EP2318546A2 - Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use - Google Patents

Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use

Info

Publication number
EP2318546A2
EP2318546A2 EP09772574A EP09772574A EP2318546A2 EP 2318546 A2 EP2318546 A2 EP 2318546A2 EP 09772574 A EP09772574 A EP 09772574A EP 09772574 A EP09772574 A EP 09772574A EP 2318546 A2 EP2318546 A2 EP 2318546A2
Authority
EP
European Patent Office
Prior art keywords
prostate
marker sequences
seq
inflammatory diseases
marker
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Withdrawn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
EP09772574A
Other languages
German (de)
French (fr)
Inventor
Helmut E. Meyer
Angelika Lueking
Jens Beator
Axel Kowald
Georg Bartsch
Helmut Klocker
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.)
Protagen GmbH
Original Assignee
Protagen 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 Protagen GmbH filed Critical Protagen GmbH
Priority to EP20130190158 priority Critical patent/EP2712933A3/en
Priority to EP20130190149 priority patent/EP2712932A3/en
Priority to EP15152989.8A priority patent/EP2902505A3/en
Priority to EP20130190162 priority patent/EP2712934A3/en
Publication of EP2318546A2 publication Critical patent/EP2318546A2/en
Withdrawn legal-status Critical Current

Links

Classifications

    • 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
    • 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
    • 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
    • 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
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57434—Specifically defined cancers of prostate
    • 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
    • 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
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • 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
    • C12Q2600/158—Expression markers
    • 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
    • C12Q2600/16—Primer sets for multiplex assays
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/34—Genitourinary disorders
    • G01N2800/342—Prostate diseases, e.g. BPH, prostatitis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/56—Staging of a disease; Further complications associated with the disease

Definitions

  • the present invention relates to novel marker sequences for prostate inflammatory diseases, prostate carcinoma and their diagnostic use, including a method for the screening of potential drugs for such prostate diseases by means of these marker sequences. Furthermore, the invention relates to a diagnostic device containing such marker sequences for prostate inflammatory diseases, prostate carcinoma, in particular a protein biochip and its use.
  • Protein biochips are gaining an increasing industrial
  • Protein biochips have become established as screening tools.
  • the vectors used for the expression are generally characterized by the fact that they carry inducible promoters, with which the timing of protein expression can be controlled.
  • expression vectors have sequences for so-called affinity epitopes or proteins, which on the one hand allow the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, on the other hand, the specific
  • the gene products of a cDNA expression library of human fetal brain tissue in the bacterial expression system Escherichia coli in high density format were placed on a membrane and successfully screened with different antibodies. It could be shown that the proportion of full-length proteins is at least 66%.
  • the recombinant proteins from expression libraries could be expressed and purified at high throughput (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J.
  • Antibody arrays An embryonic but growing technology, DDT, 7, 143-149, Kusnezow et al., (2003) Antibody microarrays: An evaluation of production parameters, proteomics, 3, 254-264).
  • indication-specific diagnostic devices such as a protein biochip.
  • Laboratory parameters include acid phosphatase (SP) and prostate specific antigen (PSA) for diagnosis of SP.
  • SP acid phosphatase
  • PSA prostate specific antigen
  • Prostate cancer Especially the PSA currently has a high priority in diagnostics. It is specific for the prostate, but not for a tumor, but may also be elevated in inflammation, benign prostatic hyperplasia, urinary retention, or for no apparent reason. A value above 4 ng / ml is already in need of clarification.
  • the object of the present invention is to provide improved marker sequences and their diagnostic use for the treatment of prostate inflammatory diseases to prostate cancer.
  • the invention relates to the use of marker sequences for the diagnosis of
  • Prostate inflammatory diseases to prostate cancer particularly preferably prostate cancer, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or each one coding protein or each a subsequence or fragment thereof (hereinafter: marker sequences according to the invention) to or to a patient to be examined is determined.
  • the marker sequences according to the invention could by means of differential screening of samples and healthy subjects with patient samples with
  • Prostate inflammatory diseases prostate cancer can be identified.
  • these marker sequences according to the invention by means of protein biochips (see examples).
  • prostate inflammatory diseases to prostate cancer includes a group of diseases from prostate to the chronic forms of all prostate inflammations and their establishment as prostate cancer or prostate cancer (definition, for example, according to Pschyrembel, de Gruyter, 261st edition (2007), Berlin).
  • At least 2 to 5 or 10 preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences are determined on or to a patient to be examined.
  • the marker sequences according to the invention can also be combined, supplemented or extended with known biomarkers for this indication.
  • the determination of the marker sequences takes place outside the human body and the determination takes place in an ex vivo / in vitro diagnosis.
  • the invention relates to the use of marker sequences as diagnostic agents, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor or in each case a partial sequence or fragment thereof.
  • the invention relates to a method for the diagnosis of prostate inflammatory diseases to prostate carcinoma, wherein a.) At least one marker sequence of a cDNA selected from the group SEQ 1-174 or each a protein coding therefor or each of a partial sequence or fragment thereof applied to a solid support and b.) are in contact with body fluid or tissue extract of a patient and c.) the detection of an interaction of the body fluid or tissue extract with the marker sequences from a. ) he follows .
  • the invention also relates to diagnostic agents for the diagnosis of prostate inflammatory diseases to
  • the detection of such an interaction can be carried out for example by a probe, in particular by an antibody.
  • the invention therefore likewise has the object of providing a diagnostic device or an assay, in particular a protein biochip, which is suitable for the
  • the invention relates to a method for stratifying, in particular for risk stratification and / or therapy control of a patient with
  • Prostate inflammatory diseases to prostate carcinoma wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor is determined on a patient to be examined.
  • Prostate inflammatory diseases to prostate cancer as well as the meaningful selection of patient groups for the clinical development of new therapeutics.
  • Therapy control also includes the classification of patients into responders and non-responders with respect to therapy or its course of therapy.
  • Diagnosis in the sense of this invention means the positive determination of the prostate inflammatory diseases up to prostate cancer by means of the marker sequences according to the invention and the assignment of the patients to the
  • diagnosis includes medical diagnostics and related investigations, in particular in vitro diagnostics and laboratory diagnostics, also proteomics and nucleic acid blots. Further investigation may be needed to secure and exclude other diseases. Therefore, the term diagnosis also includes the differential diagnosis of prostate inflammatory diseases,
  • Prostate cancer by means of the marker sequences of the invention and the prognosis of prostate inflammatory diseases, prostate cancer.
  • Stratification also: stratification or therapy control
  • stratification means that the method according to the invention allows decisions for the treatment and therapy of the patient, be it hospitalization of the patient, use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a disease course as well as the course of therapy or etiology or classification of a disease, for example into a new or existing subtype or the differentiation of diseases and their patients.
  • the term "stratification" includes in particular the
  • patient is understood to mean any subject - human or mammal - with the proviso that that the subject is examined for prostate inflammatory diseases to prostate cancer.
  • marker sequences in the sense of this invention means that the cDNA or the respectively obtainable polypeptide or protein significantly for
  • Prostate inflammatory diseases prostate cancer are.
  • the cDNA or the respective polypeptide or protein obtainable therefrom may interact with substances from the body fluid or tissue extract of a patient with prostate inflammatory diseases up to the
  • Prostate carcinoma eg, antigen (epitope) / antibody (paratope) interaction
  • at least one marker sequence of a cDNA selected from the group SEQ. 1-174 or in each case a protein coding therefor or in each case a partial sequence or fragment thereof on a patient to be examined is determined" in that an interaction between the body fluid or tissue extract
  • Such an interaction is eg a binding, in particular a binding substance on at least one marker sequence according to the invention or in the case of a cDNA hybridization with a suitable substance under selected conditions, in particular stringent conditions (eg as defined in US Pat J. Sambrook, EF Fritsch, T.
  • the marker sequences according to the invention can be present in a significantly higher or lower expression rate or concentration, which points to the prostate inflammatory diseases, prostate carcinoma.
  • the relative expression rates ill / healthy of the marker sequences according to the invention for prostate inflammatory diseases, prostate carcinoma is determined by means of proteomics or nucleic acid blots.
  • the marker sequences have a recognition signal which is addressed to the substance to be bound (for example antibodies,
  • the recognition signal for a protein is preferably an epitope and / or paratope and / or hapten and, for a cDNA, a hybridization or binding region.
  • the marker sequences according to the invention are the subject of Table A and can be clearly identified by the respective cited database entry (also by means of the Internet: http://www.ncbi.nlm.nih.gov/) (see Table A: there Accession No.), see also the associated sequence listing.
  • the invention also relates to the full-length sequences of the markers according to the invention and indeed as defined in Table 1 on the known database entry according to Table A, hereinafter called SEQ ID NO: 174a.
  • the specific marker sequences SEQ 1-174 are preferred according to the invention.
  • the marker sequences also include such modifications of the cDNA sequence and the corresponding amino acid sequence, such as chemical modification, such as citrullination, acetylation, phosphorylation,
  • partial sequences or fragments of the marker sequences according to the invention are also included.
  • those partial sequences which have an identity of 95%, 90%, in particular 80% or 70% with the marker sequences according to the invention are also included.
  • Partial sequences are also those sequences having 50 to 100 nucleotides, 70-120 nucleotides of a sequence of SEQ 1-174, or peptides obtainable therefrom.
  • the respective marker sequence may be represented in different amounts in one or more regions on a solid support.
  • the regions can each have an entirety of marker sequences, ie a sufficient number of different marker sequences, in particular 2 to 5 or 10 or more and, if appropriate, further nucleic acids and / or proteins, in particular biomarkers.
  • at least 96 to 25,000 (numerically) or more are preferred from different or identical marker sequences and further nucleic acids and / or proteins, in particular biomarkers.
  • Further preferred are more than 2,500, more preferably 10,000 or more different or the same Marker sequences and, if necessary, further nucleic acids and / or proteins, in particular biomarkers.
  • Another object of the invention relates to an array of marker sequences containing at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor.
  • the array contains at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences.
  • “arrangement” synonymously means “array” and insofar as this "array” is used to identify substances on marker sequences, this is to be understood as meaning an “assay” or a diagnostic device.
  • the arrangement is such that the marker sequences represented on the array are in the form of a grid on a solid support. Further, such arrangements are preferred which allow a high density array of protein binders and spotting the marker sequences. Such high density spotted assemblies are disclosed, for example, in WO 99/57311 and WO 99/57312, and may be advantageously used in a robotic automated high throughput method.
  • test or diagnostic device also encompasses such embodiments of a device as ELISA, bead-based assay, line assay, Western blot, immunochromatographic methods (eg, so-called lateral flow immunoassays) or similar immunological single or Multiplex detection method
  • a protein biochip in the sense of this invention is the systematic arrangement of proteins on a solid support.
  • the marker sequences of the assembly are fixed to a solid support, but preferably spotted or immobilized imprinted, ie applied reproducible.
  • One or more marker sequences may be present several times in the totality of all marker sequences and be present in different amounts relative to one spot.
  • the marker sequences can be standardized on the solid support (eg by means of serial dilution series of eg human globulins as internal calibrators for data normalization and quantitative evaluation).
  • the invention relates to an assay or protein biochip consisting of an array containing marker sequences according to the invention.
  • the marker sequences are present as clones.
  • Such clones can be obtained, for example, by means of a cDNA expression library according to the invention (Büssow et al., 1998 (supra)).
  • expression libraries containing clones are obtained by means of expression vectors from an expressing cDNA library consisting of the cDNA marker sequences. These expression vectors preferably contain inducible promoters. The induction of
  • Expression can e.g. by means of an inductor, such as IPTG.
  • IPTG inductor
  • Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol 2003 Jan; 60 (5): 523-33).
  • Expression libraries are known to the person skilled in the art, and these can be prepared according to standard works, such as Sambrook et al., Molecular Cloning, Laboratory Handbook, 2nd edition (1989), CSH press, CoId Spring Harbor, New York Further preferred are expression libraries which are tissue-specific In addition, according to the invention, expression libraries which can be obtained by exon trapping are also included in the invention, and instead of an expression library it is possible to speak synonymously of an expression library. Also preferred are protein biochips or corresponding expression libraries which have no redundancy (so-called: Uniclone® library) and can be prepared, for example, according to the teachings of WO 99/57311 and WO 99/57312. These preferred Uniclone libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.
  • the clones may not be such as transformed bacteria, recombinant phage or transformed cells of mammals, insects, fungi, yeasts or plants.
  • the clones are fixed on a solid support, spotted or immobilized.
  • the invention relates to an arrangement wherein the marker sequences are present as clones.
  • the marker sequences may be in the form of a fusion protein containing, for example, at least one affinity epitope or "tag".
  • the tag may be one such as c-myc, His-tag, Arg-tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or Strep tag, HAT tag, NusA, S-tag, SBP tag , Thioredoxin, DsbA, a fusion protein, preferably a cellulosic binding domain, green fluorescent protein, maltose binding protein, calmodulin binding protein, glutathione S-transferase or lacZ.
  • solid support includes embodiments such as a filter, a membrane, a magnetic or fluorophore-labeled bead, a silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target, or a matrix.
  • a filter is preferred according to the invention.
  • PVDF, nitrocellulose or nylon are also preferred as filters (eg Immobilon P Millipore, Protran Whatman, Hybond N + Amersham).
  • this corresponds to a grid having the size of a microtiter plate (8-12 wells strips, 96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometric target or a matrix.
  • the invention relates to an assay or protein biochip for identifying and characterizing a substance for
  • Prostate inflammatory diseases Prostate cancer, characterized in that an arrangement or assay according to the invention with a.) At least one substance to be examined is brought into contact and b.) A binding success is detected.
  • the invention further relates to a method for identifying and characterizing a substance for prostate inflammatory diseases, prostate carcinoma, characterized in that an arrangement or assay according to the invention is brought into contact with a.) At least one substance to be investigated and b.) A binding success is detected.
  • the substance to be assayed may be any native or non-native biomolecule, synthetic chemical molecule, mixture or substance library.
  • the evaluation of the binding success is carried out, for example, using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience).
  • the visualization of protein-protein interactions according to the invention can be, for example, by fluorescence labeling, biotinization, radio-isotopic labeling or colloidal gold or latex particle labeling
  • Bound antibodies are detected by secondary antibodies labeled with commercially available reporter molecules (eg Cy, Alexa, Dyomics, FITC or similar fluorescent dyes, colloidal gold or latex particles), or with reporter enzymes such as alkaline phosphatase, horseradish peroxidase, etc., and the corresponding colorimetric, fluorescent, or chemiluminescent substrates, for example, by means of a microarray laser scanner, a CCD camera, or visually.
  • reporter molecules eg Cy, Alexa, Dyomics, FITC or similar fluorescent dyes, colloidal gold or latex particles
  • reporter enzymes such as al
  • the invention relates to a drug / drug or prodrug for prostate inflammatory diseases, prostate carcinoma developed and obtainable by the use of the assay or protein biochip according to the invention.
  • the invention also relates to the use of an inventive arrangement or an assay for the screening of drugs for prostate inflammatory diseases, prostate carcinoma.
  • the invention also relates to a target for the treatment and therapy of prostate inflammatory diseases, prostate carcinoma, each selected from the group SEQ 1-174 or in each case a protein coding therefor.
  • the invention also relates to the use of the marker sequences according to the invention, preferably in the form of an arrangement, as Affinity material for performing an apheresis or iwS. a blood wash, wherein substances from body fluids of a patient with prostate inflammatory diseases, prostate cancer, such as blood or plasma, bind to the marker sequences according to the invention and thus the body fluid can be selectively withdrawn.
  • FIG. 1 shows the differential screening between two protein biochips from in each case one cDNA expression bank of a patient and one healthy subject.
  • the differential clones are detected by fluorescence labeling and evaluated bioinformatorisch.
  • Threshold method which is suitable for both classification and visual representation of the data.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Immunology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Molecular Biology (AREA)
  • Pathology (AREA)
  • Zoology (AREA)
  • Genetics & Genomics (AREA)
  • Wood Science & Technology (AREA)
  • Physics & Mathematics (AREA)
  • General Health & Medical Sciences (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Hospice & Palliative Care (AREA)
  • General Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biophysics (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The present invention relates to novel marker sequences for inflammatory prostate diseases, prostate carcinoma and the diagnostic use thereof including a method for screening potential active substances for such prostate diseases using these marker sequences. The invention furthermore relates to a diagnostic device containing such marker sequences for inflammatory prostate diseases, prostate carcinoma, in particular a protein biochip, and to its use.

Description

Markersequenzen für Prostataentzündungserkrankungen, Prostatakarzinom und deren VerwendungMarker sequences for prostate inflammatory diseases, prostate cancer and their use
Beschreibungdescription
Die vorliegende Erfindung betrifft neue Markersequenzen für Prostataentzündungserkrankungen, Prostatakarzinom und deren diagnostische Verwendung samt einem Verfahren zum Screenen von potentiellen Wirkstoffen für solche Prostata - Erkrankungen mittels dieser Markersequenzen. Ferner betrifft die Erfindung eine diagnostische Vorrichtung enthaltend solche Markersequenzen für Prostataentzündungserkrankungen, Prostatakarzinom, insbesondere ein Proteinbiochip und dessen Verwendung .The present invention relates to novel marker sequences for prostate inflammatory diseases, prostate carcinoma and their diagnostic use, including a method for the screening of potential drugs for such prostate diseases by means of these marker sequences. Furthermore, the invention relates to a diagnostic device containing such marker sequences for prostate inflammatory diseases, prostate carcinoma, in particular a protein biochip and its use.
Proteinbiochips gewinnen eine zunehmende industrielleProtein biochips are gaining an increasing industrial
Bedeutung in der Analytik und Diagnostik sowie in derMeaning in analytics and diagnostics as well as in the
Pharmaentwicklung . Proteinbiochips haben sich als Screeninginstrumente etabliert.Pharmaceutical development. Protein biochips have become established as screening tools.
Hierbei wird die schnelle und hochparallele Detektion einer Vielzahl spezifisch bindender Analysemoleküle in einem einzigen Experiment ermöglicht. Zur Herstellung von Proteinbiochips ist es erforderlich, die benötigten Proteine zur Verfügung zu haben. Hierzu haben sich insbesondere Protein-Expressionsbibliotheken etabliert. Die Hochdurchsatz- Klonierung von definierten offenen Leserahmen ist eineHere, the fast and highly parallel detection of a variety of specific binding molecules is made possible in a single experiment. For the production of protein biochips, it is necessary to have the required proteins available. In particular, protein expression libraries have been established for this purpose. The high-throughput cloning of defined open reading frames is one
Möglichkeit (Heyman, J. A., Cornthwaite, J., Foncerrada, L., Gilmore, J. R., Gontang, E., Hartman, K.J., Hernandez, CL. , Hood, R., HuIl, H. M., Lee, W. Y., Marcil, R., Marsh, E. J., Mudd, K.M., Patino, M. J., Purcell, T. J., Rowland, J. J., Sindici, M. L. and Hoeffler, J. P. (1999) Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res, 9, 383-392; Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor, M.I., Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H. and Cahill, D. J. (2003) Generation of Arabidopsis protein chip for antibody and serum Screening. Plant Molecular Biology, 52, 999-1010; Reboul, J., Vaglio, P., Rual, J. F., Lamesch, P., Martinez, M., Armstrong, CM. , Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, J. R., Jr., Hartley, J. L., Brasch, M. A., Vandenhaute, J., Boulton, S., Endress, G. A., Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, P.P., Ptacek, J., Snyder, M., Huang, R., Chance, M. R., Lee, H., Doucette-Stamm, L., Hill, D.E. and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet, 34, 35-41.; Walhout, A. J., Temple, G. F., Brasch, M. A., Hartley, J. L., Lorson, M. A., van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames or ORFeomes. MethodsPossibility (Heyman, JA, Cornthwaite, J., Foncerrada, L., Gilmore, JR, Gontang, E., Hartman, KJ, Hernandez, CL., Hood, R., Huill, HM, Lee, WY, Marcil, R ., Marsh, EJ, Mudd, KM, Patino, MJ, Purcell, TJ, Rowland, JJ, Sindici, ML and Hoeffler, JP (1999) Genome-scale cloning and expression of individual open reading frames using topoisomerase I-mediated ligation. Genome Res, 9, 383-392; Kersten, B., Feilner, T., Kramer, A., Wehrmeyer, S., Possling, A., Witt, I., Zanor, MI, Stracke, R., Lueking, A., Kreutzberger, J., Lehrach, H. and Cahill, DJ (2003) Generation of Arabidopsis protein chip for antibody and serum screening. Plant Molecular Biology, 52, 999-1010; Reboul, J., Vaglio, P., Rual, JF, Lamesch, P., Martinez, M., Armstrong, CM. , Li, S., Jacotot, L., Bertin, N., Janky, R., Moore, T., Hudson, JR, Jr., Hartley, JL, Brasch, MA, Vandenhaute, J., Boulton, S. , Endress, GA, Jenna, S., Chevet, E., Papasotiropoulos, V., Tolias, PP, Ptacek, J., Snyder, M., Huang, R., Chance, MR, Lee, H., Doucette- Stamm, L., Hill, DE and Vidal, M. (2003) C. elegans ORFeome version 1.1: experimental verification of the genome annotation and resource for proteome-scale protein expression. Nat Genet, 34, 35-41; Walhout, AJ, Temple, GF, Brasch, MA, Hartley, JL, Lorson, MA, van den Heuvel, S. and Vidal, M. (2000) GATEWAY recombinational cloning: application to the cloning of large numbers of open reading frames ORFeomes. Methods
Enzymol, 328, 575-592). Allerdings hängt ein solcher Ansatz stark mit dem Fortschritt der Genom-Sequenzierungsprojekte und der Annotierung dieser Gensequenzen zusammen. Darüber hinaus ist die Bestimmung der exprimierten Sequenz aufgrund differenzieller Spleißvorgänge nicht immer eindeutig. Dieses Problem kann durch die Anwendung von cDNA-Enzymol, 328, 575-592). However, such an approach is strongly related to the progress of genome sequencing projects and the annotation of these gene sequences. Moreover, the determination of the expressed sequence is not always clear due to differential splicing events. This problem can be overcome by the use of cDNA
Expressionsbibliotheken umgangen werden (Büssow, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach, H. and Walter, G. (1998) A method for global protein expression and antibody Screening on high-density filters of an arrayed cDNA library. Nucleic Acids Research, 26, 5007-5008; Büssow, K., Nordhoff, E., Lübbert, C, Lehrach, H. and Walter, G. (2000) A human cDNA library for high-throughput protein expression Screening. Genomics, 65, 1-8; Holz, C, Lueking, A., Bovekamp, L., Gut jähr, C, Bolotina, N., Lehrach, H. and Cahill, D. J. (2001) A human cDNA expression library in yeast enriched for open reading frames. Genome Res, 11, 1730-1735; Lueking, A., Holz, C, Gotthold, C, Lehrach, H. and Cahill, D. (2000) A System for dual protein expression in Pichia pastoris and Escherichia coli, Protein Expr. Purif., 20, 372- 378) . Hierbei wird die cDNA eines bestimmten Gewebes in einen bakteriellen oder einen eukaryotischen Expressionsvektor, wie z.B. Hefe, einkloniert. Die für die Expression verwendeten Vektoren zeichnen sich im Allgemeinen dadurch aus, dass sie induzierbare Promotoren tragen, mit denen sich der Zeitpunkt der Proteinexpression steuern lässt. Darüber hinaus weisen Expressionsvektoren Sequenzen für so genannte Affinitätsepitope oder -proteine auf, die zum einen den spezifischen Nachweis der rekombinanten Fusions-Proteine mittels eines gegen das Affinitätsepitop gerichteten Antikörpers erlauben, zum anderen wird die spezifischeBüssow, K., Cahill, D., Nietfeld, W., Bancroft, D., Scherzinger, E., Lehrach, H. and Walter, G. (1998) A method for global protein expression and antibody screening Nucleic Acids Research, 26, 5007-5008, Büssow, K., Nordhoff, E., Lübbert, C, Lehrach, H. and Walter, G. (2000) A human cDNA library for high-throughput protein expression screening, Genomics, 65, 1-8, Holz, C, Lueking, A., Bovekamp, L., Gutjahr, C, Bolotina, N., Lehrach, H. and Cahill, DJ ( Genome Res, 11, 1730-1735; Lueking, A., Holz, C, Gotthold, C, Lehrach, H. and Cahill, D. (2000) A System for dual protein expression in Pichia pastoris and Escherichia coli, Protein Expr. Purif., 20, 372-378). Here, the cDNA of a particular tissue in a bacterial or a eukaryotic expression vector, such as yeast, cloned. The vectors used for the expression are generally characterized by the fact that they carry inducible promoters, with which the timing of protein expression can be controlled. In addition, expression vectors have sequences for so-called affinity epitopes or proteins, which on the one hand allow the specific detection of the recombinant fusion proteins by means of an antibody directed against the affinity epitope, on the other hand, the specific
Aufreinigung über Affinitätschromatographie (IMAC) ermöglicht.Purification via affinity chromatography (IMAC) allows.
Beispielsweise wurden die Genprodukte einer cDNA- Expressionsbibliothek aus humanem fötalem Hirngewebe in dem bakteriellen Expressionssystem Escherichia coli im Hochdichte- Format auf einer Membran angeordnet und konnten erfolgreich mit unterschiedlichen Antikörpern gescreent werden. Es konnte gezeigt werden, dass der Anteil an Volllänge-Proteinen bei mindestens 66% liegt. Die rekombinanten Proteine aus Expressionsbibliotheken konnten darüber hinaus im Hochdurchsatz exprimiert und aufgereinigt werden (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002) Proteome-scale purification of human proteins from bacteria. Proc Natl Acad Sei U S A, 99, 2654- 2659; Büssow (2000) supra; Lueking, A., Hörn, M., Eickhoff, H., Büssow, K., Lehrach, H. and Walter, G. (1999) Protein microarrays for gene expression and antibody Screening. Analytical Biochemistry, 270, 103-111). Solche Proteinbiochips auf der Basis von cDNA-Expressionsbibliotheken sind insbesondere Gegenstand der WO 99/57311 und WO 99/57312.For example, the gene products of a cDNA expression library of human fetal brain tissue in the bacterial expression system Escherichia coli in high density format were placed on a membrane and successfully screened with different antibodies. It could be shown that the proportion of full-length proteins is at least 66%. In addition, the recombinant proteins from expression libraries could be expressed and purified at high throughput (Braun P., Hu, Y., Shen, B., Halleck, A., Koundinya, M., Harlow, E. and LaBaer, J. (2002 Proc Natl Acad., USA, 99, 2654-2659; Büssow (2000) supra; Lueking, A., Horn, M., Eickhoff, H., Büssow, K., Lehrach , H. and Walter, G. (1999) Protein microarray for gene expression and antibody screening., Analytical Biochemistry, 270, 103-111). Such protein biochips based on cDNA expression libraries are in particular the subject of WO 99/57311 and WO 99/57312.
Ferner sind neben Antigen-präsentierenden Proteinbiochips ebenfalls Antikörper-präsentierende Anordnungen beschrieben (LaI et al (2002) Antibody arrays : An embryonic but rapidly growing technology, DDT, 7, 143-149; Kusnezow et al . (2003), Antibody microarrays: An evaluation of produetion parameters, Proteomics, 3, 254-264) . Es besteht jedoch ein hohes Bedürfnis indikationsspezifische diagnostische Vorrichtungen, wie einen Proteinbiochip, bereitzustellen .In addition to antigen-presenting protein biochips, antibody-presenting arrangements are also described (LaI et al (2002) Antibody arrays: An embryonic but growing technology, DDT, 7, 143-149, Kusnezow et al., (2003) Antibody microarrays: An evaluation of production parameters, proteomics, 3, 254-264). However, there is a great need to provide indication-specific diagnostic devices, such as a protein biochip.
Zu den Laborparametern gehören die saure Phosphatase (SP) und das prostataspezifische Antigen (PSA) zur Diagnose desLaboratory parameters include acid phosphatase (SP) and prostate specific antigen (PSA) for diagnosis of
Prostatakarzinom. Vor allem das PSA hat derzeit einen hohen Stellenwert in der Diagnostik. Es ist spezifisch für die Prostata, allerdings nicht für ein Tumorleiden, sondern kann auch bei Entzündungen, benigner Prostatahyperplasie, einem Harnverhalt oder ohne ersichtlichen Grund erhöht sein. Ein Wert über 4 ng/ml gilt bereits als abklärungsbedürftig.Prostate cancer. Especially the PSA currently has a high priority in diagnostics. It is specific for the prostate, but not for a tumor, but may also be elevated in inflammation, benign prostatic hyperplasia, urinary retention, or for no apparent reason. A value above 4 ng / ml is already in need of clarification.
Die Aufgabe der vorliegenden Erfindung ist die Bereitstellung von verbesserten Markersequenzen und deren diagnostische Verwendung zur Behandlung von Prostataentzündungserkrankungen bis hin zum Prostatakarzinom.The object of the present invention is to provide improved marker sequences and their diagnostic use for the treatment of prostate inflammatory diseases to prostate cancer.
Die Bereitstellung von spezifischen Markersequenzen erlaubt eine sichere Diagnose und Stratifizierung von Patienten mit Prostataentzündungserkrankungen bis hin zum Prostatakarzinom, insbesondere mittels eines Proteinbiochips.The provision of specific marker sequences allows a reliable diagnosis and stratification of patients with prostate inflammatory diseases to prostate cancer, in particular by means of a protein biochip.
Daher betrifft die Erfindung die Verwendung von Markersequenzen zur Diagnose vonTherefore, the invention relates to the use of marker sequences for the diagnosis of
Prostataentzündungserkrankungen bis hin zum Prostatakarzinom, besonders bevorzugt Prostatakarzinom, wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon (nachstehend: erfindungsgemäße Markersequenzen) an oder zu einem zu untersuchenden Patienten bestimmt wird.Prostate inflammatory diseases to prostate cancer, particularly preferably prostate cancer, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or each one coding protein or each a subsequence or fragment thereof (hereinafter: marker sequences according to the invention) to or to a patient to be examined is determined.
Die erfindungsgemäßen Markersequenzen konnten mittels differentiellem Screenen von Proben und zwar gesunder Probanden mit Patientenproben mitThe marker sequences according to the invention could by means of differential screening of samples and healthy subjects with patient samples with
Prostataentzündungserkrankungen, Prostatakarzinom identifiziert werden. Hierbei konnte erstmals mittels Proteinbiochips (siehe Beispiele) diese erfindungsgemäßen Markersequenzen identifiziert werden.Prostate inflammatory diseases, prostate cancer can be identified. For the first time, it was possible to identify these marker sequences according to the invention by means of protein biochips (see examples).
Der Begriff „Prostataentzündungserkrankungen bis hin zum Prostatakarzinom" umfasst eine Gruppe von Erkrankungen von Prostatis bis hin zu den chronischen Formen sämtlicher Prostataentzündungen und deren Etablierung als Prostatakrebs oder Prostatakarzimon (Definition z.B. nach Pschyrembel, de Gruyter, 261. Auflage (2007), Berlin).The term "prostate inflammatory diseases to prostate cancer" includes a group of diseases from prostate to the chronic forms of all prostate inflammations and their establishment as prostate cancer or prostate cancer (definition, for example, according to Pschyrembel, de Gruyter, 261st edition (2007), Berlin).
In einer weiteren Ausführungsform werden daher mindestens 2 bis 5 oder 10, vorzugsweise 30 bis 50 Markersequenzen oder 50 bis 100 oder mehr Markersequenzen an oder zu einem zu untersuchenden Patienten bestimmt.In a further embodiment, therefore, at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences are determined on or to a patient to be examined.
In einer weiteren Ausführungsform der Erfindung können die erfindungsgemäßen Markersequenzen ebenfalls mit bekannten Biomarkern für diese Indikation kombiniert, ergänzt oder erweitert werden.In a further embodiment of the invention, the marker sequences according to the invention can also be combined, supplemented or extended with known biomarkers for this indication.
In einer bevorzugten Ausführungsform erfolgt die Bestimmung der Markersequenzen außerhalb des menschlichen Körpers und die Bestimmung erfolgt in einer ex vivo / in vitro Diagnose.In a preferred embodiment, the determination of the marker sequences takes place outside the human body and the determination takes place in an ex vivo / in vitro diagnosis.
In einer weiteren Ausführungsform der Erfindung betrifft die Erfindung die Verwendung von Markersequenzen als Diagnostika, wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon ist .In a further embodiment of the invention, the invention relates to the use of marker sequences as diagnostic agents, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor or in each case a partial sequence or fragment thereof.
Ferner betrifft die Erfindung ein Verfahren zur Diagnose von Prostataentzündungserkrankungen bis hin zum Prostatakarzinom, wobei a.) mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon auf einem festen Träger aufgebracht wird und b.) mit Körperflüssigkeit oder Gewebeauszug eines Patienten in Kontakt gebracht wird und c.) der Nachweis einer Wechselwirkung der Körperflüssigkeit oder Gewebeauszug mit den Markersequenzen aus a . ) erfolgt .Furthermore, the invention relates to a method for the diagnosis of prostate inflammatory diseases to prostate carcinoma, wherein a.) At least one marker sequence of a cDNA selected from the group SEQ 1-174 or each a protein coding therefor or each of a partial sequence or fragment thereof applied to a solid support and b.) are in contact with body fluid or tissue extract of a patient and c.) the detection of an interaction of the body fluid or tissue extract with the marker sequences from a. ) he follows .
Daher betrifft die Erfindung ebenfalls Diagnostika zur Diagnose von Prostataentzündungserkrankungen bis hin zumTherefore, the invention also relates to diagnostic agents for the diagnosis of prostate inflammatory diseases to
Prostatakarzinom jeweils ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon.Prostate carcinoma in each case selected from the group SEQ 1-174 or in each case a protein coding therefor or in each case a partial sequence or fragment thereof.
In einer besonders bevorzugten Ausführungsform sind die Markersequenzen SEQ 136, 40, 127, 83, 16, 82, 88, 152, 130,In a particularly preferred embodiment, the marker sequences SEQ 136, 40, 127, 83, 16, 82, 88, 152, 130,
138, 2, 12, 113, 20, 173, 33, 172, 52, 43, 91, 1, 32, 86, 27, 105 in dieser Reihenfolge bevorzugt.138, 2, 12, 113, 20, 173, 33, 172, 52, 43, 91, 1, 32, 86, 27, 105 are preferred in this order.
Der Nachweis einer solchen Wechselwirkung kann beispielsweise durch eine Sonde, insbesondere durch einen Antikörper erfolgen.The detection of such an interaction can be carried out for example by a probe, in particular by an antibody.
Daher betrifft die Erfindung ebenfalls die Aufgabe eine diagnostische Vorrichtung oder einen Assay, insbesondere einen Proteinbiochip, bereitzustellen, der für dieThe invention therefore likewise has the object of providing a diagnostic device or an assay, in particular a protein biochip, which is suitable for the
Prostataentzündungserkrankungen bis hin zum Prostatakarzinom eine Diagnose oder Untersuchung erlaubt.Prostate inflammatory diseases to prostate cancer diagnosis or examination allowed.
Ferner betrifft die Erfindung ein Verfahren zum Stratifizieren, insbesondere zur Risikostratifizierung und / oder Therapiesteuerung eines Patienten mitFurthermore, the invention relates to a method for stratifying, in particular for risk stratification and / or therapy control of a patient with
Prostataentzündungserkrankungen bis hin zum Prostatakarzinom, wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein an einem zu untersuchenden Patienten bestimmt wird.Prostate inflammatory diseases to prostate carcinoma, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor is determined on a patient to be examined.
Ferner umfasst ist die Stratifizierung der Patienten mit Prostataentzündungserkrankungen bis hin zum Prostatakarzinom in neue oder etablierte Subgruppen derFurthermore, the stratification of patients with prostate inflammatory diseases to prostate cancer in new or established subgroups of the
Prostataentzündungserkrankungen bis hin zum Prostatakarzinom, sowie die sinnvolle Auswahl von Patientengruppen für die klinische Entwicklung von neuen Therapeutika. Der Begriff Therapiesteuerung umfasst ebenfalls die Einteilung von Patienten in Responder und Nicht-Responder bezüglich einer Therapie oder dessen Therapieverlauf.Prostate inflammatory diseases to prostate cancer, as well as the meaningful selection of patient groups for the clinical development of new therapeutics. The term Therapy control also includes the classification of patients into responders and non-responders with respect to therapy or its course of therapy.
„Diagnose" im Sinne dieser Erfindung bedeutet die positive Feststellung der Prostataentzündungserkrankungen bis hin zum Prostatakarzinom mittels der erfindungsgemäßen Markersequenzen sowie die Zuordnung der Patienten zu den"Diagnosis" in the sense of this invention means the positive determination of the prostate inflammatory diseases up to prostate cancer by means of the marker sequences according to the invention and the assignment of the patients to the
Prostataentzündungserkrankungen bis hin zum Prostatakarzinom. Der Begriff der Diagnose umfasst die medizinische Diagnostik und diesbezügliche Untersuchungen, insbesondere die in-vitro Diagnostik und Labordiagnostik, ebenfalls Proteomics und Nukleinsäureblots . Weitere Untersuchungen können zur Absicherung und zum Ausschluss anderer Krankheiten vonnöten sein. Daher umfasst der Begriff Diagnose ebenfalls die Differentialdiagnose von Prostataentzündungserkrankungen,Prostate inflammatory diseases to prostate cancer. The term diagnosis includes medical diagnostics and related investigations, in particular in vitro diagnostics and laboratory diagnostics, also proteomics and nucleic acid blots. Further investigation may be needed to secure and exclude other diseases. Therefore, the term diagnosis also includes the differential diagnosis of prostate inflammatory diseases,
Prostatakarzinom mittels der erfindungsgemäßen Markersequenzen sowie die Prognose der Prostataentzündungserkrankungen, Prostatakarzinom.Prostate cancer by means of the marker sequences of the invention and the prognosis of prostate inflammatory diseases, prostate cancer.
„Stratifizieren (auch: Stratifikation) oder Therapiesteuerung" im Sinne dieser Erfindung bedeutet, dass das erfindungsgemäße Verfahren Entscheidungen zur Behandlung und Therapie des Patienten erlaubt, sei es Hospitalisierung des Patienten, Einsatz, Wirkung und / oder Dosierung eines oder mehrerer Arzneimittel, eine therapeutische Maßnahme oder die Überwachung eines Krankheitsverlaufes sowie Therapieverlauf bzw. Ätiologie oder Klassifizierung einer Erkrankung, z.B. in einen neuen oder bestehenden Subtyp oder die Differenzierung von Krankheiten und dessen Patienten."Stratification (also: stratification) or therapy control" in the sense of this invention means that the method according to the invention allows decisions for the treatment and therapy of the patient, be it hospitalization of the patient, use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a disease course as well as the course of therapy or etiology or classification of a disease, for example into a new or existing subtype or the differentiation of diseases and their patients.
In einer weiteren Ausführungsform der Erfindung umfasst der Begriff „Stratifizierung" insbesondere dieIn a further embodiment of the invention, the term "stratification" includes in particular the
Risikostratifizierung mit der Prognose eines „outcome" eines nachteiligen gesundheitlichen Ereignisses.Risk stratification with the prognosis of an "outcome" of an adverse health event.
Im Rahmen dieser Erfindung wird unter „Patient" ein beliebiger Proband - Mensch oder Säugetier - verstanden, mit der Maßgabe, dass der Proband auf Prostataentzündungserkrankungen bis hin zum Prostatakarzinom untersucht wird.In the context of this invention, "patient" is understood to mean any subject - human or mammal - with the proviso that that the subject is examined for prostate inflammatory diseases to prostate cancer.
Der Begriff „Markersequenzen" im Sinne dieser Erfindung bedeutet, dass die cDNA oder das jeweils daraus erhältliche Polypeptid oder Protein signifikant fürThe term "marker sequences" in the sense of this invention means that the cDNA or the respectively obtainable polypeptide or protein significantly for
Prostataentzündungserkrankungen, Prostatakarzinom sind. Beispielsweise können die cDNA oder das jeweils daraus erhältliche Polypeptid oder Protein eine Wechselwirkung mit Substanzen aus der Körperflüssigkeit oder Gewebeauszug eines Patienten mit Prostataentzündungserkrankungen bis hin zumProstate inflammatory diseases, prostate cancer are. For example, the cDNA or the respective polypeptide or protein obtainable therefrom may interact with substances from the body fluid or tissue extract of a patient with prostate inflammatory diseases up to the
Prostatakarzinom aufweisen (z.B. Antigen (Epitop) / Antikörper (Paratop) Wechselwirkung) . Im Sinne der Erfindung bedeutet „wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon an einem zu untersuchenden Patienten bestimmt wird", dass eine Wechselwirkung zwischen der Körperflüssigkeit oder Gewebeauszuges eines Patienten und den erfindungsgemäßen Markersequenzen nachgewiesen wird. Eine solche Wechselwirkung ist z.B. eine Bindung, insbesondere eine bindende Substanz an mindestens einer erfindungsgemäßen Markersequenz oder im Fall einer cDNA die Hybridisierung mit einer geeigneten Substanz unter gewählten Bedingungen, insbesondere stringenten Bedingungen (z.B. wie üblich definiert in J. Sambrook, E. F. Fritsch, T. Maniatis (1989), Molecular cloning: A laboratory manual, 2nd Edition, CoId Spring Habor Laboratory Press, CoId Spring Habor, USA oder Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, N. Y. (1989)). Ein Beispiel für stringente Hybridisierungsbedingungen ist: Hybridisierung in 4 x SSC bei 65° C (alternativ in 50% Formamid und 4 X SSC bei 42° C), gefolgt von mehreren Waschschritten in 0,1 x SSC bei 650C für insgesamt etwa eine Stunde. Ein Beispiel für wenig stringente Hybridisierungsbedingungen ist Hybridisierung in 4 x SSC bei 37° C, gefolgt von mehreren Waschritten in 1 x SSC bei Raumtemperatur . Solche Substanzen sind erfindungsgemäß Bestandteil einer Körperflüssigkeit, insbesondere Blut, Vollblut, Blutplasma, Blutserum, Patientenserum, Urin, Cerebrospinalflüssigkeit , Synovialflüssigkeit oder eines Gewebeauszuges des Patienten.Prostate carcinoma (eg, antigen (epitope) / antibody (paratope) interaction). For the purposes of the invention, "at least one marker sequence of a cDNA selected from the group SEQ. 1-174 or in each case a protein coding therefor or in each case a partial sequence or fragment thereof on a patient to be examined is determined" in that an interaction between the body fluid or tissue extract Such an interaction is eg a binding, in particular a binding substance on at least one marker sequence according to the invention or in the case of a cDNA hybridization with a suitable substance under selected conditions, in particular stringent conditions (eg as defined in US Pat J. Sambrook, EF Fritsch, T. Maniatis (1989), Molecular Cloning: A laboratory manual, 2nd Edition, CoId Spring Habor Laboratory Press, CoId Spring Habor, USA or Ausubel, "Current Protocols in Molecular Biology", Green Publishing Associates and Wiley Interscience, NY . (1989)) One example of stringent hybridization conditions is hybridization in 4 x SSC at 65 ° C (alternatively, in 50% formamide and 4 x SSC at 42 ° C), followed by several washes in 0.1 x SSC at 65 0 C for a total of about an hour. An example of less stringent hybridization conditions is hybridization in 4 x SSC at 37 ° C, followed by several washing steps in 1 x SSC at room temperature. According to the invention, such substances are constituents of a body fluid, in particular blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, synovial fluid or a tissue extract of the patient.
In einer weiteren Ausführungsform der Erfindung können jedoch die erfindungsgemäßen Markersequenzen in einer signifikant höheren oder niedrigeren Expressionsrate oder Konzentration vorliegen, dass auf die Prostataentzündungserkrankungen, Prostatakarzinom hinweist. Hierbei wird mittels Proteomics oder Nukleinsäureblots die relativen Expressionsraten krank / gesund der erfindungsgemäßen Markersequenzen für Prostataentzündungserkrankungen, Prostatakarzinom bestimmt.In a further embodiment of the invention, however, the marker sequences according to the invention can be present in a significantly higher or lower expression rate or concentration, which points to the prostate inflammatory diseases, prostate carcinoma. In this case, the relative expression rates ill / healthy of the marker sequences according to the invention for prostate inflammatory diseases, prostate carcinoma is determined by means of proteomics or nucleic acid blots.
Die Markersequenzen verfügen in einer weiteren Ausführungsform der Erfindung über ein Erkennungssignal, welches an die zu bindende Substanz adressiert ist (z.B. Antikörper,In a further embodiment of the invention, the marker sequences have a recognition signal which is addressed to the substance to be bound (for example antibodies,
Nukleinsäure) . Erfindungsgemäß bevorzugt ist für ein Protein das Erkennungssignal ein Epitop und / oder Paratop und / oder Hapten und für eine cDNA eine Hybridisierungs- oder Bindungsregion .Nucleic acid). According to the invention, the recognition signal for a protein is preferably an epitope and / or paratope and / or hapten and, for a cDNA, a hybridization or binding region.
Die erfindungsgemäßen Markersequenzen sind Gegenstand der Tabelle A und können durch den jeweilig zitierten Datenbankeintrag (auch mittels Internet: http://www.ncbi.nlm.nih.gov/) eindeutig identifiziert werden (siehe in Tabelle A: dort Accession No.), siehe ebenfalls das zugehörige Sequenzprotokoll.The marker sequences according to the invention are the subject of Table A and can be clearly identified by the respective cited database entry (also by means of the Internet: http://www.ncbi.nlm.nih.gov/) (see Table A: there Accession No.), see also the associated sequence listing.
Daher betrifft die Erfindung ebenfalls die Volllängesequenzen der erfindungsgemäßen Marker und zwar wie in Tabelle 1 über den bekannten Datenbankeintrag gemäß Tabelle A definiert, nachstehend SEQ la-174a genannt.Therefore, the invention also relates to the full-length sequences of the markers according to the invention and indeed as defined in Table 1 on the known database entry according to Table A, hereinafter called SEQ ID NO: 174a.
Weiterhin umfasst sind daher ebenfalls analogeFurthermore, therefore, also include analog
Ausführungsformen von SEQ la-174a zu den Markersequenzen SEQ 1-174, wie z.B. in den Ansprüchen dargelegt, da die erfindungsgemäßen SEQ 1-174 wiederum Teilsequenzen, zumindest mit hoher Homologie, darstellen. Die spezifischen Markersequenzen SEQ 1-174 sind jedoch erfindungsgemäß bevorzugt .Embodiments of SEQ ID NO: 174a to the marker sequences SEQ 1-174, as set forth, for example, in the claims, as the inventive SEQ ID-174 again subsequences, at least with high homology. However, the specific marker sequences SEQ 1-174 are preferred according to the invention.
Weiterhin bevorzugt sind SEQ 136a, 40a, 127a, 83a, 16a, 82a, 88a, 152a, 130a, 138a, 2a, 12a, 113a, 20a, 173a, 33a, 172a, 52a, 43a, 91a, Ia, 32a, 86a, 27a, 105a.Further preferred are SEQ 136a, 40a, 127a, 83a, 16a, 82a, 88a, 152a, 130a, 138a, 2a, 12a, 113a, 20a, 173a, 33a, 172a, 52a, 43a, 91a, Ia, 32a, 86a, 27a, 105a.
Erfindungsgemäß umfassen die Markersequenzen auch solche Modifikationen der cDNA-Sequenz und der entsprechenden Aminosäuresequenz, wie chemische Modifikation, wie Citrullinierung, Acetylierung, Phosphorylierung,According to the invention, the marker sequences also include such modifications of the cDNA sequence and the corresponding amino acid sequence, such as chemical modification, such as citrullination, acetylation, phosphorylation,
Glykosilierung oder polyA-Strang und weiteren dem Fachmann einschlägig bekannte Modifikationen.Glycation or polyA strand and other modifications known to those skilled in the art.
In einer weiteren Ausführungsform der Erfindung sind ebenfalls Teilsequenzen oder Fragmente der erfindungsgemäßen Markersequenzen umfasst. Insbesondere solche Teilsequenzen, die eine Identität von 95%, 90 %, insbesondere 80% oder 70 % mit den erfindungsgemäßen Markersequenzen aufweisen.In a further embodiment of the invention, partial sequences or fragments of the marker sequences according to the invention are also included. In particular, those partial sequences which have an identity of 95%, 90%, in particular 80% or 70% with the marker sequences according to the invention.
Teilsequenzen sind ebenfalls solche Sequenzen, die 50 bis 100 Nukleotide, 70-120 Nukleotide einer Sequenz der SEQ 1-174 aufweisen, oder davon erhältliche Peptide.Partial sequences are also those sequences having 50 to 100 nucleotides, 70-120 nucleotides of a sequence of SEQ 1-174, or peptides obtainable therefrom.
In einer weiteren Ausführungsform kann die jeweilige Markersequenz in unterschiedlichen Mengen in einen oder mehreren Bereichen auf einem festen Träger repräsentiert sein. Dies erlaubt eine Variation der Sensitivität . Die Bereiche können jeweils eine Gesamtheit von Markersequenzen aufweisen, d.h. eine genügende Zahl an verschiedenen Markersequenzen, insbesondere 2 bis 5 oder 10 oder mehr und ggfs. weiteren Nukleinsäuren und/oder Proteinen, insbesondere Biomarker. Bevorzugt sind jedoch mindestens 96 bis 25.000 (numerisch) oder mehr aus verschiedenen oder gleichen Markersequenzen und weiteren Nukleinsäuren und/oder Proteinen, insbesondere Biomarker. Weiterhin bevorzugt sind mehr als 2.500, besonders bevorzugt 10.000 oder mehr verschiedene oder gleiche Markersequenzen und ggfs. weiteren Nukleinsäuren und/oder Proteinen, insbesondere Biomarker.In a further embodiment, the respective marker sequence may be represented in different amounts in one or more regions on a solid support. This allows a variation of the sensitivity. The regions can each have an entirety of marker sequences, ie a sufficient number of different marker sequences, in particular 2 to 5 or 10 or more and, if appropriate, further nucleic acids and / or proteins, in particular biomarkers. However, at least 96 to 25,000 (numerically) or more are preferred from different or identical marker sequences and further nucleic acids and / or proteins, in particular biomarkers. Further preferred are more than 2,500, more preferably 10,000 or more different or the same Marker sequences and, if necessary, further nucleic acids and / or proteins, in particular biomarkers.
Ein weiterer Gegenstand der Erfindung betrifft eine Anordnung von Markersequenzen enthaltend mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein. Vorzugsweise enthält die Anordnung mindestens 2 bis 5 oder 10, vorzugsweise 30 bis 50 Markersequenzen oder 50 bis 100 oder mehr Markersequenzen.Another object of the invention relates to an array of marker sequences containing at least one marker sequence of a cDNA selected from the group SEQ 1-174 or in each case a protein coding therefor. Preferably, the array contains at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences.
Im Rahmen dieser Erfindung bedeutet „Anordnung" synonym „Array" und sofern dieser „Array" zur Identifizierung von Substanzen an Markersequenzen verwendet wird, ist hierunter ein „Assay" oder eine diagnostische Vorrichtung zu verstehen. In einer bevorzugten Ausführungsform ist die Anordnung derart gestaltet, dass die auf der Anordnung repräsentierten Markersequenzen in Form eines Gitters auf einem festen Träger vorliegen. Ferner sind solche Anordnungen bevorzugt, die eine hochdichte (high-density) Anordnung von Proteinbindern erlauben und die Markersequenzen gespottet werden. Solche hochdichte gespotteten Anordnungen sind beispielsweise in der WO 99/57311 und WO 99/57312 offenbart und können vorteilhaft in einem robotergestützten automatisierten High-Throughput Verfahren zur Anwendung kommen.In the context of this invention, "arrangement" synonymously means "array" and insofar as this "array" is used to identify substances on marker sequences, this is to be understood as meaning an "assay" or a diagnostic device. In a preferred embodiment, the arrangement is such that the marker sequences represented on the array are in the form of a grid on a solid support. Further, such arrangements are preferred which allow a high density array of protein binders and spotting the marker sequences. Such high density spotted assemblies are disclosed, for example, in WO 99/57311 and WO 99/57312, and may be advantageously used in a robotic automated high throughput method.
Im Rahmen dieser Erfindung umfasst jedoch der Begriff „Assay" oder diagnostische Vorrichtung ebenfalls solche Ausführungsformen einer Vorrichtung, wie ELISA, Bead-based Assay, Line Assay, Western Blot, immunchromatographische Verfahren (z.B. so genannte Lateral Flow Immunoassays) oder ähnliche immunologische Single- oder Multiplex- Nachweisverfahren . Ein Proteinbiochip im Sinne dieser Erfindung ist die systematische Anordnung von Proteinen auf einem festen Träger.In the context of this invention, however, the term "assay" or diagnostic device also encompasses such embodiments of a device as ELISA, bead-based assay, line assay, Western blot, immunochromatographic methods (eg, so-called lateral flow immunoassays) or similar immunological single or Multiplex detection method A protein biochip in the sense of this invention is the systematic arrangement of proteins on a solid support.
Die Markersequenzen der Anordnung sind auf einen festen Träger fixiert, vorzugsweise jedoch gespottet oder immobilisiert gar aufgedruckt, d.h. reproduzierbar aufgebracht. Ein oder mehrere Markersequenzen können mehrfach in der Gesamtheit aller Markersequenzen präsent sein und in unterschiedlichen Mengen bezogen auf einen Spot vorliegen. Ferner können die Markersequenzen auf dem festen Träger standardisiert sein (z.B. mittels serieller Verdünnungsreihen von z.B. Humanglobulinen als interne Kalibratoren zur Datennormalisierung und quantitativen Auswertung) .The marker sequences of the assembly are fixed to a solid support, but preferably spotted or immobilized imprinted, ie applied reproducible. One or more marker sequences may be present several times in the totality of all marker sequences and be present in different amounts relative to one spot. Furthermore, the marker sequences can be standardized on the solid support (eg by means of serial dilution series of eg human globulins as internal calibrators for data normalization and quantitative evaluation).
Daher betrifft die Erfindung einen Assay oder Proteinbiochip bestehend aus einer Anordnung enthaltend erfindungsgemäße Markersequenzen .Therefore, the invention relates to an assay or protein biochip consisting of an array containing marker sequences according to the invention.
In einer weiteren Ausführungsform liegen die Markersequenzen als Clone vor. Solche Clone können beispielsweise mittels einer erfindungsgemäßen cDNA-Expressionsbibliothek erhalten werden (Büssow et al . 1998 (supra) ) . In einer bevorzugten Ausführungsform werden solche Expressionsbibliotheken enthaltend Clone mittels Expressionsvektoren aus einer exprimierenden cDNA Bibliothek bestehend aus den cDNA Markersequenzen erhalten. Diese Expressionsvektoren enthalten vorzugsweise induzierbare Promotoren. Die Induktion derIn a further embodiment, the marker sequences are present as clones. Such clones can be obtained, for example, by means of a cDNA expression library according to the invention (Büssow et al., 1998 (supra)). In a preferred embodiment, such expression libraries containing clones are obtained by means of expression vectors from an expressing cDNA library consisting of the cDNA marker sequences. These expression vectors preferably contain inducible promoters. The induction of
Expression kann z.B. mittels eines Induktors, solche wie IPTG, erfolgen. Geeignete Expressionsvektoren sind beschrieben in Terpe et al . (Terpe T Appl Microbiol Biotechnol. 2003 Jan; 60 (5) :523-33) .Expression can e.g. by means of an inductor, such as IPTG. Suitable expression vectors are described in Terpe et al. (Terpe T Appl Microbiol Biotechnol 2003 Jan; 60 (5): 523-33).
Expressionsbibliotheken sind dem Fachmann bekannt, diese können nach Standardwerken, wie Sambrook et al, "Molecular Cloning, A laboratory handbook, 2nd edition (1989), CSH press, CoId Spring Harbor, New York hergestellt werden. Weiterhin bevorzugt sind solche Expressionsbibliotheken, die gewebespezifisch sind (z.B. humanes Gewebe, insbesondere humane Organe) . Ferner sind erfindungsgemäß ebenfalls solche Expressionsbibliotheken mit eingeschlossen, die mittels exon- trapping erhalten werden können. Statt Expressionsbibliothek kann synonym von einer Expressionsbank gesprochen werden. Weiterhin bevorzugt sind Proteinbiochips oder entsprechende Expressionsbibliotheken, die keine Redundanz aufweisen (so genannte: Uniclone®-Bibliothek) und nach den Lehren der WO 99/57311 und WO 99/57312 beispielsweise hergestellt werden können. Diese bevorzugten Uniclone- Bibliotheken weisen einen hohen Anteil an nicht-fehlerhaften vollständig exprimierten Proteinen einer cDNA-Expressionsbibliothek auf.Expression libraries are known to the person skilled in the art, and these can be prepared according to standard works, such as Sambrook et al., Molecular Cloning, Laboratory Handbook, 2nd edition (1989), CSH press, CoId Spring Harbor, New York Further preferred are expression libraries which are tissue-specific In addition, according to the invention, expression libraries which can be obtained by exon trapping are also included in the invention, and instead of an expression library it is possible to speak synonymously of an expression library. Also preferred are protein biochips or corresponding expression libraries which have no redundancy (so-called: Uniclone® library) and can be prepared, for example, according to the teachings of WO 99/57311 and WO 99/57312. These preferred Uniclone libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.
Im Rahmen dieser Erfindung können die Clone ebenfalls nicht abschließend solche sein, wie transformierte Bakterien, rekombinante Phagen oder transformierte Zellen von Säugern, Insekten, Pilzen, Hefen oder Pflanzen.Also within the scope of this invention, the clones may not be such as transformed bacteria, recombinant phage or transformed cells of mammals, insects, fungi, yeasts or plants.
Die Clone werden auf einen festen Träger fixiert, gespottet oder immobilisiert.The clones are fixed on a solid support, spotted or immobilized.
Daher betrifft die Erfindung eine Anordnung, wobei die Markersequenzen als Clone vorliegen.Therefore, the invention relates to an arrangement wherein the marker sequences are present as clones.
Zusätzlich können die Markersequenzen in der jeweiligen Form in Form eines Fusionsproteins vorliegen, welches beispielsweise mindestens ein Affinitätsepiptop oder "Tag" enthält. Der Tag kann ein solcher sein wie wie c-myc, His-Tag, Arg-tag, FLAG, alkalische Phosphatase, V5-Tag, T7-Tag oder Strep-Tag, HAT-tag, NusA, S-tag, SBP-tag, Thioredoxin, DsbA, ein Fusionsprotein, vorzugsweise eine Cellulose-bindende Domäne, grünfluoreszierendes Protein, Maltose bindendes Protein, calmodulin-bindendes Protein, Glutathione S- transferase oder lacZ enthalten.In addition, the marker sequences may be in the form of a fusion protein containing, for example, at least one affinity epitope or "tag". The tag may be one such as c-myc, His-tag, Arg-tag, FLAG, alkaline phosphatase, V5 tag, T7 tag or Strep tag, HAT tag, NusA, S-tag, SBP tag , Thioredoxin, DsbA, a fusion protein, preferably a cellulosic binding domain, green fluorescent protein, maltose binding protein, calmodulin binding protein, glutathione S-transferase or lacZ.
In sämtlichen Ausführungsformen umfasst der Begriff "fester Träger" Ausführungen wie einen Filter, eine Membran, ein magnetisches oder Fluorophor-markiertes Kügelchen, ein Silizium-Wafer, Glas, Metall, Kunststoff, ein Chip, ein massenspektrometrisches Target oder eine Matrix. Ein Filter ist jedoch erfindungsgemäß bevorzugt. Als Filter ist weiterhin PVDF, Nitrocellulose oder Nylon bevorzugt (z.B. Immobilon P Millipore, Protran Whatman, Hybond N+ Amersham) .In all embodiments, the term "solid support" includes embodiments such as a filter, a membrane, a magnetic or fluorophore-labeled bead, a silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target, or a matrix. However, a filter is preferred according to the invention. PVDF, nitrocellulose or nylon are also preferred as filters (eg Immobilon P Millipore, Protran Whatman, Hybond N + Amersham).
In einer weiteren bevorzugten Ausführungsform der erfindungsgemäßen Anordnung entspricht diese einem Gitter, dass die Größenordnung einer Mikrotiterplatte (8-12 Wells Streifen, 96 Wells, 384 Wells oder mehr) , eines Silizium- Wafers, eines Chips, eines massenspektrometrischen Targets oder einer Matrix besitzt.In a further preferred embodiment of the arrangement according to the invention, this corresponds to a grid having the size of a microtiter plate (8-12 wells strips, 96 wells, 384 wells or more), a silicon wafer, a chip, a mass spectrometric target or a matrix.
In einer weiteren Ausführungsform betrifft die Erfindung einen Assay oder Proteinbiochip zum Identifizieren und Charakterisieren einer Substanz fürIn a further embodiment, the invention relates to an assay or protein biochip for identifying and characterizing a substance for
Prostataentzündungserkrankungen, Prostatakarzinom, dadurch gekennzeichnet, dass eine erfindungsgemäße Anordnung oder Assay mit a.) mindestens einer zu untersuchenden Substanz in Kontakt gebracht wird und b.) ein Bindungserfolg nachgewiesen wird.Prostate inflammatory diseases, prostate cancer, characterized in that an arrangement or assay according to the invention with a.) At least one substance to be examined is brought into contact and b.) A binding success is detected.
Ferner betrifft die Erfindung ein Verfahren zum Identifizieren und Charakterisieren einer Substanz für Prostataentzündungserkrankungen, Prostatakarzinom, dadurch gekennzeichnet, dass eine erfindungsgemäße Anordnung oder Assay mit a.) mindestens einer zu untersuchenden Substanz in Kontakt gebracht wird und b.) ein Bindungserfolg nachgewiesen wird.The invention further relates to a method for identifying and characterizing a substance for prostate inflammatory diseases, prostate carcinoma, characterized in that an arrangement or assay according to the invention is brought into contact with a.) At least one substance to be investigated and b.) A binding success is detected.
Die zu untersuchende Substanz kann ein beliebiges natives oder nicht-natives Biomolekül, ein synthetisches chemisches Molekül, eine Mischung oder eine Substanzbibliothek sein.The substance to be assayed may be any native or non-native biomolecule, synthetic chemical molecule, mixture or substance library.
Nachdem die zu untersuchende Substanz eine Markersequenz kontaktiert, erfolgt die Auswertung des Bindungserfolges, die beispielsweise unter Verwendung mit handelsüblicher Image- Analyse Software (GenePix Pro (Axon Laboratories) , Aida (Raytest), ScanArray (Packard Bioscience) erfolgt. Die Visualisierung erfindungsgemäßer Protein-Protein- Wechselwirkungen (z.B. Protein an Markersequenz, wie Antigen/Antikörper) oder entsprechende „Mittel zum Nachweis des Bindungserfolges" kann beispielsweise mittels Fluoresenzmarkierung, Biotiniylierung, Radio-Isotopen- Markierung oder kolloidale Gold- oder Latex-Partikel- Markierung in üblicher Weise erfolgen. Ein Nachweis von gebundenen Antikörpern erfolgt mit Hilfe von sekundären Antikörpern, die mit handelsüblichen Reportermolekülen markiert sind (z.B. Cy-, Alexa-, Dyomics, FITC- oder ähnliche Fluoreszenzfarbstoffe, , kolloidale Gold- oder Latex- Partikel) , oder mit Reporter-Enzymen wie alkalischer Phosphatase, Meerrettichperoxidase, usw. und den entsprechenden colorimetrischen, fluoreszenten oder chemolumineszenten Substraten. Eine Auslesung erfolgt z.B. mittels eines Microarray-Laserscanners, einer CCD-Kamera oder visuell .After the substance to be investigated contacts a marker sequence, the evaluation of the binding success is carried out, for example, using commercially available image analysis software (GenePix Pro (Axon Laboratories), Aida (Raytest), ScanArray (Packard Bioscience). The visualization of protein-protein interactions according to the invention (eg protein to marker sequence, such as antigen / antibody) or corresponding "means for detecting the binding success" can be, for example, by fluorescence labeling, biotinization, radio-isotopic labeling or colloidal gold or latex particle labeling Bound antibodies are detected by secondary antibodies labeled with commercially available reporter molecules (eg Cy, Alexa, Dyomics, FITC or similar fluorescent dyes, colloidal gold or latex particles), or with reporter enzymes such as alkaline phosphatase, horseradish peroxidase, etc., and the corresponding colorimetric, fluorescent, or chemiluminescent substrates, for example, by means of a microarray laser scanner, a CCD camera, or visually.
In einer weiteren Ausführungsform betrifft die Erfindung ein Arzneimittel / Wirkstoff oder Prodrug für Prostataentzündungserkrankungen, Prostatakarzinom entwickelt und erhältlich durch den Einsatz des erfindungsgemäßen Assays oder Proteinbiochip.In a further embodiment, the invention relates to a drug / drug or prodrug for prostate inflammatory diseases, prostate carcinoma developed and obtainable by the use of the assay or protein biochip according to the invention.
Daher betrifft die Erfindung ebenfalls die Verwendung einer erfindungsgemäßen Anordnung oder einem Assay zum Screenen von Wirkstoffen für Prostataentzündungserkrankungen, Prostatakarzinom.Therefore, the invention also relates to the use of an inventive arrangement or an assay for the screening of drugs for prostate inflammatory diseases, prostate carcinoma.
Daher betrifft die Erfindung in einer weiteren Ausführungsform ebenfalls ein Target zur Behandlung und Therapie von Prostataentzündungserkrankungen, Prostatakarzinom, jeweils ausgewählt aus der Gruppe SEQ 1 - 174 oder jeweils ein dafür kodierendes Protein.Therefore, in a further embodiment, the invention also relates to a target for the treatment and therapy of prostate inflammatory diseases, prostate carcinoma, each selected from the group SEQ 1-174 or in each case a protein coding therefor.
In einer weiteren Ausführungsform betrifft die Erfindung ebenfalls die Verwendung der erfindungsgemäßen Markersequenzen, vorzugsweise in Form einer Anordnung, als Affinitätsmaterial zur Durchführung einer Apherese bzw. iwS . einer Blutwäsche, wobei Substanzen aus Körperflüssigkeiten eines Patienten mit Prostataentzündungserkrankungen, Prostatakarzinom, wie Blut oder Plasma, an die erfindungsgemäßen Markersequenzen binden und folglich der Körperflüssigkeit selektiv entzogen werden können.In a further embodiment, the invention also relates to the use of the marker sequences according to the invention, preferably in the form of an arrangement, as Affinity material for performing an apheresis or iwS. a blood wash, wherein substances from body fluids of a patient with prostate inflammatory diseases, prostate cancer, such as blood or plasma, bind to the marker sequences according to the invention and thus the body fluid can be selectively withdrawn.
Beispiele und Figuren:Examples and figures:
Zehn oder mehr Patientenproben wurden individuell gegen eine cDNA Expressionsbibliothek gescreent. Die Prostataentzündungserkrankungen, Prostatakarzinom - spezifischen Expressionsklone wurden ermittelt durch einen Vergleich mit zehn oder mehr gesunden Proben. Die Identität der Markersequenzen wurde durch DNA-Sequenzierung ermittelt.Ten or more patient samples were individually screened against a cDNA expression library. Prostate inflammatory, prostate carcinoma - specific expression clones were identified by comparison with ten or more healthy specimens. The identity of the marker sequences was determined by DNA sequencing.
In Figur 1 wird das differentielle Screenen zwischen zwei Proteinbiochips aus jeweils einer cDNA-Expressionsbank eines Patienten und einem gesunden Probanden gezeigt. Die differentiellen Clone werden mittels Fluoresenzmarkierung nachgewiesen und bioinformatorisch ausgewertet.FIG. 1 shows the differential screening between two protein biochips from in each case one cDNA expression bank of a patient and one healthy subject. The differential clones are detected by fluorescence labeling and evaluated bioinformatorisch.
Im Rahmen der Biomarkeridentifizierung werden verschiedene bioinformatische Analysen durchgeführt. Für jedes Serum werden mittels Microarray Reaktivitäten gegen ca. 2000 unterschiedliche Antigene gemessen. Diese Daten werden für ein Ranking der gespotteten Antigene bzgl. ihrer Differenzierungsfähigkeit zwischen gesunden und erkrankten Seren benutzt. Diese Auswertung wird mittels des nicht parametrischen Mann-Whitney Tests auf normalisierten Intensitätsdaten durchgeführt. Zur Normalisierung wird ein interner Standard benutzt, der auf jedem Chip mitgespottet wird. Da für jedes Antigen ein p-Wert berechnet wird, werden Methoden zur Korrektur des multiples Testens eingesetzt. Als sehr konservativer Ansatz wird eine Bonferroni Korrektur durchgeführt und zusätzlich wird die weniger restriktive False Discovery Rate (FDR) nach Benjamini & Hochberg berechnet. Desweiteren werden die Daten zur Klassifikation der Seren benutzt. Hierbei kommen unterschiedliche multivariate Methoden zum Einsatz. Dies sind Methoden aus den statistischen Lernverfahren wie Support Vector Machines (SVM), Neuronale Netze oder Klassifikationsbäume, sowie eineIn the context of biomarker identification various bioinformatic analyzes are carried out. Microarray reactivities against approximately 2000 different antigens are measured for each serum. These data are used to rank the spotted antigens for their ability to differentiate between healthy and diseased sera. This evaluation is performed using the non-parametric Mann-Whitney test on normalized intensity data. For normalization, an internal standard is used, which is spotted on each chip. Since a p-value is calculated for each antigen, methods for correcting the multiple testing are used. As a very conservative approach, a Bonferroni correction is performed and, in addition, the less restrictive False Discovery Rate (FDR) is calculated according to Benjamini & Hochberg. Furthermore, the data are used to classify the sera. Here, different multivariate methods are used. These are methods from the statistical learning methods such as Support Vector Machines (SVM), neural networks or classification trees, as well as a
Schwellenwertmethode, welche sowohl zur Klassifikation als auch zur visuellen Repräsentation der Daten geeignet ist.Threshold method, which is suitable for both classification and visual representation of the data.
Zur Vermeidung von Overfitting wird eine lOfache Cross- Validierung der Daten durchgeführt.To avoid overfitting, a 10-fold cross-validation of the data is performed.
Tabelle A:Table A:

Claims

Patentansprüche claims
1. Verwendung der Markersequenzen zur Diagnose von Prostataentzündungserkrankungen, Prostatakarzinom, wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon an oder zu einem zu untersuchenden Patienten bestimmt wird.1. Use of the marker sequences for the diagnosis of prostate inflammatory diseases, prostate carcinoma, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 and / or SEQ.LAMB-174a or in each case a protein coding therefor or in each case a partial sequence or fragment thereof on or to a to be examined.
2. Verwendung der Markersequenzen zur Diagnose von Prostataentzündungserkrankungen, Prostatakarzinom nach Anspruch 1, dadurch gekennzeichnet, mindestens 2 bis 5 oder 10, vorzugsweise 30 bis 50 Markersequenzen oder 50 bis 100 oder mehr Markersequenzen an oder zu einem zu untersuchenden Patienten bestimmt wird.2. Use of the marker sequences for the diagnosis of prostate inflammatory diseases, prostate cancer according to claim 1, characterized in that at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences is determined on or to a patient to be examined.
3. Verwendung der Markersequenzen zur Diagnose von Prostataentzündungserkrankungen, Prostatakarzinom nach Anspruch 1 oder 2, dadurch gekennzeichnet, dass die Bestimmung mittels in-vitro Diagnose erfolgt.3. Use of the marker sequences for the diagnosis of prostate inflammatory diseases, prostate cancer according to claim 1 or 2, characterized in that the determination is carried out by means of in-vitro diagnosis.
4. Verwendung einer Markersequenz einer cDNA jeweils ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon als Diagnostikum.4. Use of a marker sequence of a cDNA in each case selected from the group SEQ 1 - 174 and / or SEQ-174a or in each case a protein coding therefor or in each case a partial sequence or fragment thereof as a diagnostic agent.
5. Verwendung der Markersequenzen zur Diagnose von Prostataentzündungserkrankungen, Prostatakarzinom nach einem der vorhergehenden Ansprüche, dadurch gekennzeichnet, dass die Markersequenzen auf einem festen Träger aufgebracht werden, insbesondere einen5. Use of the marker sequences for the diagnosis of prostate inflammatory diseases, prostate cancer according to one of the preceding claims, characterized in that the marker sequences are applied to a solid support, in particular a
Filter, eine Membran, ein magnetisches oder Fluorophor- markiertes Kügelchen, ein Silizium-Wafer, Glas, Metall, Kunststoff, ein Chip, ein massenspektrometrisches Target oder eine Matrix.Filter, a membrane, a magnetic or fluorophore-labeled bead, a silicon wafer, glass, metal, Plastic, a chip, a mass spectrometric target or a matrix.
6. Verfahren zur Diagnose von6. Method of Diagnosing
Prostataentzündungserkrankungen, Prostatakarzinom, wobei a.) mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon auf einem festen Träger aufgebracht wird und b.) mit Körperflüssigkeit oder Gewebeauszug eines Patienten in Kontakt gebracht wird und c.) der Nachweis einer Wechselwirkung der Körperflüssigkeit oder Gewebeauszug mit den Markersequenzen aus a.) erfolgt.Prostate inflammatory diseases, prostate carcinoma, wherein a.) At least one marker sequence of a cDNA selected from the group SEQ 1-174 and / or SEQ-174a or a protein coding therefor or each of a partial sequence or fragment thereof is applied to a solid support and b. ) is brought into contact with body fluid or tissue extract of a patient and c.) the detection of an interaction of the body fluid or tissue extract with the marker sequences from a.).
7. Verfahren zum Stratifizieren, insbesondere zur Risikostratifizierung, oder zur Therapiesteuerung eines Patienten mit Prostataentzündungserkrankungen, Prostatakarzinom, wobei mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon an einem zu untersuchenden Patienten bestimmt wird.7. A method for stratifying, in particular for the risk stratification, or for the therapy control of a patient with prostate inflammatory diseases, prostate carcinoma, wherein at least one marker sequence of a cDNA selected from the group SEQ 1-174 and / or SEQ.LAMB-174a or a protein coding therefor or each one Partial sequence or fragment thereof is determined on a patient to be examined.
8. Verfahren nach Anspruch 7, wobei das Stratifizieren oder die Therapiesteuerung Entscheidungen zur Behandlung und Therapie des Patienten, insbesondere Hospitalisierung des Patienten, Einsatz, Wirkung und / oder Dosierung eines oder mehrerer Arzneimittel, eine therapeutische Maßnahme oder die Überwachung eines8. The method of claim 7, wherein the stratification or therapy control decisions for treatment and therapy of the patient, in particular hospitalization of the patient, use, effect and / or dosage of one or more drugs, a therapeutic measure or the monitoring of a
Krankheitsverlaufes sowie Therapieverlauf, Ätiologie oder Klassifizierung einer Erkrankung samt Prognose umfasst . Disease progression as well as course of therapy, etiology or classification of a disease including prognosis.
9. Anordnung von Markersequenzen enthaltend mindestens eine Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein.9. Arrangement of marker sequences containing at least one marker sequence of a cDNA selected from the group SEQ 1 - 174 and / or SEQ-174a or in each case a protein coding therefor.
10. Anordnung nach Anspruch 9, dadurch gekennzeichnet, dass mindestens 2 bis 5 oder 10, vorzugsweise 30 bis 50 Markersequenzen oder 50 bis 100 oder mehr Markersequenzen enthalten sind.10. Arrangement according to claim 9, characterized in that at least 2 to 5 or 10, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences are included.
11. Anordnung nach Anspruch 9, dadurch gekennzeichnet, dass die Markersequenzen als Clone vorliegen.11. Arrangement according to claim 9, characterized in that the marker sequences are present as clones.
12. Assay, Proteinbiochip bestehend aus einer Anordnung nach Anspruch 9, dadurch gekennzeichnet, dass die Markersequenzen auf einem festen Träger aufgebracht sind.12. Assay, protein biochip consisting of an arrangement according to claim 9, characterized in that the marker sequences are applied to a solid support.
13. Verwendung einer Anordnung nach einem der Ansprüche 9 bis 11 oder einem Assay nach Anspruch 12 zum Identifizieren und Charakterisieren einer Substanz für Prostataentzündungserkrankungen, Prostatakarzinom enthaltend Mittel zum Nachweis eines Bindungserfolges, dadurch gekennzeichnet, dass eine Anordnung oder Assay mit a.) mindestens einer zu untersuchenden Substanz in Kontakt gebracht wird und b.) ein Bindungserfolg nachgewiesen wird.Use of a device according to any one of claims 9 to 11 or an assay according to claim 12 for identifying and characterizing a substance for prostate inflammatory diseases, prostate cancer containing means for detecting a binding success, characterized in that an arrangement or assay with a.) At least one Binding substance is brought into contact and b.) A binding success is detected.
14. Verwendung einer Anordnung nach einem der Ansprüche 9 bis 11 oder einem Assay nach Anspruch 12 zum Screenen von Wirkstoffen für Prostataentzündungserkrankungen, Prostatakarzinom.14. Use of an arrangement according to one of claims 9 to 11 or an assay according to claim 12 for the screening of drugs for prostate inflammatory diseases, prostate cancer.
15. Diagnostika zur Diagnose von15. Diagnostics for the diagnosis of
Prostataentzündungserkrankungen, Prostatakarzinom, jeweils ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon . Prostate inflammatory diseases, prostate cancer, each selected from the group SEQ 1-174 and / or SEQ-174a or in each case a protein coding therefor or in each case a partial sequence or fragment thereof.
16. Target zur Behandlung und Therapie von16. Target for treatment and therapy of
Prostataentzündungserkrankungen, Prostatakarzinom, jeweils ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon .Prostate inflammatory diseases, prostate cancer, each selected from the group SEQ 1-174 and / or SEQ-174a or in each case a protein coding therefor or in each case a partial sequence or fragment thereof.
17. Verwendung einer Markersequenz einer cDNA ausgewählt aus der Gruppe SEQ 1 - 174 und / oder SEQ la-174a oder jeweils ein dafür kodierendes Protein oder jeweils einer Teilsequenz oder Fragment davon als17. Use of a marker sequence of a cDNA selected from the group SEQ 1-174 and / or SEQ-174a or in each case a protein coding therefor or in each case a partial sequence or fragment thereof as
Affinitätsmaterial zur Durchführung einer Apherese oderAffinity material for performing an apheresis or
Blutwäsche für Patienten mitBlood washing for patients with
Prostataentzündungserkrankungen, Prostatakarzinom. Prostate inflammatory diseases, prostate cancer.
EP09772574A 2008-07-04 2009-07-06 Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use Withdrawn EP2318546A2 (en)

Priority Applications (4)

Application Number Priority Date Filing Date Title
EP20130190158 EP2712933A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190149 EP2712932A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP15152989.8A EP2902505A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190162 EP2712934A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE200810031699 DE102008031699A1 (en) 2008-07-04 2008-07-04 Marker sequences for prostate inflammatory diseases, prostate cancer and their use
PCT/EP2009/058534 WO2010000874A2 (en) 2008-07-04 2009-07-06 Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use

Related Child Applications (4)

Application Number Title Priority Date Filing Date
EP20130190149 Division EP2712932A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190158 Division EP2712933A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP15152989.8A Division EP2902505A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190162 Division EP2712934A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same

Publications (1)

Publication Number Publication Date
EP2318546A2 true EP2318546A2 (en) 2011-05-11

Family

ID=41412566

Family Applications (5)

Application Number Title Priority Date Filing Date
EP15152989.8A Withdrawn EP2902505A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190162 Withdrawn EP2712934A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP09772574A Withdrawn EP2318546A2 (en) 2008-07-04 2009-07-06 Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use
EP20130190149 Withdrawn EP2712932A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190158 Withdrawn EP2712933A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same

Family Applications Before (2)

Application Number Title Priority Date Filing Date
EP15152989.8A Withdrawn EP2902505A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190162 Withdrawn EP2712934A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same

Family Applications After (2)

Application Number Title Priority Date Filing Date
EP20130190149 Withdrawn EP2712932A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same
EP20130190158 Withdrawn EP2712933A3 (en) 2008-07-04 2009-07-06 Marker sequences for prostate inflammation illnesses, prostate carcinoma and use of same

Country Status (4)

Country Link
US (3) US20110275528A1 (en)
EP (5) EP2902505A3 (en)
DE (1) DE102008031699A1 (en)
WO (1) WO2010000874A2 (en)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2487251A1 (en) 2011-02-13 2012-08-15 Protagen AG Marker sequences for the diagnosis of prostate carcinoma and use of same
US10443102B2 (en) 2011-02-24 2019-10-15 Cornell University Recurrent SPOP mutations in prostate cancer
US10060911B2 (en) 2011-08-19 2018-08-28 Protagen Aktiengesellschaft Method for diagnosis of high-affinity binders and marker sequences
WO2013072393A2 (en) 2011-11-14 2013-05-23 Protagen Ag Novel method for identifying specific marker sequences for prostate cancer
US9782418B2 (en) * 2013-12-03 2017-10-10 Celestra Life Science Llc Rationale-based design of a targeted therapy for cancer

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE69921982T2 (en) 1998-04-30 2005-12-29 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. NOVEL METHOD FOR IDENTIFYING CLONES WITH A DESIRED BIOLOGICAL PROPERTY, EXPRESSED FROM AN EXPRESSION BENCH
EP1073771B1 (en) 1998-04-30 2004-12-01 Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. New method for the selection of clones of an expression library involving rearraying
CA2359816C (en) * 1999-01-06 2010-08-03 Genenews Inc. Method for the detection of gene transcripts in blood and uses thereof
WO2001060860A2 (en) * 2000-02-17 2001-08-23 Millennium Predictive Medicine, Inc. Genes differentially expressed in human prostate cancer and their use
AU2001253079A1 (en) * 2000-04-03 2001-10-15 Corixa Corporation Methods, compositions and kits for the detection and monitoring of breast cancer
US20060141493A1 (en) * 2001-11-09 2006-06-29 Duke University Office Of Science And Technology Atherosclerotic phenotype determinative genes and methods for using the same
MXPA05005283A (en) * 2002-11-21 2005-07-25 Wyeth Corp Methods for diagnosing rcc and other solid tumors.
RU2473555C2 (en) * 2006-12-19 2013-01-27 ДжинГоу, Инк. New method for functional analysis of body of experimental data and gene groups identified from said data
US20110159498A1 (en) * 2008-04-11 2011-06-30 China Synthetic Rubber Corporation Methods, agents and kits for the detection of cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO2010000874A2 *

Also Published As

Publication number Publication date
EP2712932A2 (en) 2014-04-02
US20110275528A1 (en) 2011-11-10
EP2902505A3 (en) 2015-09-30
EP2712933A3 (en) 2014-07-30
WO2010000874A2 (en) 2010-01-07
US20150197820A1 (en) 2015-07-16
EP2712934A3 (en) 2014-07-30
EP2902505A2 (en) 2015-08-05
EP2712934A2 (en) 2014-04-02
EP2712933A2 (en) 2014-04-02
WO2010000874A3 (en) 2010-07-22
EP2712932A3 (en) 2014-07-30
DE102008031699A1 (en) 2010-01-14
US20130217591A1 (en) 2013-08-22

Similar Documents

Publication Publication Date Title
EP2483690A1 (en) Marker sequences for pancreatic cancer diseases, pancreatic carcinoma and use thereof
WO2013093115A2 (en) Marker sequences for breast cancer and the use thereof
WO2013087929A2 (en) Method for identifying marker sequences for gynaecological malignant tumours
EP2318546A2 (en) Marker sequences for inflammatory prostate diseases, prostate carcinoma and their use
WO2009030226A2 (en) Marker sequences for rheumatoid arthritis and use thereof
EP2441848A1 (en) Marker sequences for systematic lupus erythematodes and use of same
WO2009080017A2 (en) Marker sequence for neurodegenerative diseases and the use thereof
EP2622350A2 (en) Marker sequences for multiple sclerosis and use thereof
EP2201132A2 (en) Marker sequences for multiple sclerosis and use thereof
EP2745115B1 (en) Novel method for diagnosis of high-affinity binders and marker sequences
DE102007041656A1 (en) Use of marker sequences for the diagnosis of rheumatoid arthritis, where the marker sequences of a complementary DNA (cDNA) from the specific sequence is determined in a patient
EP3436828A1 (en) Marker sequences for rheumatoid arthritis
EP2627783A2 (en) Marker sequences for multiple sclerosis and the use thereof
WO2012107596A2 (en) Marker sequences for diagnosing prostate cancer, and use thereof
WO2008092442A1 (en) Stratification and therapy control of a patient by means of protein biochips
EP2780471A2 (en) Novel method for identifying specific marker sequences for prostate cancer
WO2011006909A2 (en) Marker sequences for adiposity
EP2644704A1 (en) Marker sequences for rheumatoid arthritis
DE102015108523A1 (en) Peptide biomarker for cancer
DE102007041654A1 (en) Use of marker sequences for the diagnosis of rheumatoid arthritis, where the marker sequences of a complementary DNA (cDNA) from the specific sequence is determined in a patient

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20110203

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO SE SI SK SM TR

AX Request for extension of the european patent

Extension state: AL BA RS

DAX Request for extension of the european patent (deleted)
17Q First examination report despatched

Effective date: 20111014

RIC1 Information provided on ipc code assigned before grant

Ipc: G01N 33/68 20060101AFI20131121BHEP

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20150203