchor as an aid for cleaning
  • a large number of protocols can be found, for example, in the “Current Protocols in Molecular Biology (John Wiley & Sons Ltd, Wiley InterScience).
  • a cell line is available which endogenously synthesizes the desired protein, this cell line can also be used to produce the specific antiserum.
  • the immunization is carried out in 1-3 injections, each with approx. 1-5 x 10 7 cells.
  • the immunization can also be carried out by injection of DNA (DNA immunization).
  • the target gene is first cloned into an expression vector so that the target sequence is under the control of a strong eukaryotic promoter (eg CMV promoter). Subsequently, 5-100 ⁇ g of DNA are transferred as immunogen with a “gene gun” into highly perfused, capillary areas of an organism (eg mouse, rabbit). The transferred DNA is taken up by cells of the animal, the target gene is expressed and the animal finally develops an immune response against the target gene (Jung et al, Mol Cells 12: 41-49, 2001; Kasinrerk et a., Hybrid Hybridomics 21: 287-293, 2002).
  • a strong eukaryotic promoter eg CMV promoter
  • the purification of the polyclonal sera was carried out entirely in the case of the peptide antibodies or partially in the case of the antibodies against recombinant proteins as a service by the contracted companies.
  • the corresponding peptide or recombinant protein was covalently bound to a matrix, this was equilibrated after coupling with a native buffer (PBS: phosphate buffered saline) and then incubated with the raw serum. After a further washing step 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.
  • the antibodies thus purified could then be used for the specific detection of the target proteins both by Western blotting and by immunofluorescence.
  • the Boyden Chamber is used to quantify cell migration in response to chemotactic stimuli.
  • the chamber consists of two compartments separated by a micropore membrane.
  • the cells to be examined are placed in minimal medium in the upper compartment, while the lower compartment is filled with medium which contains the corresponding chemotactic.
  • the cells move according to the gradient through the membrane and adhering to the underside of the membrane. After fixation, the transmigrated cells can be counted under the microscope.
  • the migration assay was performed to determine the promigratory potential of TPTE.
  • NIH3T3 fibroblasts transfected with TPTE-eGFP, c-erbB2 transformed were used.
  • Non-transfected cells and cells transfected with eGFP-N3 empty vector were used as control cells.
  • Transwell chambers (Becton Dickinson) with a pore size of 8.0 ⁇ M in the membrane were used for the assay.
  • 4 ⁇ 10 4 cells in 400 ⁇ l of serum-free DMEM medium were added to the upper compartment.
  • the lower compartment was filled with 800 ⁇ l DMEM medium, to which 10% FCS or PDGF-BB was added in increasing concentrations (10-300ng / ⁇ l).
  • the chambers were incubated at 37 ° for 40 h.
  • the transmigrated cells were then fixed in ice-cold methanol, the membranes were cut out and covered on slides with Hoechst core dye (DAKO) for fluorescence microscopy. Cells in five visual fields (20x magnification) per membrane were counted. All experiments were carried out in triplicate.
  • DAKO Hoechst core dye
  • RNA interference RNA interference
  • 5x10 6 cells were taken up in 250 ⁇ l serum-free X-VIVO 15 medium and electroporated with l ⁇ M of the corresponding siRNA duplexes (200V, 250 ⁇ F).
  • the TPTE mRNA expression was quantified 24 hours later using real-time RT-PCR. methylation Analysis
  • TPTE-negative cell lines BT549 mimma-Ca
  • HCTl 16 colon-Ca
  • the TPTE expression was then quantified using real-time RT-PCR.
  • DNMT DNA methyl transferase
  • DNMT1 HCT116 DNMTW -
  • DNMT3b H CT116 DNMT3W -
  • TPTE-eGFP transfected cells were incubated for 12 h in serum-free DMEM medium.
  • FCS serum-free DMEM medium.
  • pseudopodia and filopodia the cells were stimulated by adding FCS before the analysis. Images of the vital cells were taken at 30sec intervals with a üivert Olympus microscope (1X70) with a TILL IMAGO-VGA CCD camera.
  • TPTE immunofluorescence microscopy of heterologously expressed TPTE
  • the complete ORF of TPTE was cloned into pEGFP-Cl and pEGFP-N3 vectors (Clontech).
  • LDH C (SEQ ID NO: 1) and its translation product (SEQ ID NO: 6) have been described as the testis-specific isoenzyme of the lactate dehydrogenase family.
  • the sequence is published in GenBank under accession number NM_017448.
  • the enzyme forms a homotetramer with a molecular weight of 140 kDa (Goldberg, E. et al, Contraception 64 (2): 93-8, 2001; Cooker et al, Biol. Reprod. 48 (6): 1309-19, 1993; Gupta, GS, Grit. Rev. Biochem. Mol. Biol. 34 (6): 361-85, 1999).
  • the expected size of the amphication product with the above-mentioned PCR primers is 824 bp. According to the invention, however, the amplification of multiple additional bands was observed in tumors, but not in testis. Since this is indicative of the presence of alternative splice variants, the entire open reading frame was amplified with LDH-C specific primers (5'-TAGCGCCTCAACTGTCGTTGG-3 ', 5'-CAACATCTGAGACACCATTCC-3') and independent full-length clones were sequenced. Alignments with the prototype ORF of the described sequence of LDH C (SEQ ID NO: 1) and the genomic sequence on chromosome 11 confirm additional splice variants (SEQ ID NO: 2-5).
  • SEQ ID NO: 14 GAVGMACAISILLKITVYLQTPE faus SEQ ID NO: 7
  • SEQ ID NO: 15 GAVGMACAISILLKWJF (from SEQ ID NO: 9)
  • SEQ ID NO: 16 GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand SEQ ID NO: 16: GWIIGEHGDSSGIIWNKRRTLSQYPLCLGAEWCLRCCEN Thousand
  • SEQ ID NO: 17 MVGLLENMVILVGLYGIKEELFL (from SEQ ID NO: 12)
  • SEQ ID NO: 18 EHWKNIHKOVIORDYME thousand SEQ ID NO: 13)
  • expression levels were quantified by real-time PCR using a specific primer probe set.
  • the amplicon lies in the transition between exon 1 and 2 and thus detects all variants (SEQ ID NO: 1-5). Detect these tests too no transcripts in normal tissues other than testis. They confirm significant expression levels in tumors (Fig. 4).
  • the data should be presented for antibodies that are directed against SEQ ID NO: 80. These recognize LDHC protein in Testis tissue but not in other normal tissues in the Western blot examination (Fig. 15) and thus confirm the RT-PCR data at the transcript level.
  • the specific antibody can also be used under various fixation conditions for immunofluorescence studies. RT-PCR studies showed that the colon carcinoma cell line HCT 116 P does not express LDHC. However, their DNA methyltransferase deleted variant HCT 116 DKO is positive for LDHC. Comparative staining of both cell lines with the above-mentioned antibody specifically detected the corresponding protein in an easily detectable amount in the cell lines typed as positive (Fig. 16). Accordingly, this antibody also qualifies for immunohistochemical staining of normal and tumor tissue sections by the expert. The staining of testis tissue e.g. shows a clear positivity of germ cells (Fig. 16).
  • spermatids prefer to use the citrate cycle as the main source of energy (Storey BT Biol. Reprod. 16: 549-556, 1977). It has long been known that tumor cells often cover their energy requirements through anaerobic glycolysis, ie they increasingly produce lactate, and do not use the citrate cycle and the respiratory chain even under aerobic conditions. The molecular basis of this phenomenon, which is described as the Warburg effect, has not yet been elucidated. Caused by enzyme defects in the mitochondria and the overexpression of key enzymes in glycolysis.
  • sequences of the transcript of TPTE (SEQ ID NO: 19) and its translation product (SEQ ID NO: 22) are published in GenBank under accession number NM_013315 (Walker, SM et al., Biochem. J. 360 (Pt 2): 277 -83, 2001; Guipponi M. et al., Hum. Genet. 107 (2): 127-31, 2000; Chen H. et al, Hum. Genet. 105 (5): 399-409, 1999).
  • TPTE has been described as a gene coding for a possible transmembrane tyrosine phosphatase with Testis-specific expression that is located in the pericentromeric region of chromosomes 21, 13, 15, 22, and Y (Chen, H. et al., Hum. Genet 105: 399-409, 1999). Alignment studies according to the invention additionally show homologous genomic sequences on chromosomes 3 and 7.
  • the membrane localization e.g. A prerequisite for the accessibility of therapeutic antibodies could be proven beyond any doubt by transfection of TPTE-negative cells with a fusion construct of TPTE and green fluorescent protein (Fig. 18, right). This accumulates on the membrane surface and can be shown to be colocalizing with other known membrane markers such as HLA molecules.
  • PCR primers were generated according to the invention (5 * -TGGATGTCACTCTCATCCTTG-3 'and 5'-
  • TPTE variants are activated ectopically in a number of tumor tissues; see. Table 2.
  • SEQ ID NO: 105 MNESPDPTDLAGVIIELGPNDSPQTSEFKGATEEAPAKESPHTS EFKGAARVSP (rec. Prot. Aa 1-54)
  • Fig. 21 The localization of the protein on the membrane of the cells is also confirmed here (Fig. 21).
  • the specificity of the staining was confirmed by competition experiments.
  • the specific antibody was first pre-incubated with recombinant TPTE protein and then placed on the tissue sections (eg Testis as a positive control, Fig. 20). This can successfully block the reactivity.
  • This antibody can be used to successfully stain many different primary tumor types (including breast tumors, melanomas, etc.) but not the associated normal tissue.
  • a staining of prostate tissue is also shown as an example. Normal prostate tissue is negative, while invasive as well as not yet invaded prostate tumors are positive for this molecule.
  • TPTE TPTE
  • SEQ ID NO: 20 SEQ ID NO: 21, SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57
  • TPTE TPTE
  • the TPTE genomic sequence consists of 24 exons (accession number NT_029430).
  • the transcript shown in SEQ ID NO: 19 contains all of these exons.
  • the splice variant shown in SEQ ID NO: 20 results from splicing out exon 7.
  • the splicing variant shown in SEQ ID NO: 21 shows a partial incoherence of an intron that follows exon 15.
  • SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, exons 18, 19, 20 and 21 can alternatively be spliced out. These alternative splicing events result in changes in the encoded protein, whereby the reading frame is basically retained (Fig. 6).
  • the translation product encoded by the sequence shown in SEQ ID NO: 20 has a deletion of 13 amino acids compared to the sequence shown in SEQ ID NO: 22.
  • the translation product which is encoded by the sequence shown in SEQ ID NO: 21 (SEQ ID 24) carries an additional insertion in the central region of the molecule and thus differs from the other variants by 14 amino acids.
  • the translation products of the variants SEQ ID NO: 54, SEQ ID NO: 55, SEQ ID NO: 56, SEQ ID NO: 57, namely the proteins SEQ ID NO: 58, SEQ ID NO: 59, SEQ ID NO: 60, SEQ ID NO: 61 are also changed.
  • TPTE is localized with class I histocompatibility antigens on the cell surface of tumor cells.
  • TPTE had only been described as a Golgi-associated protein. Due to the expression of TPTE on the cell surface of tumor cells, this tumor antigen is suitable according to the invention as an excellent target for the development of diagnostic and therapeutic monoclonal antibodies. Because of the predicted Membrane topology of TPTE are particularly suitable for this purpose according to the invention in the extracellularly exposed areas.
  • this includes the peptides FTDSKLYIPLEYRS (SEQ ID NO: 81) and FDIKLLRNIPRWT (SEQ ID NO: 82). It was also shown that TPTE promotes the migration of tumor cells. Cells that are cultivated for several hours in minimal medium without FCS tend to spontaneously develop pseudopodia and membrane protrusions. A pronounced accumulation of the protein on the membrane of this structure was demonstrated both in heterologously expressing cells (TPTE-eGFP) and in spontaneously expressing cell lines (AK staining). Furthermore, co-staining with rhodamine-phalloidin resulted in clear co-localization of TPTE with F-actin in these areas.
  • TPTE as a cell membrane-bound lipid phosphatase with substrate specificity for P ⁇ P 3,4,5 has a regulating function on the RTK-mediated signal transduction and thus exerts a modulating effect on the membrane dynamics in tumor cells.
  • TPTE-eGFP was expressed in NIH3T3 fibroblasts which, by transformation with c-erbB-2, have a constitutively activated PI-3 kinase signaling and thus an overproduction of the second messenger PIP 3.4> 5 .
  • RNA interference RNA interference
  • TPTE is a cytoplasmic protein that can be directed to the plasma membrane using PH domain-recruiting second messengers such as PIP 3,4,5
  • TPTE could perceive chemotactic gradients directly on the membrane by blocking signal transduction from modulate activated RTKs, which are often overexpressed in cancer cells, thereby conveying a spatial gradient perception.
  • Phosphatases are often involved in cell motility and migration.
  • TPTE was introduced into a TPTE-negative cell line as a fusion protein with green fluorescence protein and the distribution of this protein was observed in vivo using real-time microscopy. Cells form membrane extensions (protrusions) as a precursor to migration.
  • TPTE apparently imparts promigratory properties to a cell
  • Brast or lung tumors of a total of 58 patients were examined for expression of TPTE and compared statistically with the course of the patient's disease.
  • Those tumors that express TPTE are significantly more likely to have lymph nodes, but also distant metastases, than the TPTE-negative ones (Fig. 24).
  • TPTE plays an essential role in the metastasis of tumors. Accordingly, TPTE can be used as a marker for prognostically worse progression and tendency to metastasis. Furthermore, methods which inhibit the endogenous activity of TPTE in tumor cells, e.g. through the use of antisene RNA, different methods of RNA interference (RNAi) using expression vectors or retroviruses, as well as through the use of small molecules, lead to reduced breast stasis and are therefore therapeutically very important.
  • RNAi RNA interference
  • the electronic cloning method used according to the invention gave TSBP (SEQ ID NO: 29) and the protein derived therefrom (SEQ ID NO: 30).
  • the gene is described as Testis-specifically regulated (accession number NM_006781).
  • the gene was predicted to encode a basic protein and to be located on chromosome 6 in the vicinity of a sequence coding for an MHC complex (C6orfl0) (Stammers M. et al., Immuno genetics 51 (4-5): 373-82, 2000). According to the invention, it was shown that the sequence described above is incorrect.
  • the sequence according to the invention has significant sequence differences from the known sequence. According to the invention, 3 different splicing variants were cloned.
  • TSBP tumor marker or tumor-associated antigen
  • TSBP can therefore serve as a potential marker for tumor endothelia and for neovascular targeting.
  • the TSBP promoter can thus be cloned to another gene product whose selective expression in the lymph node is desired.
  • Analysis of TSBP antigen expression with specific antibodies confirmed the selective localization of the protein in testis and lymph nodes as well as in melanoma and bronchial carcinoma.
  • immunohistological examinations with GFP-tagged TSBP showed a pronounced perinuclear accumulation. Table 3. Expression of TSBP in tumors
  • SEQ ID NO: 106 CYQHKVTLHMITERDP (aa 60-74)
  • SEQ ID NO: 107 CRIPQVHTMDSSGKI (aa 191-204)
  • the data should be shown as an example for antibodies which are directed against SEQ ID NO: 108.
  • the specific antibody can be used under various fixation conditions for immunofluorescence studies.
  • RT-PCR studies showed that the colon carcinoma cell line HCT 116 P does not express TSBP.
  • their DNA methyltransferase deleted variant HCT 116 DKO is positive for TSBP. Comparative staining of both cell lines with the above-mentioned antibody specifically detected the corresponding protein in an easily detectable amount in the cell lines typed as positive (Fig. 25).
  • the endogenous protein is primarily localized on the core and ER membrane with a slight staining of the plasma membrane.
  • MS4A12 (SEQ ID NO: 37, accession number NM) 17716) and its translation product (SEQ ID NO: 38) are members of a multigen family related to the B cell-specific antigen CD20, the hematopoiesis cell-specific protein HTm4, and the ß - Chain of the high-affinity IgE receptor described above. All family members have at least four potential transmembrane domains as characteristics, both the C- and the N-terminus are cytoplasmic (Liang Y. et al., Immunogenetics 53 (5): 357-68, 2001; Liang Y. & Tedder, Genomics 72 (2): 119-27, 2001). According to the invention, RT-PCR tests for MS4A12 were carried out.
  • MS4A12 is therefore a cell-membrane-based differentiation antigen for normal colonic epithelium, which is also expressed in colorectal tumors and metastases.
  • a quantitative real-time PCR (40 cycles, initial denaturation 15 min 95 ° C, 30 sec 94 ° C, 30 sec 62 ° C, and 30 sec 72 ° C) was carried out with the specific primers (CTGTGTCAGCATCCAAGGAGC and TTCACCTTTGCCAGCATGTAG) (Fig . 26). This not only confirms the absence of MS4A12 in the majority of normal tissues except testis and colorectal normal tissues. Rather, it reveals large amounts of transcripts in, for example, primary intestinal tumors and their metastases.
  • SEQ ID NO: 109 MMSSKPTSHAEVNETC (aa 1 - 15)
  • SEQ ID NO: 110 CGVAGQDYWAVLSGKG (aa 64 - 73)
  • SEQ ID NO: lll MMSSKPTSHAEVNETC (aa 1 - 15)
  • the data should be shown as an example for antibodies which are directed against SEQ ID NO: 11.
  • a specific band can be detected in the Western blot at the expected level in normal intestine, but in colon carcinomas but not in other normal tissues (Fig. 27).
  • the specific antibody can be used under various fixation conditions for immunofluorescence studies.
  • the corresponding protein can be detected specifically in a well-detectable amount specifically in the cell lines typified as positive and MS4A12 also expressed in a eukaryotic recombinant manner is specifically recognized (Fig. 28).
  • These experiments also confirm membrane localization.
  • This antibody was also used for immunohistochemical staining of tissue sections. As expected, the color
  • BRCOl and its translation product are not previously described.
  • the data mining method according to the invention resulted in the EST (expressed sequence tag) AI668620.
  • specific primers sense: CTTGCTCTGAGTCATCAGATG
  • antisense CACAGAATATGAGCCATACAG
  • RT-PCR studies were carried out for expression analysis.
  • specific expression was found in testicular tissue and additionally in normal mammary gland (Table 5). This antigen is repressed in all other tissues. It is also found in mammary gland tumors (20 out of 20). In breast tumors, BRCO1 is clearly overexpressed compared to the expression in normal breast gland tissue (Fig. 10).
  • BRCOl is thus a new differentiation antigen for normal mammary gland epithelium that is overexpressed in breast tumors.
  • TPX1 Acc No. NM_003296; SEQ ID NO: 40
  • SEQ ID NO: 41 The sequence of TPX1 (Acc No. NM_003296; SEQ ID NO: 40) and its translation product (SEQ ID NO: 41) are known.
  • the antigen has so far been described as only Testis-specific, namely as an element of the outer fibers and the acromosome of sperm. He has previously been assigned a role as an adhesion molecule in the attachment of sperm to Sertoli cells (O'Bryan, MK et al., Mol. Reprod. Dev. 58 (l): 116-25, 2001; Maeda, T. et al., Dev. Growth Differ. 41 (6): 715-22, 1999).
  • TPX1-specific protein sequences which are suitable for the production of diagnostic and therapeutic molecules according to the invention Peptide SREVTTNAQR (SEQ ID NO: 84).
  • BRCO2 and its translation product are not previously described.
  • the method according to the invention gave the ESTs (expressed sequence tag) BE069341, BF330573 and AA601511.
  • RT-PCR studies for expression analysis were carried out with specific primers (sense: AGACATGGCTCAGATGTGCAG, antisense: GGAAATTAGCAAGGCTCTCGC).
  • specific expression was found in testicular tissue and additionally in normal breast gland (Table 7). This gene product is transcriptionally repressed in all other tissues. It is also found in mammary tumors.
  • BRCO2 is a new differentiation gene product for normal mammary gland epithelium, which is also expressed in breast tumors.
  • Example 8 Identification of PCSC as a new tumor gene product
  • PCSC (SEQ_ID_63) and its translation product are not previously described.
  • the data mining method according to the invention resulted in the EST (expressed sequence tag) BF064073.
  • RT-PCR studies for expression analysis were carried out with specific primers (sense: TCAGGTATTCCCTGCTCTTAC, antisense: TGGGCAATTCTCTCAGGCTTG).
  • specific expression was found in normal colon and also in colon carcinoma (Table 5). This gene product is transcriptionally repressed in all other tissues.
  • PCSC encodes three putative ORFs (SEQ_ID_64, SEQ_ID_65 and SEQ_ID_117). Sequence analysis of SEQ_ID_64 revealed structural homology to CXC cytokines.
  • each cDNA contains 3 putative ORFs which code for the polypeptides shown in SEQ ID NO: 89-100.
  • PCSC is therefore a differentiation antigen for normal colon epithelium, which is also expressed in colorectal tumors and in all colon metastases investigated.
  • the PCSC expression detected according to the invention in all colorectal metastases make this tumor antigen a very interesting target for the prophylaxis and treatment of metastatic colon tumors.
  • PCR primers were generated according to the invention (5'-CTCCTATCCATGATGCTGACG-3 'and 5'-CCTGAGGATGTACAGTAAGTG-3 *) and for RT-PCR analyzes (95 ° 15min; 94 ° 1 min; 63 ° 1 min; 72 ° 1 min; 35 cycles) in a number of human tissues. It was shown that expression in normal tissues is limited to testis. According to the invention, as described for the other eCTs, SGY-1 was shown to be activated ectopically in a number of tumor tissues; see. Table 9.
  • MORC The sequences of the transcript of MORC (SEQ ID NO: 74) and its translation product (SEQ ID NO: 75) are published in GenBank under accession number XM_037008 (Inoue et al., Hum Mol Genet. Jul; 8 (7): 1201- 7, 1999). MORC is originally described as being involved in spermatogenesis. Mutation of this protein in the mouse system leads to gonadal underdevelopment.
  • PCR primers were generated according to the invention (5'-CTGAGTATCAGCTACCATCAG-3 'and 5'-TCTGTAGTCCTTCACATATCG-3') and for RT-PCR analyzes (95 ° 15min; 94 ° 1 min; 63 ° 1 min; 72 ° 1 min; 35 cycles) in a number of human tissues. It was shown that the expression in normal tissues is limited to testis. As described for the other eCT, it was possible to show according to the invention that MORC is activated in a number of tumor tissues; see. Table 10.

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Abstract

Selon l'invention, des produits géniques exprimés en association avec des tumeurs et les acides nucléiques codant pour ces derniers ont été identifiés. L'invention concerne la thérapie et le diagnostic de maladies, dans lesquelles ces produits géniques exprimés en association avec des tumeurs sont exprimés de manière aberrante. L'invention concerne en outre des protéines, des polypeptides et des peptides, qui sont exprimés en association avec des tumeurs, ainsi que les acides nucléiques codant pour ces derniers.
EP04765088A 2003-09-10 2004-09-10 Produits geniques exprimes de maniere differentielle dans des tumeurs et leur utilisation Withdrawn EP1664103A2 (fr)

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EP10011193.9A EP2327721B1 (fr) 2003-09-10 2004-09-10 Produits géniques exprimés de manière différentielle dans des tumeurs et leur utilisation
EP10011190A EP2336156A3 (fr) 2003-09-10 2004-09-10 Produits génique exprimés de manière differentielle dans des tumeurs et leurs utilisation
EP10011191A EP2336157A3 (fr) 2003-09-10 2004-09-10 Produits génique exprimés de manière differentielle dans des tumeurs et leurs utilisation
EP10011194A EP2322544A3 (fr) 2003-09-10 2004-09-10 Produits géniques d'expression différentielle dans les tumeurs et leur utilisation
EP10011192A EP2314613A3 (fr) 2003-09-10 2004-09-10 Produits géniques exprimes de maniere différentielle dans des tumeurs et leur utilisation
EP16176546.6A EP3095791B1 (fr) 2003-09-10 2004-09-10 Produits geniques exprimes de maniere differentielle dans des tumeurs et leur utilisation

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EP3095791B1 (fr) 2020-06-10
JP5711698B2 (ja) 2015-05-07
EP2327721A2 (fr) 2011-06-01
EP2314613A2 (fr) 2011-04-27
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AU2004272270B2 (en) 2011-07-21
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EP3095791A1 (fr) 2016-11-23
EP2327721B1 (fr) 2017-12-27
ES2663242T3 (es) 2018-04-11
JP2007526759A (ja) 2007-09-20
WO2005026205A3 (fr) 2005-07-07
EP2336156A3 (fr) 2011-11-16
EP2336157A3 (fr) 2011-11-16
CA2538528C (fr) 2020-03-10
JP2012050441A (ja) 2012-03-15
US8765389B2 (en) 2014-07-01
EP2336157A2 (fr) 2011-06-22
EP2322544A3 (fr) 2011-11-16
EP2314613A3 (fr) 2011-11-16
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WO2005026205A2 (fr) 2005-03-24
CA2538528A1 (fr) 2005-03-24

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