EP2780471A2 - Nouveau procédé d'identification de séquences de marqueurs spécifiques pour le cancer de la prostate - Google Patents

Nouveau procédé d'identification de séquences de marqueurs spécifiques pour le cancer de la prostate

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Publication number
EP2780471A2
EP2780471A2 EP12809624.5A EP12809624A EP2780471A2 EP 2780471 A2 EP2780471 A2 EP 2780471A2 EP 12809624 A EP12809624 A EP 12809624A EP 2780471 A2 EP2780471 A2 EP 2780471A2
Authority
EP
European Patent Office
Prior art keywords
sequences
seq
prostate cancer
marker sequences
homo sapiens
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Withdrawn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
EP12809624.5A
Other languages
German (de)
English (en)
Inventor
Peter Amersdorfer
Angelika Lueking
Axel Kowald
Bettina SCHLICK
Petra MASSONER
Christof SEIFART
Georg Schäfer
Helmut Klocker
Peter Schulz-Knappe
Klaus MARQUART
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
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Filing date
Publication date
Application filed by Protagen GmbH filed Critical Protagen GmbH
Priority to EP12809624.5A priority Critical patent/EP2780471A2/fr
Publication of EP2780471A2 publication Critical patent/EP2780471A2/fr
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • G01N33/575—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57555—Immunoassay; Biospecific binding assay; Materials therefor for cancer of the prostate
    • 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
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to a novel method for the identification of specific marker sequences for the diagnosis of prostate cancer and / or prognosis in prostate cancer and the use of the identified specific
  • Protein biochips are gaining an increasing industrial
  • Protein biochips require the necessary proteins to be available. In particular,
  • the cDNA of a particular tissue in a bacterial or a eukaryotic expression vector, such as yeast, is 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.
  • expression vectors have sequences for so-called
  • Affinity epitopes or proteins on the one hand for the specific detection of the recombinant fusion proteins by means of a directed against the affinity epitope
  • Antibody on the other hand becomes the specific one
  • 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).
  • Prevalence, incidence and mortality from prostate cancer are increasing worldwide.
  • Prostate cancer is the second most common lethal cancer in men.
  • the incidence of prostate carcinoma is much higher than that
  • prostatitis is a potential risk factor for prostate cancer, which may also influence the progression of the disease.
  • Prostatitis diagnosis is currently performed exclusively by biopsy and histo-pathological analysis.
  • Applicant's WO2010 / 000874 describes e.g. the diagnosis of prostate cancer and prostate infections by means of a
  • Marker sequences for prostate cancer available. For the first time, these marker sequences could be sensitively identified by protein biochips for the respective indications.
  • Robert et al. discloses that CCR4, CCR7, CD40LG, CTLA4, ICOS, IL17, PTPRC, SELP and TFRC
  • the invention relates to a method for the identification of specific marker sequences for the diagnosis of
  • the method according to the invention can be used to identify simple, non-invasive biomarkers (specific marker sequences) which can be used as an indicator of inflammation of the prostate,
  • Prostate cancer can be used.
  • the provision of specific marker sequences allows a reliable diagnosis, prognosis and stratification of
  • Marker sequences located on a solid support are particularly preferably used (presented) in the method according to the invention by means of a protein biochip.
  • Prostate cancer for example, the formation of antigens, especially prostate cancer-specific antigens - are recognized as "foreign" by the immune system, whereupon autoantibodies become targeted and specific to B cells produced. Both the antigens and the autoantibodies that are formed in the course of prostate cancer development and the progression of prostate cancer can be specific
  • Inflammation or an inflammatory process in the prostate / prostate tissue formed antigens and also the autoantibodies then formed are inflammatory markers in the context of this invention.
  • the invention relates to the
  • the selected marker sequences differ at high levels
  • the marker sequences to be investigated, the selected sequences, the specific marker sequences are inflammatory markers and / or autoantigens and / or autoantibodies.
  • a particularly preferred embodiment of the invention relates to methods for the identification of specific
  • Marker sequences which are determined by histological methods, whether the selected marker sequences between progressive (malignant) and non-progressive
  • prostate cancer differentiate (benign) prostate cancer, for example by means of immunohistochemistry on prostate tissue.
  • the invention enables the identification of antigens and autoantigens as specific marker sequences for the
  • Prostate cancer genesis and / or progression The invention also allows a demarcation of prostate cancer
  • the invention thus also relates to a correlation of inflammatory reactions in the prostate with antibodies and / or auto-antibodies, for example in serum.
  • the selected patients or test subjects belong to a uniform population group (population).
  • the procedure can be applied to different populations
  • An essential embodiment of the invention therefore relates
  • a method for identifying specific marker sequences wherein the selected patients belong to a population.
  • a population is an organism belonging to a particular species, preferably Homo sapiens, living in a specific geographical area. Examples of populations are "Europeans, Americans, Asians.”
  • a population may also mean a match in certain genetic parameters, for example one
  • the method according to the invention is therefore also suitable for applications in the context of personalized medicine.
  • the sample of the selected patients or test persons is one
  • Body fluid or a tissue extract in particular blood, whole blood, blood plasma, blood serum, patient serum, urine, cerebrospinal fluid, synovial fluid.
  • Inflammation-specific autoantibodies for example, from blood serum of prostate cancer patients may e.g. in
  • Another embodiment of the invention relates to methods for identifying specific marker sequences wherein the marker sequences mRNA, si-RNA, microRNA, cDNA, peptide or
  • Protein in particular antigens or autoantigens or are derived from an expression library, in particular an mRNA, si-RNA, microRNA, cDNA, peptide or protein expression library.
  • an expression library in particular an mRNA, si-RNA, microRNA, cDNA, peptide or protein expression library.
  • patient samples may be divided into high and low-lit specimens based on the number of tissue-infiltrating lymphocytes.
  • serum samples can be analyzed on protein microarrays.
  • these proteins exist Microarrays of over 3,000 to 5,000 cancer and
  • the marker sequences to be investigated are selected from the group SEQ ID No. 1-176 (proteins), SEQ ID No. 177-352 (DNA cloning sequences) and SEQ ID No. 353-528 (corresponding RNA sequences), partial sequences of SEQ ID No. 1-528 with at least 90%, preferably at least 95% of the length of SEQ ID No. 1 - 528 and homologues of SEQ ID no. 1 - 528 with an identity of at least 95%, preferably at least 98% or more, to the corresponding marker sequences and partial sequences of the homologs of SEQ ID No. 1-528 with at least 90%,
  • the marker sequences to be investigated are presented on a protein microarray.
  • the invention also provides the use of one or more specific marker sequences obtainable by a method according to the invention for the diagnosis of prostate cancer, preferably for the diagnosis of prostate cancer.
  • the invention also provides the use of one or more specific marker sequences obtainable by a method according to the invention for prognosis in prostate cancer and / or for stratification, in particular for
  • the invention also relates to the use of SPOP and / or partial sequences and / or homologs of SPOP
  • SPOP Homo sapiens speckle-type POZ protein
  • the invention also relates to the use of STX18 and / or partial sequences and / or homologs of STX18 for
  • STX18 Homo sapiens syntaxin 18
  • Gl accession number gi / 39725935 has the Gl accession number gi / 39725935.
  • the invention also relates to the use of SPAST and / or partial sequences and / or homologs of SPAST
  • SPAST Homo sapiens spastin
  • the invention therefore also relates to the use of the specific marker sequences obtainable by the method according to the invention, in particular of the specific marker sequences SEQ ID Nos. 1 to 528, for example SPOP, STX18 and / or SPAST for distinguishing benign prostate cancer from malignant prostate cancer.
  • the invention is also an arrangement of
  • Marker sequences obtainable by the method according to the invention include or consist of.
  • the invention is also an arrangement of
  • Method according to the invention for the diagnosis of prostate cancer and / or prognosis in prostate cancer and / or for Stratification in prostate cancer comprising or consisting of one or more specific marker sequences and wherein the specific marker sequences are selected from the group SEQ ID No 1-176 (proteins), SEQ ID No. 177-352 (DNA clone sequences) and SEQ ID No. 353-528 (related RNA
  • the invention is also an arrangement of
  • inventive method comprising or consisting of SPOP and / or partial sequences of SPOP and / or homologs of SPOP and / or STX18 and / or partial sequences of STX18 and / or
  • the invention also provides an inventive arrangement of specific marker sequences for the diagnosis of prostate cancer and / or
  • Prognosis in prostate cancer and / or stratification in prostate cancer Prognosis in prostate cancer and / or stratification in prostate cancer.
  • the subject of the invention is also an assay or protein
  • Microarray comprising an inventive arrangement of specific marker sequences and optionally further additives and excipients.
  • the invention also relates to an assay or protein microarray (protein biochip) comprising a
  • Prostate cancer in particular prostate cancer containing means for detecting a binding success, wherein a.) The arrangement or the assay or the protein microarray is brought into contact with at least one substance to be examined and b.) A binding success is detected.
  • the invention also provides a diagnostic agent for
  • Diagnosis of and / or prognosis in prostate cancer comprising an arrangement according to the invention and / or one or more specific marker sequences obtainable by a
  • the invention also provides a kit for the diagnosis or prognosis or stratification of prostate cancer disorders containing one or more specific marker sequences obtainable by a method according to the invention and / or one or more of the marker sequences selected from the group SEQ ID No 1-176 (proteins), SEQ ID No. 177-352 (DNA cloning sequences) and SEQ ID No. 353-528 (related RNA sequences),
  • SEQ ID No. 1 - 528 preferably at least 95% of the length of SEQ ID No. 1 - 528, for example SPOP and / or STX18 and / or SPAST and / or a partial sequence and / or a homologous sequence thereof.
  • the invention is also the use of a
  • inventive method or selected from one of the sequences SEQ ID No. 1-528 or SPOP or STX18 or SPAST or a partial sequence or a homologous sequence as
  • the invention also provides a target for the treatment and therapy of prostate cancer obtained by
  • the invention is also the use of a
  • the invention also provides a method for the diagnosis of prostate cancer or for prognosis in prostate cancer, wherein a.) One or more specific marker sequences obtained by a method according to the invention and / or one or more of the selected marker sequences SEQ ID. No. 1-528 and / or SPOP and / or STX18 and / or SPAST and / or a partial sequence and / or a homologous sequence is applied to a solid support and b. ) is brought into contact with the body fluid or tissue extract of a subject or patient, and c. ) the detection of an interaction of the body fluid or tissue extract with the marker sequences from a.).
  • a particular embodiment of the invention relates
  • a method for the early detection and diagnosis of prostate cancer wherein the interaction with c.) Represents a prostate cancer-associated autoantibody profile of the patient or a cohort or a population group or a particular disease course (prognosis) or a specific response to a therapy / drug.
  • Diagnostic agent a protein microarray or an array
  • one or more specific marker sequences are used.
  • at least 2, for example 3, 4, 5, 6, 7, 8, 9, 10, preferably 15 to 20 marker sequences or 30 to 50 or 100 or more specific marker sequences are used together or in combination, for example directly after one another or parallel.
  • Marker sequences can be detected, for example, by a probe,
  • Predicting the course and / or early diagnosis of prostate cancer or prostate cancer may allow the patients to be treated and / or monitored early if it is determined that a severe course is likely. In such a case, the patient can be closely monitored and / or treated early. On the other hand, patients may be identified who have a mild course and / or
  • the invention relates to embodiments in which 2 or more specific marker sequences, for example 3.4 or 5 or more, 10 to or more, preferably 30 to 50 marker sequences or 50 to 100 or more marker sequences on a
  • the specific marker sequences according to the invention can also be combined, supplemented or extended with known biomarkers for this indication.
  • the stratification of the patients is included
  • Prostate inflammatory diseases to prostate cancer as well as the meaningful selection of patient groups for the clinical development of new therapeutic substances.
  • Therapy control also includes the classification of patients into responders and non-responders with regard to a 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
  • 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
  • diagnosis also includes the
  • Prostate cancer by means of the marker sequences of the invention and the prognosis of prostate inflammatory diseases,
  • the term “stratification” includes in particular the Risk stratification with the prognosis of an "outcome” of an adverse health event.
  • patient is understood to mean any test subject - human or mammal - with the proviso that the subject is examined for prostate inflammatory diseases up to prostate cancer.
  • Prostate cancer include 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, Pschyrembel, de Gruyter, 261st edition (2007), Berlin).
  • Prostate cancer includes all cancers of the prostate, especially prostate carcinoma. Prostate cancer includes all forms of progression, progressive / aggressive and non-progressive.
  • Prostate cancer specific or “specific” means that the marker sequence, for example the nucleic acid or the respectively obtainable polypeptide or protein from a
  • markers from the body fluid or tissue extract of a patient with prostate cancer Interact with substances from the body fluid or tissue extract of a patient with prostate cancer (e.g., antigen (epitope) / antibody (paratope) interaction). These substances from the body fluid or tissue extract occur either only or at least increased in prostate cancer or are expressed, whereas these substances are not present in patients without prostate cancer or at least to a lesser extent (smaller amount, lower concentration).
  • specific marker sequences can also be characterized in that they have a
  • Marker sequences may also be present in healthy subjects, but their amount (concentration), for example, in the development, establishment and therapy of prostate cancer changes.
  • the specific marker sequences are therefore biomarkers for prostate cancer. The specific ones
  • Marker sequences can in this way have a profile of
  • Imaging substances from body fluid and tissue extract such as a prostate cancer associated
  • compositions thus include on the one hand the composition (one or more
  • the specific marker sequence is an antigen or part of an antigen or encodes an antigen or part of an antigen.
  • the specific marker sequence recognizes / binds to autoantibodies which in the
  • Prostate cancer are present or to a lesser extent (or not) are available.
  • Autoantibodies are formed by the body against the body's own antigens, which arise for example in prostate cancer. Autoantibodies are from Body formed against different substances and pathogens.
  • Marker sequences are detected and thus serve as an indication for prostate cancer.
  • Patients may use for early detection, diagnosis and / or
  • Marker sequences may be necessary to map a prostate cancer-associated autoantibody profile.
  • these autoantibodies can be detected with specific marker sequences derived from another individual, for example, from a commercial cDNA library.
  • these autoantibodies may contain specific marker sequences
  • cDNA library originates.
  • homologues of said specific marker sequences are used, for example sequences which have non-synonymous mutations in the specific marker sequences.
  • Autoantibodies can be formed by the patient many years before the onset of the first disease symptoms. This would be an early detection, diagnosis and also prognosis and
  • Devices and means thus allow for very early intervention compared to known methods, which significantly improves prognosis and survival rates. Since the prostate cancer-associated autoantibody profiles during the establishment and treatment / therapy of
  • the invention also enables the detection and monitoring of prostate cancer in each
  • the agents of the invention also allow for easy home handling by the patient and cost-effective routine screening for early detection.
  • Each patient may have one or more different prostate cancer-associated
  • Autoantibodies in the course of the development of prostate cancer and the progression of the disease ie also different Autoantibody profiles, form.
  • the composition and / or the amount of specific autoantibodies formed may change in the course of the onset and progression of the disease, so that a quantitative evaluation becomes necessary.
  • the treatment / treatment of prostate cancer also leads to changes in the composition and / or the amount of prostate cancer-associated autoantibodies.
  • the wide range of specific marker sequences according to the invention enable the individual composition of specific marker sequences in an arrangement for individual patients,
  • Serum / plasma has the advantage over other biomarkers of high stability and storability and good detectability. Also subject to the presence of
  • Autoantibodies do not have a circadian rhythm, so the sampling is independent of time of day, food intake and the like.
  • prostate cancer-associated autoantibodies can be detected using the corresponding antigens / autoantigens in
  • ELISA ELISA
  • Western Blot a known assays such as e.g. ELISA or Western Blot can be detected and the results are checked.
  • the cDNA or the respectively obtainable polypeptide or protein significantly for Prostate inflammatory diseases and / or prostate cancer, such as prostate cancer are.
  • the cDNA or the respectively obtainable polypeptide or protein interact with substances from the
  • Body fluid or tissue extract of a patient with
  • prostate cancer e.g., antigen (epitope) / antibody (paratope)
  • Essential to the invention is that an interaction between the body fluid or tissue extract of a patient and the specific marker sequences is detected.
  • Such an interaction is e.g. a bond, in particular a binding substance to at least one invention
  • Marker sequence or, in the case of a cDNA, hybridization with a suitable substance under selected conditions, in particular stringent conditions (for example, as usual
  • Hybridization conditions is hybridization in 4 x SSC at 37 ° C followed by several washing steps in 1 x SSC
  • Marker sequences have in a further embodiment of the Invention via a detection signal, which to the
  • binding substance e.g., antibodies
  • the recognition signal is preferably an epitope and / or paratope and / or hapten and, for a cDNA, a hybridization or hybridization protein
  • the invention also relates to the full-length sequences of the marker sequences of the invention as shown in Table 1 on the known database entry according to Table 1
  • the marker sequences also include such
  • Amino acid sequence such as chemical modification, such as
  • partial sequences (partial sequences also include fragments) or homologs of the marker sequences according to the invention are likewise included.
  • Marker sequences according to the invention within the meaning of the invention are specific marker sequences, marker sequences with SEQ ID No. 1-528 according to the attached Sequence Listing, SPOP, STX18 and SPAST.
  • the invention likewise encompasses the full-length sequences of the specific marker sequences of the invention SEQ ID o. 1-528 mentioned.
  • the invention also relates to homologs of the specific
  • Marker sequences and partial sequences for example fragments of specific marker sequences.
  • Homologs are, for example, nucleic acid and / or nucleic acid
  • marker sequences of at least 70% or 80%, preferably 90% or 95%, more preferably 96% or 97% or more, for example 98% or 99% or more.
  • marker sequences of at least 70% or 80%, preferably 90% or 95%, more preferably 96% or 97% or more, for example 98% or 99% or more.
  • Sequence area in which the antigen-antibody or antigen-autoantibody interaction takes place at least 95%, preferably at least 97%, particularly preferably at least 99%.
  • homologs comprising mutations such as base exchange mutations, raster mutations, base insertion mutations,
  • Partial sequences also comprise fragments of the marker sequences according to the invention, partial sequences are those nucleic acids or proteins / peptides which are opposite to the complete nucleic acid or the complete
  • Protein / peptide are shortened.
  • the deletion may be at or near the end and / or within the sequence.
  • partial sequences and / or fragments are the 50 to 100 nucleotides, 70-120 nucleotides one
  • sequences 1 - 528 have complete sequence, for example of SEQ 1-528. Homologs of partial sequences and fragments are also included in the invention. In a particular embodiment, the specific marker sequences compared to the sequences 1 - 528 are so far shortened that they only from the / the
  • the invention also encompasses specific marker sequences which differ from the
  • Sequences SEQ ID no. 1 - 528 in that they contain one or more insertions, wherein the insertions are, for example, 1 to 100 or more nucleotides / amino acids, preferably 5 to 50, particularly preferably 10 to 20
  • Nucleotides / amino acids are long and the sequences are otherwise identical or homologous to the sequences 1-1578. Particularly preferred are partial sequences which are at least 90%, preferably at least 95%, particularly preferred
  • the length of the specific marker sequences of the invention is at least 97% or 98% of the length of the specific marker sequences of the invention. Also included according to the invention are homologs of the partial sequences. Particularly preferred are homologs of the specific marker sequences which have one or more non-synonymous point mutations.
  • the marker protein SPOP for example, shows mutations of the SPOP gene. The described mutations were detected in 6-13% of prostate cancer cases and lead to the loss of the
  • Prostate cancer-specific autoantibodies differ from
  • specific marker sequence may be represented in different amounts in one or more regions on the support. This allows a variation of the sensitivity.
  • the areas can each be a set of specific ones
  • Marker sequence can be represented in different amounts in one or more areas on a solid support. This allows a variation of the sensitivity.
  • the regions may each comprise a total of marker sequences, i. a sufficient number of different marker sequences, in particular 2 to 5 or 10 or more and optionally further nucleic acids and / or proteins, in particular biomarkers.
  • Biomarkers Further preferred are more than 2,500, more preferably 10,000 or more different or the same
  • Marker sequences and optionally other nucleic acids and / or proteins, in particular biomarkers are optionally other nucleic acids and / or proteins, in particular biomarkers.
  • the invention also relates to arrangements of marker proteins.
  • 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 designed such that those represented on the assembly
  • 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 includes such
  • Embodiments of a device such as ELISA, bead-based assay, line assay, Western blot, immunochromatographic
  • 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 even printed, i. applied reproducibly.
  • One or more marker sequences can be duplicated in the totality of all
  • Marker sequences are present and available in different quantities based on a spot. Furthermore, the
  • Marker sequences on the solid support e.g., by serial dilution series of e.g.
  • 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 for example by means of a cDNA expression library according to the invention (Büssow et al., 1998 (supra)).
  • such expression libraries become
  • 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, these can be prepared according to standard works, such as Sambrook et al., Molecular Cloning, Laboratory Laboratory, 2nd edition (1989), CSH press, Cold Spring Harbor, New York Further preferred are such expression libraries
  • tissue specific e.g., human tissue, especially human organs.
  • expression libraries are also included according to the invention, which can be obtained by exon trapping. Instead of expression library can be spoken synonymously from an expression bank.
  • Uniclone® library protein biochips or corresponding expression libraries which have no redundancy
  • Uniclone® library protein biochips or corresponding expression libraries which have no redundancy
  • WO 99/57311 and WO 99/57312 preferred Uniclone libraries have a high content of non-defective, fully expressed proteins of a cDNA expression library.
  • the clones may not be those 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. Therefore, the invention relates to an arrangement, wherein the
  • Marker sequences are present as clones.
  • marker sequences may be in the form of a fusion protein in the particular form
  • At least one affinity epipope or "tag” contains.
  • 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 cellulose-binding one
  • Protein 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
  • Silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target or a matrix Silicon wafer, glass, metal, plastic, a chip, a mass spectrometric target or a matrix.
  • a filter is preferred according to the invention.
  • the filter is PVDF, nitrocellulose or nylon (e.g., Immobilon P Millipore, Protran Whatman, Hybond N + Amersham).
  • this arrangement corresponds to a grid such that the order of magnitude of a microtiter plate (8-12 wells strips, 96 wells, 384 wells or more), of a silicon plate. Wafers, a chip, a mass spectrometric target or a matrix has.
  • a substance to be tested may be any native or non-native biomolecule, synthetic chemical molecule, mixture or substance library.
  • Protein-protein interactions e.g. protein to be examined or specific marker sequence, such as antigen / antibody
  • proteins for detecting binding success can be, for example, by fluorescence labeling, biotinization, radioisotope labeling or colloidal gold or latex particles
  • detection of bound antibodies takes place, for example, with the aid of
  • Reporter molecules e.g., Cy, Alexa, Dyomics
  • FITC fluorescent dyes
  • colloidal gold or latex particles colloidal gold or latex particles
  • reporter enzymes such as
  • alkaline phosphatase horseradish peroxidase, etc. and the corresponding colorimetric, fluorescent or
  • a readout is e.g. by means of a microarray laser scanner, a CCD camera or visually.
  • Figure 1 Volkano plot Representation of the 176 marker sequences (see Example 2).
  • Patients studied were divided into a low-inflammatory group and a high-inflammatory group, and characterized for age, carcinoma size, Gleason score, C-ractives protein, prostate volume, prostate weight, PSA and fPSA%.
  • Prostate tissue of 70 patients with prostate cancer was assessed.
  • tumor and benign prostate are present.
  • the distinction between tumor and benign prostate can be made by p63 staining (benign in p63 positive).
  • HistoFaxs presents a p63 staining to differentiate tumor / benign tissue in red (FastRed) and a CD45 staining for the detection of leukocytes in brown (DAP). Infiltrating immune cells can be detected by the pan-
  • Leukocyte marker CD45 can be detected in prostate tissue. This color combination clearly distinguishes p63 and CD45 from hematoxylin counterstaining. The evaluation in the HistoFAXS was done using the "single reference shade"
  • Zyototoxic T lymphocytes can be detected by the surface marker CD8 in prostate tissues. Counterstaining (tissue recognition) is carried out with hematoxylin.
  • Marker sequences determined (p ⁇ 0.05; Mann-Whitney test; fold change> 2).
  • Table 2 gives a selection of 30 protein sequences which, interestingly, have already been identified as prostate cancer as marker sequences in the course of this study (see WO2010 / 000874).
  • TTC5 Homo sapiens tetratricopeptide repeat domain 5
  • IKBE_HUMAN NF-kappaB inhibitorepsilon (NF-kappa-BIE) (l-kappa-B-epsilon) gi 114548073 0.007 3.59 (IkappaBepsilon) (IKB-epsilon) (IKBE)
  • cyclin-dependent kinase 7 (M015 homologous; Xenopus laevis; cdk-activating gi 116950659 0.008 22.02 kinase) (CDK7); mRNA
  • G protein-coupled reeeptor 161 (GPR161); transcript variant 2; mRNA gi 124476015 0.009 4.12
  • RNA polymerase I UBTF
  • mRNA gi 7657670 0.012 10.36
  • TNFRSF16 nerve growth factor reeeptor (TNFRSF16) associated protein 1 (NGFRAP1); gi
  • IHC reagents ie, commercially available primary antibodies
  • TTLL12 which plays a role in prostate tumor genesis
  • SPAST, SPOP, STX19 are already immunogenic.
  • SPOP and STX18 indicate differentiated staining and are thus useful for distinguishing between benign ones and cancer in areas in the prostate with low inflammation.
  • SPOP (speckle-type POZ protein) modulates the transcriptional repression activity of the death-associated protein 6 (DAXX): E3-ubiquitin ligase genes.
  • DAXX death-associated protein 6
  • SPOP plays a role in TNF-mediated JNK signaling (kidney cancer). SPOP is mutated in prostate cancer. SPOP is localized in the nucleos.
  • ICH Immunohistochemistry
  • Tissue sections with prostate cancer is stronger than in benign prostate cells.
  • SPOP does not show autoantibody staining in prostate cells in low-inflammatory processes (Be greater Ca).
  • STX18 (Syntaxin 18)
  • Stxl8 is a Q-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein associated with the Q-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein associated with the Q-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein associated with the Q-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein associated with the Q-SNARE (soluble N-ethylmaleimide-sensitive factor attachment receptor) protein associated with the
  • Stxl8 is a cell growth inhibiting gene 9 protein. For Stxl8 implications in breast cancer and moderate expression in prostate cancer are described. Stxl8 stains the plasma membrane in ICC.
  • Stxl8 shows a strong staining (high autoantibody levels) with strong inflammation, but no difference between
  • SPAST proteose
  • ATPase Prostate cells (Be larger Ca).
  • SPAST proteose
  • ATPase belongs to the family of proteins that share an ATPase domain and play a role in diverse cellular processes, including membrane transport, intracellular mobility, organelle biogenesis, protein folding, and proteolysis. ATPase may be under construction
  • SPAST moderately stains the cytoplasm in protate cells and positive membranes.
  • SPAST shows strong staining (high autoantibody levels) in strong inflammation but no difference between benign cells and cancer cells. SPAST shows little
  • TTLL12 tubulin tyrosine ligase-like family, member 12
  • AAB no AAB
  • TTLL12 is expressed in the proliferating layer of benign prostate cells. Expression of TTLL12 increases as cancer progresses to metastasis. In many cell lines that are different from metastatic
  • TTLL12 is highly regulated. Downregulation of TTLL12 expression has an effect on various posttranslational Modifications of tubulin. Overexpression of TTLL12 alters the chromosomal ploidy.
  • TTLL12 shows strong staining (high autoantibody levels) in cases of severe inflammation, with benign cells showing markedly higher staining than cancer cells. TTLL12 does not show autoantibody staining in low-inflammatory processes
  • Prostate cells All investigated primary antibodies detect autoantibodies in prostate cells. They show a significantly stronger staining in highly inflammatory processes than in low-inflammatory processes.
  • the targets of three autoantibodies found in blood serum samples from subjects with highly inflammatory prostate cancer are expressed in prostate tissue.
  • Lymphocytes infiltrating the prostate tissue show more staining than the prostate tissue itself. This effect was seen in all four primary antibodies tested
  • STX18 and SPOP showed different amounts in areas with prostate cancer and areas with benign epithelial cells.
  • STXT18 and SPOP might differentiate between benign and prostate cancer areas with highly inflammatory prostate cancer.
  • areas of easily inflamed tissue with and without autoantibodies in blood serum samples were examined.
  • Extremely flammable areas are characterized by a high concentration of infiltrating lymphocytes.
  • Light Inflammatory areas are characterized by low or no infiltration of lymphocytes.
  • SPOP and STX18 both show a stronger staining in non-invasive ⁇ / benign Protatakrebs
  • ribose 5-phosphate isomerase A ribose 5-phosphate epimerase [Homo sapiens]
  • RNA polymerase 1 isoform a [Homo sapiens]
  • TNFRSF16 nerve growth factor receptor
  • nucleotide binding protein 2 (MinD homologue, E. coli) [Homo sapiens]

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Abstract

L'invention concerne un nouveau procédé d'identification de séquences de marqueurs spécifiques pour établir un diagnostic de cancer de la prostate et/ou un pronostic lors d'un cancer de la prostate, et l'utilisation de séquences de marqueurs spécifiques identifiées à l'aide de ce procédé.
EP12809624.5A 2011-11-14 2012-11-14 Nouveau procédé d'identification de séquences de marqueurs spécifiques pour le cancer de la prostate Withdrawn EP2780471A2 (fr)

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EP11189067 2011-11-14
EP12809624.5A EP2780471A2 (fr) 2011-11-14 2012-11-14 Nouveau procédé d'identification de séquences de marqueurs spécifiques pour le cancer de la prostate
PCT/EP2012/072658 WO2013072393A2 (fr) 2011-11-14 2012-11-14 Nouveau procédé d'identification de séquences de marqueurs spécifiques pour le cancer de la prostate

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EP2678450A4 (fr) * 2011-02-24 2015-07-29 Univ Cornell Mutations spop récurrentes dans le cancer de la prostate
WO2018234577A1 (fr) * 2017-06-23 2018-12-27 Protagen Ag Immuno-oncologie destinée au traitement du cancer

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EP1073771B1 (fr) 1998-04-30 2004-12-01 Max-Planck-Gesellschaft Zur Förderung Der Wissenschaften E.V. Nouveau procede permettant la selection de clones dans une banque d'expression et comprenant un rearrangement
ATE282717T1 (de) 1998-04-30 2004-12-15 Max Planck Gesellschaft Neuartiges verfahren zur identifizierung von klonen mit einer gewünschten biologischen eigenschaft, ausgehend von einer expressionsgenbank
US20040029114A1 (en) * 2001-01-24 2004-02-12 Eos Technology, Inc. Methods of diagnosis of breast cancer, compositions and methods of screening for modulators of breast cancer
US20060019256A1 (en) * 2003-06-09 2006-01-26 The Regents Of The University Of Michigan Compositions and methods for treating and diagnosing cancer
DE102008031699A1 (de) * 2008-07-04 2010-01-14 Protagen Ag Markersequenzen für Prostataentzündungserkrankungen, Prostatakarzinom und deren Verwendung
CN102308212A (zh) * 2008-12-04 2012-01-04 加利福尼亚大学董事会 用于确定前列腺癌诊断和预后的材料和方法

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