US20130251751A1 - Method for identifying immunoreactive peptides - Google Patents

Method for identifying immunoreactive peptides Download PDF

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US20130251751A1
US20130251751A1 US13/775,095 US201313775095A US2013251751A1 US 20130251751 A1 US20130251751 A1 US 20130251751A1 US 201313775095 A US201313775095 A US 201313775095A US 2013251751 A1 US2013251751 A1 US 2013251751A1
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peptides
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Toni Weinschenk
Hans Georg Rammensee
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Immatics Biotechnologies GmbH
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    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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    • 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
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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    • C12Q1/6813—Hybridisation assays
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K39/00—Medicinal preparations containing antigens or antibodies
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    • A—HUMAN NECESSITIES
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    • A61K38/00—Medicinal preparations containing peptides
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • C—CHEMISTRY; METALLURGY
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
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    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids

Definitions

  • the invention relates to a method for identifying and to a method for preparing immunoreactive peptides and to the immunoreactive peptides identified/prepared thereby.
  • Such peptides are being used—for example—in immunotherapy of tumor-associated diseases.
  • TAA tumor-associated antigens
  • the identification of tumor-associated antigens (TAA) by components of the immune system plays a pivotal role. This mechanism is based upon the fact that there exist qualitative or quantitative differences between tumor cells and normal cells.
  • TAA tumor-associated antigens
  • the tumor cells have to express antigens which induce an immune response being sufficient for the elimination of the tumor.
  • CD8 expressing cytotoxic T-lymphocytes are involved in rejection of tumors.
  • foreign proteins/peptides have to be presented to T-cells.
  • Antigens are recognized as peptide fragments by T-cells only if they are presented by MHC-molecules on cell surfaces.
  • MHC major histocompatibility complex
  • MHC major histocompatibility complex
  • Human MHC-molecules are also designated as human leukocyte antigens (HLA).
  • TAA tumor-associated antigens
  • TAA TAA
  • This immunological approach is combined either with a gene expression approach or with a mass-spectrometry (MS)-assisted sequencing of the recognized peptides (see van der Bruggen, et al., 1991, A gene encoding an antigen recognized by cytolytic Tlymphocytes on a human melanoma, Science 254: 1643-1647, and Cox et al., 1994, Identification of a peptide recognized by five melanoma-specific human cytotoxic T-cell lines, Science 264: 716-719).
  • Methods for identifying TAA which are based on comparative transcription profiling of tumorous and corresponding normal tissue are—for example—hybridization and the use of DNA microarray technology.
  • T-cell-independent approach also known in the art combines epitope prediction and screening for predicted peptides in complex peptide mixtures, the peptides being identified by highly sensitive capillary liquid chromatography mass spectrometry (LC-MS) (see Schirle et al., 2000, Identification of tumor-associated MHC class I-ligands by a novel T-cell independent approach, Eur. J. Immunol. 30: 2216-2225).
  • LC-MS capillary liquid chromatography mass spectrometry
  • DNA microarray technology provides a new approach using comparative expression profiling of tumorous and corresponding autologous normal tissue.
  • Young et al., 2001 Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, Am. J. Pathol. 158: 1639-1651, disclosed that by using this technique a large number of tumor-associated antigens from individual tumor samples can be identified.
  • MHC-I-ligands derived from overexpressed or selectively expressed proteins provide possible targets for specific CTL recognition of tumors.
  • Mathiassen et al., 2001 Tumor-associated antigens identified by mRNA expression profiling induce protective antitumor immunity, Eur. J. Immuno. 31: 1239-1246, demonstrated, in a mouse model, that by combining expression analysis with epitope prediction a successful vaccine can be prepared.
  • this object is achieved by means of a method for identifying immunoreactive peptides comprising:
  • the inventors revealed that by combining an expression analysis with antigenic tumor peptides, which have been isolated and analyzed, specific candidates for an individual vaccine can be identified.
  • the new method is applicable within the scope of a mere service as well as in combination with the supply of the identified immunoreactive peptide.
  • a further object is the method according to the invention, wherein the peptides isolated in step (c) are MHC-ligands.
  • step (b) is performed by means of a microarray-analysis and/or reverse transcription-polymerase chain reaction.
  • microarray analysis With microarray analysis the expression profile of tumorous tissue is compared with corresponding normal tissue by using certain DNA- or gene-chips, and selectively expressed or overexpressed genes are identified. This method is well known in the art and is, for example, disclosed in Schena et al., 1995, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270: 467-470.
  • Reverse transcription-polymerase chain reaction in the following RT-PCT can be utilized to quantify expression of a gene.
  • cDNA is generated from RNA, which—for example—has been isolated from tumor cells, and the cDNA is consequently used as template for PCR. In that way, on the basis of the amplified DNA one can compare which genes are being transcribed with which intensity.
  • step (c) is performed by means of a mass spectrometer.
  • each peptide can be identified precisely and efficiently with high performance.
  • Schirle et al., 2000, Identification of tumor-associated MHC class I ligands by a novel T-cell-independent approach, Eur. J. Immunol. 30: 2216-2225 describe the use of mass spectrometry to identify peptides derived from tumor tissue.
  • a further object is the method according to the invention, wherein in step (c) candidate antigenic peptides are predicted on the basis of the expression profile using suitable databases, and the mass spectrometer is calibrated with respect to the predicted antigens.
  • step (c) is followed by a further step, in which the reactivity of peripheral leukocytes, preferably of T-leukocytes, against the isolated antigenic peptides, is tested.
  • a further object is the method according to the invention, wherein the reactivity of peripheral leukocytes against the isolated antigenic peptides is tested by means of measuring ⁇ -Interferon-mRNA and/or cytokin-mRNA synthesized by the leukocytes.
  • ⁇ -Interferon- or cytokin-mRNA By detecting ⁇ -Interferon- or cytokin-mRNA it is possible to precisely prove the specific reactivity of leukocytes, preferably of T-lymphocytes against antigenic peptides. Both substances are secreted by activated T-lymphocytes after their activation by corresponding antigenic peptides.
  • Yet another object is the method according to the invention, wherein, following step (c), a further step is performed, in which the presence of the T-lymphocytes is detected.
  • a further object is the method according to the invention, wherein the detection of specific pre-existing T-lymphocytes is performed by labeling the leukocytes with reconstituted complexes of antigen-presenting molecules and antigenic peptide.
  • tetramers A method for generating such reconstituted complexes (“tetramers”) and for utilizing them is disclosed, for example, in Altman et al., 1996, Phenotypic analysis of antigen-specific T-lymphocytes, Science 274: 94-96.
  • immunoreactive peptides which are identified and prepared by the method according to the invention.
  • Another object of the invention is a pharmaceutical composition comprising one or more peptides which have been identified and/or prepared by the method according to the invention.
  • the composition may be applied, for example, parenterally, for example subcutaneously, intradermally or intramuscularly, or may be administered orally.
  • the peptides are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier; the composition can further comprise additives, for example buffers, binders, etc.
  • the peptides can also be administered together with immunostimulating substances, for example cytokins
  • An extensive description of additives which can be used in compositions of this nature is given, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed., 2000, American Pharmaceutical Association and Pharmaceutical Press.
  • the peptide may be used for treatment of tumor diseases and for preparing a medicament for treatment of tumor diseases.
  • Tumor diseases to be treated comprise renal, breast, pancreas, gastric, testis and/or skin cancer. Listing of tumor diseases is supposed to be merely illustrative and shall not limit the scope of usage.
  • the peptide can further be used for assessment of the therapy-course of a tumor disease.
  • the peptide can also be used for monitoring a therapy in other immunizations or therapies. In that way the peptide according to the invention may not only be used in a therapeutical way but also in a diagnostic way.
  • a further embodiment object of the invention is using the peptides for generating an antibody.
  • Polyclonal antibodies can be obtained, in a general manner, by immunization of animals by means of injection of the peptides and subsequent purification of the immunoglobulin.
  • Monoclonal antibodies can be generated according to standardized protocols, for example as described in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies.
  • nucleic acid molecules coding for the peptide isolated with the method according to the invention are nucleic acid molecules coding for the peptide isolated with the method according to the invention.
  • the nucleic acid molecules can be DNA- or RNA-molecules and can be used for immune therapy of cancer as well.
  • the nucleic acid molecules can be provided in a vector.
  • a further object of the invention is a cell genetically modified by means of the nucleic acid molecule, such, that the cell is producing a peptide identified according to the invention.
  • Another object of the invention is a method for preparing an immunoreactive peptide with which a peptide is identified according to the disclosed method and the identified peptide is synthesized chemically, in vitro or in vivo.
  • Peptides can be prepared by chemical linkage of amino acids, for example by the method of Merrifield, which is known in the art (see Merrifield R B, 1963, J. Am. Chem. Soc. 85 :2149-2154).
  • Peptides can be prepared in vitro, for example, in cell-free systems, and in vivo by using cells.
  • Another object of the present invention is a method for preparing a vaccine comprising the steps of
  • FIG. 1 shows the expression analysis of selected genes by means of quantitative RT-PCR
  • FIG. 2 shows the detection of keratin 18-specific CD8+ T-lymphocytes.
  • Samples of patients having histologically confirmed renal cell carcinoma were obtained from the department of urology, University of Tubingen. Both patients had not received preoperative therapy.
  • Patient No. 1 (in the following designated RCC01) had the following HLA-typing: HLA-A*02 A68 B*18 B*44; patient No 2 (in the following designated RCCI3) HLA-A*02 A*24 B*07 B*40.
  • Peptides from tumor tissue of patient RCC13 were identified by online capillary LCMS as mentioned above with minor modifications: Sample volumes of about 100 ⁇ l were loaded, desalted and preconcentrated on a 300 pm*5 mm C18 ⁇ -precolumn (LC packings). A syringe pump (PHD 2000, Harvard Apparatus, Inc.) equipped with a gastight 100 p ⁇ l-syringe (1710 RNR, Hamilton), delivered solvent and sample at 2 ⁇ l/min. For peptide separation, the preconcentration column was switched in line with a 75*250 mm C-18-column (LC packings).
  • Double-stranded DNA was synthesized from 40 ⁇ g of total RNA using superscript RT II reverse transcriptase (Life Technologies).
  • the primer Eurogentec
  • In vitro transcription was performed using the BioArrayTM High YieldTM RNA Transcript Labeling Kit (ENZO Diagnostics, Inc.); subsequently, fragmentation and hybridization were carried out on Affymetrix HuGeneFL gene chips, and staining with a streptavidin-phycoerythrin and biotinylated anti-streptavidin-antibody followed the manufacturer's protocols (Affymetrix).
  • the Affymetrix GeneArray Scanner was used and data were analyzed with the Microarray Analysis Suite 4.0 Software.
  • cDNA generated for microarray analysis was used for quantitative PCR analysis.
  • PCR products were analyzed on 3% agarose gels for purity and sequence-verified after cloning into pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen). Data analysis involved the delta CT method for relative quantification.
  • Embedded frozen tissue specimens were cut at 6 ⁇ m thickness and transferred in 70% ethanol for about 15 min. Slides were incubated 90 seconds in Mayer's hematoxylin (Merck), rinsed in water, incubated for 1 min in 70% ethanol, 1 min in 95% ethanol, 30 seconds in 1% alcoholic eosin Y (Sigma), 2 ⁇ 2 min in 95% ethanol, 2 ⁇ 2 min in 100% ethanol and finally 2 ⁇ 2 min in xylene. After air drying for 15 minutes, slides were stored under dry conditions. Normal malignant epithelial tubular cells and carcinoma cells were isolated by a Laser Capture Microdissection (LCM) using the PixCell II LCM system (Arcturus Engineering). Total RNA was extracted in 400 ⁇ l TRIZOL.
  • LCM Laser Capture Microdissection
  • PBMC peripheral blood mononuclear cells
  • HLA-A*0201 tetrameric complexes were produced as described by Altman et al., 1996, Phenotypic analysis of antigen-specific T-lymphocytes, Science 274: 94-96, as follows:
  • the HLA-A2-binding peptides used for the refolding were ALLNIKVKL from keratin 18 and NLVPMVATV from pp65 HCMVA.
  • Tetramers were assembled by mixing biotinylated monomers with streptavidin-PE or streptavidin-APC and 2-3 ⁇ 10 6 cells were incubated 30 min at 4° C.
  • the expression of approximately 7,000 genes in tumors and corresponding normal tissue of two renal cell carcinoma was analyzed. Between 400 and 500 genes were found to be overexpressed or selectively expressed in the tumor. 70 genes were overexpressed in the tumors of both patients. In patient I, 268 overexpressed and 129 exclusively expressed genes were found. Most of the overexpressed genes are cancer-related, i.e., either oncogenes, tumor suppressor genes or genes already described as overexpressed in cancer, such as CCND1, CA9, cerebrosidesulfotransferase and parathyroid hormone-like hormone.
  • the cancer-associated adipose differentiation-related protein (ADFP) or adipophilin showed the second highest degree of overexpression. In addition, this protein was shown to be highly overexpressed in tumorous tissue in comparison to normal tissue of other organs, that is not only in comparison to normal renal tissue.
  • FIG. 1 the expression-analysis of selected genes by means of RT-PCR is shown.
  • the RT-PCR was performed using the same cDNA as generated for microarray analysis.
  • the black bars correspond to the numbers in tumor tissue of patient RCC01, the white bars represent the numbers of normal tissues and the angular-striped gray bars the numbers in tumor tissue of patient RCC13.
  • ADFP adipophilin
  • CCND1 cycline D1
  • ETS1 ets-1
  • adipophilin was overexpressed in tumor tissue of patient RCCOI by a factor of 29.1 as proven by means of microarray, compared to 18.1 as measured by means of quantitative PCR (see FIG. 1 ).
  • patient RCC13 by means of microarray analysis a factor of 11.4 and by means of quantitative PCR a factor of 6.7 could be demonstrated (see FIG. 1 ).
  • Galectin 2 (LGALS2) was overexpressed in patient RCCO1 and keratin-18 (KRT18) in patient RCC13.
  • An exception to the congruence between microarray and quantitative PCR was the overexpression of KIAA0367 and met proto-oncogene (MET) in patient RCC13.
  • a total of 85 ligands could be obtained from tumor tissue, which were bound to HLA-subtypes HLA-A*02, HLA-A*68, HLA-B*18 or HLA-B*44.
  • Peptides that bind to HLAA*02 reflected the allele-specific peptide motif (Leucine, Valine, Isoleucine, Alanine, Methionine on position 2, Leucine, Valine, Isoleucine or Alanine at the C-Terminus).
  • ligands were derived from abundantly expressed housekeeping proteins, but ligands from proteins with reported tumor association could be detected also, for example, YVDPVITSI derived from met proto-oncogene, ALLNIKVKL derived from keratin 18, and SVASTITGV from adipophilin.
  • HLA-A*68 ligands were identified by their anchor amino acids Threonine, Isoleucine, Valine, Alanine or Leucine on position 2 and arginine or lysine at the C-terminus. Two other ligands from adipophilin were found among HLA-A*68-presented peptides: MTSALPIIQK and MAGDIYSVFR. Ligands carrying glutamic acid on position 2 were assigned to HLA-B*44; since the peptide motif of HLA-B*18 is unknown, a distinction between ligands of these two HLA-B-molecules was not possible.
  • a most interesting ligand could be identified from patient RCC13 (ALAAVVTEV) encoded by a “Reading frame” shifted by one nucleotide compared to DEAD/H-box polypeptide 3 (DDX3).
  • ALAAVVTEV is encoded by the nucleotides 317 to 343 of the coding strand of DDX3, whereas nucleotides 316 to 342 are coding for GIGSRGDRS of the DDX3 protein.
  • PBMC from 6 HLA-A2 positive renal cell carcinoma patients were tested for reaction against four of the relevant peptides: HLA-A*02-restricted ligands from adipophilin, keratin 18, K1AA0367 and met-proto-oncogene.
  • HLA-A*02-restricted ligands from adipophilin HLA-A*02-restricted ligands from adipophilin
  • keratin 18, K1AA0367 met-proto-oncogene.
  • a very sensitive quantitative PCT assay was carried out to detect ⁇ -Interferon-mRNA production by CD8 + T-cells following a 7 day-in vitro-sensitization with peptide. Sporadic responses were seen after stimulation with met-proto-oncogene or keratin 18 or adipophilin peptides.
  • FIG. 2 shows the detection of keratin 18-specific T-lymphocytes.
  • PBMC from four healthy HLA-A*02 + donors HD 1, 2, 4, 6) were simultaneously stained with HLA-A2/keratin 18-PE tetramers, HLA-A2/CMV-APC tetramers, CD8-PerCP and CD4-FITC.
  • Dot plots show samples from one of three independent experiments for 1 ⁇ 10 6 PBMC. In the plots, percentage of tetramer + -cells within the CD8 + CD4 ⁇ population are indicated.
  • CD8 + T-lymphocytes specific for keratin 18 was found in 4 out of 22 healthy individuals. This population did not stain with a CMV tetramer showing that the binding of keratin 18 tetramer was specific.
  • CD8 + T-lymphocytes specific for the keratin 18-peptide are contained in the human T-cell repertoire.

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Abstract

The invention relates to a method for identifying immunoreactive peptides. According to said method, a sample of tumorous and corresponding healthy tissue is first provided, the tumor-specific expression profile is subsequently determined and antigenic peptides are isolated from the tumorous tissue and analyzed. The respective data that has been obtained is then matched and peptides are identified on the basis of said data.

Description

    CROSS-REFERENCE TO RELATED APPLICATIONS
  • This application is a Continuation of U.S. application Ser. No. 11/000,474, filed Nov. 29, 2004, incorporated herein by reference in its entirety, which is a Continuation of International Application No. PCT/EP03/05038, filed May 14, 2003, designating the United States and published in German as WO 03/100432, which claims priority to German Application No. 102 25 139.8, filed May 29, 2002.
    • TECHNICAL FIELD
  • The invention relates to a method for identifying and to a method for preparing immunoreactive peptides and to the immunoreactive peptides identified/prepared thereby.
  • Such peptides are being used—for example—in immunotherapy of tumor-associated diseases. When tumor cells are eliminated by the immune system the identification of tumor-associated antigens (TAA) by components of the immune system plays a pivotal role. This mechanism is based upon the fact that there exist qualitative or quantitative differences between tumor cells and normal cells. To induce an anti-tumor response, the tumor cells have to express antigens which induce an immune response being sufficient for the elimination of the tumor.
  • CD8 expressing cytotoxic T-lymphocytes (in the following CTL), in particular, are involved in rejection of tumors. To induce such an immune reaction by cytotoxic T-cells foreign proteins/peptides have to be presented to T-cells. Antigens are recognized as peptide fragments by T-cells only if they are presented by MHC-molecules on cell surfaces. These MHC (“major histocompatibility complex”) molecules are peptide receptors which normally bind peptides intracellularly and transport them to the cell surface. This complex of peptide and MHC-molecule is recognized by T-cells. Human MHC-molecules are also designated as human leukocyte antigens (HLA).
  • In the past, antigen-specific immunotherapy based on T-cells has proven successful in the treatment of cancer.
  • Induction of a specific CTL response directed against a tumor is dependent on identification of MHC class I-ligands derived from tumor-associated antigens (TAA). Such TAA can be exclusively present in malignant cells, for example as products of mutated genes. Other important classes of tumor-associated antigens are tissue-specific structures such as the cancer-testis antigens, and a third class of tumor-associated antigens are proteins overexpressed in tumors.
    • BACKGROUND ART
  • The methods for identification and characterization of TAA, which represent the starting point for a tumor vaccine, are—on the one hand—based on the use of patient derived CTL or antibodies. This immunological approach is combined either with a gene expression approach or with a mass-spectrometry (MS)-assisted sequencing of the recognized peptides (see van der Bruggen, et al., 1991, A gene encoding an antigen recognized by cytolytic Tlymphocytes on a human melanoma, Science 254: 1643-1647, and Cox et al., 1994, Identification of a peptide recognized by five melanoma-specific human cytotoxic T-cell lines, Science 264: 716-719). Methods for identifying TAA which are based on comparative transcription profiling of tumorous and corresponding normal tissue are—for example—hybridization and the use of DNA microarray technology.
  • Celis et al., 1994, Induction of anti-tumor cytotoxic T-Iymphocytes in normal humans using primary cultures and synthetic peptide epitopes, Proc. Natl. Acad. Sci. USA 91: 2105-2109, applied a method which takes advantage of the prediction of MHC class I-ligands derived from a selected tumor-associated antigen, and in which these ligands were verified as Tcell epitopes in a next step.
  • Drawbacks of patients-derived T-cell-based approaches are the extensive culture techniques and their restriction to the frequency of pre-existing T-cells.
  • Another T-cell-independent approach also known in the art combines epitope prediction and screening for predicted peptides in complex peptide mixtures, the peptides being identified by highly sensitive capillary liquid chromatography mass spectrometry (LC-MS) (see Schirle et al., 2000, Identification of tumor-associated MHC class I-ligands by a novel T-cell independent approach, Eur. J. Immunol. 30: 2216-2225).
  • DNA microarray technology provides a new approach using comparative expression profiling of tumorous and corresponding autologous normal tissue. Young et al., 2001, Expression profiling of renal epithelial neoplasms: a method for tumor classification and discovery of diagnostic molecular markers, Am. J. Pathol. 158: 1639-1651, disclosed that by using this technique a large number of tumor-associated antigens from individual tumor samples can be identified. MHC-I-ligands derived from overexpressed or selectively expressed proteins provide possible targets for specific CTL recognition of tumors. Mathiassen et al., 2001, Tumor-associated antigens identified by mRNA expression profiling induce protective antitumor immunity, Eur. J. Immuno. 31: 1239-1246, demonstrated, in a mouse model, that by combining expression analysis with epitope prediction a successful vaccine can be prepared.
  • However, a disadvantage is that epitope prediction even for only a few target genes results in the identification of a vast number of candidate peptides, the majority of which are actually not presented by MHC molecules and thus do not induce a CTL-response.
    • DISCLOSURE OF THE INVENTION
  • Therefore, it is an object of the present invention to provide a new method for selective and simple identification of immunoreactive peptides.
  • According to the invention, this object is achieved by means of a method for identifying immunoreactive peptides comprising:
  • (a) providing a sample of tumorous and corresponding healthy tissue;
  • (b) determining tumor-specific expression profile of the provided sample,
  • (c) isolating and analyzing of antigenic peptides in the sample of tumorous tissue,
  • (d) matching data obtained in step (b) and (c), and
  • (e) identifying peptides on basis of the matched data.
  • The inventors revealed that by combining an expression analysis with antigenic tumor peptides, which have been isolated and analyzed, specific candidates for an individual vaccine can be identified.
  • By isolating antigenic peptides and matching them with gene expression profiles of tumorous tissue it can be avoided that a vast number of possible immunoreactive peptides is obtained. Rather, specific peptides are identified, which are actually presented by MHC molecules and which are thus suitable as immunoreactive peptides.
  • With the method according to the invention it is, respectively, possible to identify patient-specific peptides, i.e. it is possible to precisely match peptides, which are to be used as vaccine, to the patient, in order to induce a specific immune response.
  • For example, industrial laboratories—after having received patient samples—can systematically and efficiently perform this method, and can—after having identified suitable immunoreactive peptides—provide clinics in charge with the peptide sequences; the clinics can then synthesize and administer the peptides. Nevertheless, it is also possible that a laboratory is carrying out identification as well as production of the peptides suitable for the respective patient.
  • Therefore, the new method is applicable within the scope of a mere service as well as in combination with the supply of the identified immunoreactive peptide.
  • A further object is the method according to the invention, wherein the peptides isolated in step (c) are MHC-ligands.
  • Only peptides which are bound to MHC-molecules can induce a cellular immune response. Peptides which are derived, for example, from overexpressed genes of a tumor but which are not bound to MHC-molecules, do not induce a CTL immune reaction. Therefore, not all peptides, for example, identified solely by epitope prediction are immunoreactive.
  • A further object is the method according to the invention, wherein step (b) is performed by means of a microarray-analysis and/or reverse transcription-polymerase chain reaction.
  • With microarray analysis the expression profile of tumorous tissue is compared with corresponding normal tissue by using certain DNA- or gene-chips, and selectively expressed or overexpressed genes are identified. This method is well known in the art and is, for example, disclosed in Schena et al., 1995, Quantitative monitoring of gene expression patterns with a complementary DNA microarray, Science 270: 467-470.
  • Reverse transcription-polymerase chain reaction (in the following RT-PCT) can be utilized to quantify expression of a gene.
  • For this purpose cDNA is generated from RNA, which—for example—has been isolated from tumor cells, and the cDNA is consequently used as template for PCR. In that way, on the basis of the amplified DNA one can compare which genes are being transcribed with which intensity.
  • Yet a further object is the method according to the invention, wherein step (c) is performed by means of a mass spectrometer.
  • Using this technique each peptide can be identified precisely and efficiently with high performance. For example, Schirle et al., 2000, Identification of tumor-associated MHC class I ligands by a novel T-cell-independent approach, Eur. J. Immunol. 30: 2216-2225, describe the use of mass spectrometry to identify peptides derived from tumor tissue.
  • A further object is the method according to the invention, wherein in step (c) candidate antigenic peptides are predicted on the basis of the expression profile using suitable databases, and the mass spectrometer is calibrated with respect to the predicted antigens.
  • Use of databases for prediction of candidate antigens and utilizing the obtained data is—for example—disclosed in Schirle et al., 2001, Combining computer algorithms with experimental approaches permits rapid and accurate identification of T-cell epitopes from defined antigens, J. Immunol. Methods, 257: 1-16.
  • Yet a further object is the method according to the invention, wherein step (c) is followed by a further step, in which the reactivity of peripheral leukocytes, preferably of T-leukocytes, against the isolated antigenic peptides, is tested.
  • A further object is the method according to the invention, wherein the reactivity of peripheral leukocytes against the isolated antigenic peptides is tested by means of measuring □-Interferon-mRNA and/or cytokin-mRNA synthesized by the leukocytes.
  • By detecting □-Interferon- or cytokin-mRNA it is possible to precisely prove the specific reactivity of leukocytes, preferably of T-lymphocytes against antigenic peptides. Both substances are secreted by activated T-lymphocytes after their activation by corresponding antigenic peptides.
  • With this additional step candidates of the already identified peptides can be identified even more precisely.
  • Yet another object is the method according to the invention, wherein, following step (c), a further step is performed, in which the presence of the T-lymphocytes is detected.
  • Using this method it is possible to specifically detect to what extent T-lymphocytes directed against isolated and identified peptides are pre-existing in patients. By performing this step it is possible to apply, as a vaccine, only those peptides against which T-lymphocytes are already pre-existing in the patient. The peptides can then be used to activate these specific T-lymphocytes.
  • A further object is the method according to the invention, wherein the detection of specific pre-existing T-lymphocytes is performed by labeling the leukocytes with reconstituted complexes of antigen-presenting molecules and antigenic peptide.
  • With this method the so-called tetramer-technology is utilized. A method for generating such reconstituted complexes (“tetramers”) and for utilizing them is disclosed, for example, in Altman et al., 1996, Phenotypic analysis of antigen-specific T-lymphocytes, Science 274: 94-96.
  • Further objects of the invention are immunoreactive peptides, which are identified and prepared by the method according to the invention.
  • After identification these peptides can be selectively and specifically prepared for each patient.
  • Another object of the invention is a pharmaceutical composition comprising one or more peptides which have been identified and/or prepared by the method according to the invention.
  • The composition may be applied, for example, parenterally, for example subcutaneously, intradermally or intramuscularly, or may be administered orally. In doing so, the peptides are dissolved or suspended in a pharmaceutically acceptable carrier, preferably an aqueous carrier; the composition can further comprise additives, for example buffers, binders, etc. The peptides can also be administered together with immunostimulating substances, for example cytokins An extensive description of additives which can be used in compositions of this nature is given, for example, in A. Kibbe, Handbook of Pharmaceutical Excipients, 3. Ed., 2000, American Pharmaceutical Association and Pharmaceutical Press.
  • According one object of the invention the peptide may be used for treatment of tumor diseases and for preparing a medicament for treatment of tumor diseases.
  • Tumor diseases to be treated comprise renal, breast, pancreas, gastric, testis and/or skin cancer. Listing of tumor diseases is supposed to be merely illustrative and shall not limit the scope of usage.
  • The peptide can further be used for assessment of the therapy-course of a tumor disease.
  • The peptide can also be used for monitoring a therapy in other immunizations or therapies. In that way the peptide according to the invention may not only be used in a therapeutical way but also in a diagnostic way.
  • A further embodiment object of the invention is using the peptides for generating an antibody.
  • Polyclonal antibodies can be obtained, in a general manner, by immunization of animals by means of injection of the peptides and subsequent purification of the immunoglobulin.
  • Monoclonal antibodies can be generated according to standardized protocols, for example as described in Methods Enzymol. (1986), 121, Hybridoma technology and monoclonal antibodies.
  • Further objects of the invention are nucleic acid molecules coding for the peptide isolated with the method according to the invention.
  • The nucleic acid molecules can be DNA- or RNA-molecules and can be used for immune therapy of cancer as well.
  • According to one object of the invention the nucleic acid molecules can be provided in a vector.
  • A further object of the invention is a cell genetically modified by means of the nucleic acid molecule, such, that the cell is producing a peptide identified according to the invention.
  • Another object of the invention is a method for preparing an immunoreactive peptide with which a peptide is identified according to the disclosed method and the identified peptide is synthesized chemically, in vitro or in vivo.
  • Peptides can be prepared by chemical linkage of amino acids, for example by the method of Merrifield, which is known in the art (see Merrifield R B, 1963, J. Am. Chem. Soc. 85 :2149-2154).
  • Peptides can be prepared in vitro, for example, in cell-free systems, and in vivo by using cells.
  • Another object of the present invention is a method for preparing a vaccine comprising the steps of
  • (a) performing the disclosed method,
  • (b) preparing the identified immunoreactive peptides, and
  • (c) formulating the prepared immunoreactive peptides.
  • It will be understood that the features which are mentioned above and the features still to be explained below can be used not only in the combinations which are in each case specified but also in other combinations or on their own without departing from the scope of the present invention.
    • BRIEF DESCRIPTION OF THE DRAWINGS
  • Embodiments of the invention are displayed and explained in the figures and the example below.
  • FIG. 1 shows the expression analysis of selected genes by means of quantitative RT-PCR;
  • FIG. 2 shows the detection of keratin 18-specific CD8+ T-lymphocytes.
    •  MODES OF CARRYING OUT THE INVENTION EXAMPLE 1 Patient Samples
  • Samples of patients having histologically confirmed renal cell carcinoma were obtained from the department of urology, University of Tubingen. Both patients had not received preoperative therapy. Patient No. 1 (in the following designated RCC01) had the following HLA-typing: HLA-A*02 A68 B*18 B*44; patient No 2 (in the following designated RCCI3) HLA-A*02 A*24 B*07 B*40.
    • Isolation of MHC Class 1-Bound Peptides
  • Shock-frozen tumor samples were processed as described in Schirle, M. et al., Identification of tumor-associated MHC-class I ligands by a novel T-cell-independent approach, 2000, European Journal of Immunology, 30: 2216-2225. Peptides were isolated according to standard protocols using monoclonal antibody W6/32 being specific for HLA class I or monoclonal antibody BB7.2 being specific for HLA-A2. Production and utilization of these antibodies is described by Barnstable, C. J. et al., Production of monoclonal antibodies to group A erythrocytes, HLA and other human cell surface antigens—New tools for genetic analysis, 1978, Cell, 14:9-20 and Parham, P. & Brodsky, F. M., Partial purification and some properties of BB7.2. A cytotoxic monoclonal antibody with specificity for HLA-A2 and a variant of HLAA28, 1981, Hum. Immunol., 3: 277-299.
    • Mass Spectrometry
  • Peptides from tumor tissue of patient RCCO1 were separated by reversed phase HPLC (SMART-system, μRPC C2/C18 SC 2.1/19, Amersham Pharmacia Biotech) and fractions were analyzed by nanoESI MS. In doing so it was proceeded as described in Schirle, M. et al., Identification of tumor-associated MHC class I ligands by a novel T-cell-independent approach, 2000, European Journal of Immunology, 30: 2216-2225.
  • Peptides from tumor tissue of patient RCC13 were identified by online capillary LCMS as mentioned above with minor modifications: Sample volumes of about 100 μl were loaded, desalted and preconcentrated on a 300 pm*5 mm C18 μ-precolumn (LC packings). A syringe pump (PHD 2000, Harvard Apparatus, Inc.) equipped with a gastight 100 pμl-syringe (1710 RNR, Hamilton), delivered solvent and sample at 2 μl/min. For peptide separation, the preconcentration column was switched in line with a 75*250 mm C-18-column (LC packings). Subsequently a binary gradient of 25%-60% B within 70 min was performed, applying a 12 μl/min flow rate reduced to approximately 300 nl/min with a precolumn using a TEE-piece (ZT1C, Valco) and a 300 μm*150 mm C-18-column.
  • A blank run was always included to ensure that the system was free of residual peptides. On-line fragmentation was performed as described and fragment spectra were analyzed manually.
  • Database searches (NCBInr, EST) were made using MASCOT.
    • Preparation of RNA
  • Fragments of normal and malignant renal tissue were dissected, shock-frozen, ground by a mortar and pestle under liquid nitrogen and homogenized with a rotary homogenizer (Heidolph instruments) in TRIZOL (Life Technologies). Total RNA was prepared according to the manufacturer's protocol followed by a clean-up with RNeasy (QIAGEN). Total RNA from human tissues were obtained commercially (Human total RNA Master Panel II, Clontech).
    • High-Density Oligonucleotide Micro-Array Analysis
  • Double-stranded DNA was synthesized from 40 μg of total RNA using superscript RT II reverse transcriptase (Life Technologies). The primer (Eurogentec) were given by the Affymetrix manual. In vitro transcription was performed using the BioArray™ High Yield™ RNA Transcript Labeling Kit (ENZO Diagnostics, Inc.); subsequently, fragmentation and hybridization were carried out on Affymetrix HuGeneFL gene chips, and staining with a streptavidin-phycoerythrin and biotinylated anti-streptavidin-antibody followed the manufacturer's protocols (Affymetrix). The Affymetrix GeneArray Scanner was used and data were analyzed with the Microarray Analysis Suite 4.0 Software.
    • Real Time RT-PCR
  • cDNA generated for microarray analysis was used for quantitative PCR analysis.
  • Each gene was run in duplicates (40 cycles, 95° C.×15 s, 60° C.×1 min) using SYBRGreen chemistry on the ABI PRISM 7700 sequence detection system (Applied Biosystems). Samples were independently analyzed two to three times. Primers (MWGBiotech) were selected to flank an Intron and PCR efficiencies were tested for all primer pairs and found to be close to 1.
  • PCR products were analyzed on 3% agarose gels for purity and sequence-verified after cloning into pCR4-TOPO vector using the TOPO TA Cloning Kit (Invitrogen). Data analysis involved the delta CT method for relative quantification.
    • Laser Capture Microdissection
  • Embedded frozen tissue specimens were cut at 6 μm thickness and transferred in 70% ethanol for about 15 min. Slides were incubated 90 seconds in Mayer's hematoxylin (Merck), rinsed in water, incubated for 1 min in 70% ethanol, 1 min in 95% ethanol, 30 seconds in 1% alcoholic eosin Y (Sigma), 2×2 min in 95% ethanol, 2×2 min in 100% ethanol and finally 2×2 min in xylene. After air drying for 15 minutes, slides were stored under dry conditions. Normal malignant epithelial tubular cells and carcinoma cells were isolated by a Laser Capture Microdissection (LCM) using the PixCell II LCM system (Arcturus Engineering). Total RNA was extracted in 400 μl TRIZOL.
    • PBMC, Tetramer Production and Flow Cytometry
  • Peripheral blood mononuclear cells (in the following PBMC) from two healthy donors (HDI and HD2), which were serologically typed as CMV-positive, were isolated by gradient centrifugation (FicoLite H) and frozen.
  • HLA-A*0201 tetrameric complexes were produced as described by Altman et al., 1996, Phenotypic analysis of antigen-specific T-lymphocytes, Science 274: 94-96, as follows: The HLA-A2-binding peptides used for the refolding were ALLNIKVKL from keratin 18 and NLVPMVATV from pp65 HCMVA. Tetramers were assembled by mixing biotinylated monomers with streptavidin-PE or streptavidin-APC and 2-3×106 cells were incubated 30 min at 4° C. with both tetramers: 10 μg/ml for each monomer in PBS, 0.01% NaN3, 2 mM EDTA, 50% fetal calf serum). Then, monoclonal antibodies Anti-CD4-FITC (Coulter-Immunotech) and Anti-CD8-PerCP (Becton Dickinson) were added for 20 min. After three washes, samples were fixed in FACS buffer, 1% formaldehyde. Four-color analysis was performed on a FACScalibur cytometer (Becton Dickinson).
    • Results
  • The expression of approximately 7,000 genes in tumors and corresponding normal tissue of two renal cell carcinoma was analyzed. Between 400 and 500 genes were found to be overexpressed or selectively expressed in the tumor. 70 genes were overexpressed in the tumors of both patients. In patient I, 268 overexpressed and 129 exclusively expressed genes were found. Most of the overexpressed genes are cancer-related, i.e., either oncogenes, tumor suppressor genes or genes already described as overexpressed in cancer, such as CCND1, CA9, cerebrosidesulfotransferase and parathyroid hormone-like hormone. The cancer-associated adipose differentiation-related protein (ADFP) or adipophilin, showed the second highest degree of overexpression. In addition, this protein was shown to be highly overexpressed in tumorous tissue in comparison to normal tissue of other organs, that is not only in comparison to normal renal tissue.
  • To verify data obtained by microarray analysis the expression of selected genes was analyzed by quantitative PCR. In FIG. 1, the expression-analysis of selected genes by means of RT-PCR is shown. The RT-PCR was performed using the same cDNA as generated for microarray analysis. Copy numbers are relative to 18S rRNA and normalized to the normal tissue (=1) of each patient. The black bars correspond to the numbers in tumor tissue of patient RCC01, the white bars represent the numbers of normal tissues and the angular-striped gray bars the numbers in tumor tissue of patient RCC13.
  • It was shown that overexpression of adipophilin (ADFP) and cycline D1 (CCND1) as proven by microarray could be confirmed by quantitative PCR. Further, it was demonstrated that ets-1 (ETS1) was expressed equally both in normal and in tumor tissue. Further, relative expression levels detected by both techniques were roughly comparable.
  • For example, adipophilin was overexpressed in tumor tissue of patient RCCOI by a factor of 29.1 as proven by means of microarray, compared to 18.1 as measured by means of quantitative PCR (see FIG. 1). With patient RCC13, by means of microarray analysis a factor of 11.4 and by means of quantitative PCR a factor of 6.7 could be demonstrated (see FIG. 1). Galectin 2 (LGALS2) was overexpressed in patient RCCO1 and keratin-18 (KRT18) in patient RCC13. An exception to the congruence between microarray and quantitative PCR was the overexpression of KIAA0367 and met proto-oncogene (MET) in patient RCC13.
    • Identification of MHC Class 1-Ligands
  • A total of 85 ligands could be obtained from tumor tissue, which were bound to HLA-subtypes HLA-A*02, HLA-A*68, HLA-B*18 or HLA-B*44. Peptides that bind to HLAA*02 reflected the allele-specific peptide motif (Leucine, Valine, Isoleucine, Alanine, Methionine on position 2, Leucine, Valine, Isoleucine or Alanine at the C-Terminus). Most ligands were derived from abundantly expressed housekeeping proteins, but ligands from proteins with reported tumor association could be detected also, for example, YVDPVITSI derived from met proto-oncogene, ALLNIKVKL derived from keratin 18, and SVASTITGV from adipophilin.
  • HLA-A*68 ligands were identified by their anchor amino acids Threonine, Isoleucine, Valine, Alanine or Leucine on position 2 and arginine or lysine at the C-terminus. Two other ligands from adipophilin were found among HLA-A*68-presented peptides: MTSALPIIQK and MAGDIYSVFR. Ligands carrying glutamic acid on position 2 were assigned to HLA-B*44; since the peptide motif of HLA-B*18 is unknown, a distinction between ligands of these two HLA-B-molecules was not possible.
  • Comparison of microarray data with the isolated ligands indicated 10 overexpressed genes as sources for MHC-ligands: adipophilin, KIAA0367, SEC14-like 1, B-cell translocation gene 1, aldolase A, cycline D1, annexin A4, catenin alpha 1, galectin 2 and LMP2. Three of them were also included in the SEREX database: KIAA0367, aldolase A and catenin alpha 1.
  • A most interesting ligand could be identified from patient RCC13 (ALAAVVTEV) encoded by a “Reading frame” shifted by one nucleotide compared to DEAD/H-box polypeptide 3 (DDX3). ALAAVVTEV is encoded by the nucleotides 317 to 343 of the coding strand of DDX3, whereas nucleotides 316 to 342 are coding for GIGSRGDRS of the DDX3 protein.
    • Detection of Specific T-ceHs in Normal CD8+ T-Cell Repertoire
  • PBMC from 6 HLA-A2 positive renal cell carcinoma patients were tested for reaction against four of the relevant peptides: HLA-A*02-restricted ligands from adipophilin, keratin 18, K1AA0367 and met-proto-oncogene. In doing so, a very sensitive quantitative PCT assay was carried out to detect γ-Interferon-mRNA production by CD8+ T-cells following a 7 day-in vitro-sensitization with peptide. Sporadic responses were seen after stimulation with met-proto-oncogene or keratin 18 or adipophilin peptides.
  • Staining of PBMC of tumor patients and healthy individuals with HLA-A*0201 tetramers was performed with tetramers reconstituted either with adipophilin, keratin 18 or met-proto-oncogene.
  • FIG. 2 shows the detection of keratin 18-specific T-lymphocytes. For this purpose, PBMC from four healthy HLA-A*02+ donors (HD 1, 2, 4, 6) were simultaneously stained with HLA-A2/keratin 18-PE tetramers, HLA-A2/CMV-APC tetramers, CD8-PerCP and CD4-FITC. Dot plots show samples from one of three independent experiments for 1×106 PBMC. In the plots, percentage of tetramer+-cells within the CD8+ CD4− population are indicated.
  • Unexpectedly, a significant population of CD8+ T-lymphocytes specific for keratin 18 (between 0.02% and 0.2% of CD8+ T-cells) was found in 4 out of 22 healthy individuals. This population did not stain with a CMV tetramer showing that the binding of keratin 18 tetramer was specific.
  • To summarize, it can be concluded that CD8+ T-lymphocytes specific for the keratin 18-peptide are contained in the human T-cell repertoire.

Claims (53)

1. Method for identifying immunoreactive peptides comprising the following steps:
a) providing a sample of tumorous and corresponding healthy tissue,
b) determining the tumor-specific expression profile in the provided sample,
c) isolation and analysis of antigenic peptides in the sample of tumorous tissue,
d) matching the data obtained in steps b) and c), and
e) identification of peptides on the basis of the matched data.
2. Method according to claim 1, wherein the antigenic peptides are MHC-ligands.
3. Method according to claim 1, wherein step b) is performed by microarray analysis and/or reversed transcriptase-polymerase chain reaction.
4. Method according to claim 2, wherein step b) is performed by microarray analysis and/or reversed transcriptase-polymerase chain reaction.
5. Method according to claim 1, wherein in step c) analysis is performed by mass spectrometry.
6. Method according to claim 2, wherein in step c) analysis is performed by mass spectrometry.
7. Method according to claim 3, wherein in step c) analysis is performed by mass spectrometry.
8. Method according to claim 5, wherein in step c) candidate antigenic peptides are predicted on basis of the expression profile using suitable databases, and that the mass spectrometer is calibrated in view of the peptides.
9. Method according to claim 6, wherein in step c) candidate antigenic peptides are predicted on basis of the expression profile using suitable databases, and that the mass spectrometer is calibrated in view of the peptides.
10. Method according to claim 7, wherein in step c) candidate antigenic peptides are predicted on basis of the expression profile using suitable databases, and that the mass spectrometer is calibrated in view of the peptides.
11. Method according to claim 1, wherein after step c) a further step is performed, in which the reactivity of peripheral leukocytes, preferably T-lymphocytes, is tested against the isolated antigenic peptides.
12. Method according to claim 2, wherein after step c) a further step is performed, in which the reactivity of peripheral leukocytes, preferably T-lymphocytes, is tested against the isolated antigenic peptides.
13. Method according to claim 3, wherein after step c) a further step is performed, in which the reactivity of peripheral leukocytes, preferably T-lymphocytes, is tested against the isolated antigenic peptides.
14. Method according to claim 4, wherein after step c) a further step is performed, in which the reactivity of peripheral leukocytes, preferably T-lymphocytes, is tested against the isolated antigenic peptides.
15. Method according to claim 5, wherein after step c) a further step is performed, in which the reactivity of peripheral leukocytes, preferably T-lymphocytes, is tested against the isolated antigenic peptides.
16. Method according to claim 11, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or γ-Interferon mRNA synthesized by the leukocytes.
17. Method according to claim 1, wherein after step c) a further step is performed, in which the presence of specific T-lymphocytes is detected.
18. Method according to claim 2, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or γ-Interferon mRNA synthesized by the leukocytes.
19. Method according to claim 3, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or γ-Interferon mRNA synthesized by the leukocytes.
20. Method according to claim 4, wherein the reactivity test is performed by means of measuring cytokine-mRNA and/or γ-Interferon mRNA synthesized by the leukocytes.
21. Method according to claim 17, wherein detection of specific T-lymphocytes is carried out by means of labeling leukocytes with reconstituted complexes of antigen-presenting molecules and antigenic peptide.
22. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 1 and that the identified peptide is synthesized chemically, in vitro or in vivo.
23. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 2 and that the identified peptide is synthesized chemically, in vitro or in vivo.
24. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 3 and that the identified peptide is synthesized chemically, in vitro or in vivo.
25. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 4 and that the identified peptide is synthesized chemically, in vitro or in vivo.
26. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 5 and that the identified peptide is synthesized chemically, in vitro or in vivo.
27. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 6 and that the identified peptide is synthesized chemically, in vitro or in vivo.
28. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 7 and that the identified peptide is synthesized chemically, in vitro or in vivo.
29. Method for preparing an immunoreactive peptide, in which a peptide is identified according to the method of claim 8 and that the identified peptide is synthesized chemically, in vitro or in vivo.
30. Immunoreactive peptide identified by a method according to claim 1.
31. Immunoreactive peptide, said peptide being prepared by a method according to claim 10.
32. Immunoreactive peptide identified by a method according to claim 2.
33. Immunoreactive peptide identified by a method according to claim 3.
34. Immunoreactive peptide identified by a method according to claim 4.
35. Immunoreactive peptide identified by a method according to claim 5.
36. Immunoreactive peptide identified by a method according to claim 6.
37. Immunoreactive peptide identified by a method according to claim 7.
38. Immunoreactive peptide identified by a method according to claim 8.
39. Pharmaceutical composition comprising one or more peptides according to claim 30.
40. Method for treating a tumor disease comprising the step of administering a peptide according to claim 11.
41. Method for assessing the therapy-course of a tumor disease, the method comprising the step of using a peptide according to claim 30.
42. Method for preparing an antibody, wherein a peptide according to claim 30 is used.
43. Nucleic acid molecule encoding a peptide according to claim 30.
44. Vector comprising a nucleic acid molecule according to claim 42.
45. Cell genetically modified by a nucleic acid molecule according to claim 43, which is producing a peptide according to claim 30.
46. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 1;
b) preparing the identified immunoreactive peptides; and
c) formulating the prepared immunoreactive peptides.
47. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 2, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
48. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 3, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
49. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 4, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
50. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 5, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
51. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 6, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
52. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 7, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
53. Method for preparing a vaccine comprising the steps of:
a) performing a method according to claim 8, and
b) preparing the identified immunoreactive peptides and
c) formulating the prepared immunoreactive peptides.
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