WO2003056341A2 - Methode de diagnostic de cancers du sein, peptides associes et leurs utilisations - Google Patents

Methode de diagnostic de cancers du sein, peptides associes et leurs utilisations Download PDF

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Publication number
WO2003056341A2
WO2003056341A2 PCT/DE2002/004708 DE0204708W WO03056341A2 WO 2003056341 A2 WO2003056341 A2 WO 2003056341A2 DE 0204708 W DE0204708 W DE 0204708W WO 03056341 A2 WO03056341 A2 WO 03056341A2
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Prior art keywords
bcap
peptides
peptide
breast cancer
seq
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PCT/DE2002/004708
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German (de)
English (en)
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WO2003056341A3 (fr
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Norbert Lamping
Hans Kreipe
Rüdiger HESS
Markus Kellemann
Richard Lilischkis
Harald Tammen
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Biovision Ag
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Priority to AU2002365104A priority Critical patent/AU2002365104A1/en
Priority to DE10296200T priority patent/DE10296200D2/de
Publication of WO2003056341A2 publication Critical patent/WO2003056341A2/fr
Publication of WO2003056341A3 publication Critical patent/WO2003056341A3/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • 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
    • G01N33/57415—Specifically defined cancers of breast

Definitions

  • the invention relates to a method by the detection and quantification of defined peptide fragments in a biological sample of a patient, which make it possible to detect the presence of breast cancer.
  • the presence of increased concentrations of these peptides or the absence of these peptides in the sample enables early diagnosis and an assessment of the severity / stage of the disease, as well as stratification of the patients.
  • the invention also relates to the use of these peptides for the preparation of diagnostic reagents such as e.g. Antibodies and the use of these peptides or agents made using these peptides, e.g. Antibodies, for the therapy of tumor diseases of the human breast.
  • breast cancer is the most common cancer in women. Only about 1% of all breast cancers occur in men, and breast cancer therefore does not play a major role in men.
  • Various genes, presumably closely related to breast cancer, have been identified in recent years. For example, about 5% of women with breast cancer have one of the two breast cancer genes BRCA1 or BRCA2 [1].
  • BRCA1 or BRCA2 [1] the exact cause of the majority of breast cancers is unclear and presumably the disease is often based on several gradually occurring mutations. These mutations can lead to changes in the expression rates, the tissue specificity or the processing of numerous other proteins, even if the genes which code for these proteins are not directly affected by the mutations.
  • transcription factors for example, transcription factors, structural proteins, protease inhibitors or proteases can be mutated and these mutations thus indirectly affect expression, tissue specificity or processing affect other proteins.
  • the complexity of these processes and the previously limited knowledge of mutations that are causally linked to breast cancer make it currently impossible to predict from which proteins, for example, new peptide fragments that may be suitable as markers will increase or decrease in breast cancer.
  • peptide fragments in breast cancer are causally related to the tumor disease, e.g. Regulate functions of signal transduction chains and thus influence cell proliferation or because they e.g. Influence the activity of proteases.
  • Proteases in turn, are related to the metastasis of tumors and local inflammatory reactions that occur [1].
  • breast cancer Most cases of breast cancer are discovered by the patients themselves, especially by palpating the tumors, whereby tumors can only be felt from a certain size. However, as the size of the tumor increases, the prognosis worsens, reducing the likelihood that the breast can be preserved by surgical removal of the tumor. The preservation of the breast is very often of great psychological importance for the patient.
  • X-ray examinations of the breast are another important aid for the diagnosis of breast cancer and can be used routinely Reduce breast cancer mortality by up to 25 to 35%, but also have a high rate of false positive diagnoses at 15%. In routine breast cancer examinations, 40% of breast cancer diseases are discovered not by palpation but by mammography.
  • breast cancer markers that can be measured, for example, in plasma or in tissue samples (biopsies, tissue sections, etc.) is of no importance in routine examinations and also plays only a minor role in examinations where there is a specific suspicion of breast cancer.
  • the "Carcinoembryonic antigen” (CEA) and the cancer antigen 15-3 (CA 15-3) are among the best investigated experimental markers to date. Both markers indicate with a greater than 50% probability that the breast tumor has already metastasized [ 1], but are not suitable for the diagnosis of early stages of breast cancer [2].
  • Cancer is one of the most common human diseases. Breast cancer is the most common with more than 1 million new cases
  • the treatment of breast cancer usually includes the surgical removal of the malignant tissue in the breast and the removal of the axillary lymph nodes on the same side of the body.
  • a breast-conserving operation is usually carried out in the meantime, but in these cases any remaining tumor tissue is destroyed with subsequent radiation therapy.
  • the treated breast is usually irradiated 5 times a week on an outpatient basis over a period of 6 weeks.
  • chemotherapy is carried out before or after the operation in order to reduce the tumor mass before the operation, or in order to destroy any remaining tumor cells and possibly undetected metastases after the operation.
  • therapy with estrogen-blocking drugs such as tamoxifen (trade name: Noivadex TM), can be carried out for estrogen receptor-positive tumors which often require estrogen for growth.
  • Chemotherapy is urgently needed.
  • An example of this would be e.g. a
  • BCAP Breast Cancer Eeptides
  • These intermediate filament proteins include cytokeratin-19 (CK19), of which BCAP-5 and -6, cytokeratin-18 (CK18), of which BCAP-7 to -9, cytokeratin-17 ( CK17), of which BCAP-37 and -38, cytokeratin-1 (CK14), of which BCAP-10 and 1 1, cytokeratin-5 (CK5), of which BCAP-39 and -40, and vimentin (VIME), derived from BCAP-12 to -15, another protein, thymosin-beta 10 (Tb-10), derived from BCAP-16 to 22, binds to actin and actin is also a central building block from which intermediate filaments are built BCAP-3 to BCAP-22 and BCAP-37 to BCAP-40 all directly with the Structure of intermediate filaments, a central structural element of the cytoskeleton of the cell in the cytosol, in connection.
  • CK19 cytokeratin-19
  • CK18 cytokeratin-18
  • BCAP-7 to -9
  • BCAP-23 to BCAP-28 are derived exclusively from histone proteins.
  • BCAP-23 and BCAP-24 are derived from histone H2B, BCAP-25 and BCAP-26 from histone H3B and BCAP-27 and BCAP-28 from histone H2A.
  • UDP-glucose 6-dehydrogenase (UGDH) and glyceraldehyde 3-phosphate dehydrogenase (G3P2) are two enzymes that play an important role in glycogen metabolism and both are dehydrogenases.
  • BCAP-1, -2, -31 and -32 can thus be assigned to a group of BCAP peptides.
  • hAG-2 human anterior gradient 2 homolog
  • TAG2 transgelin 2
  • VIME 444-465 2534.3 IKTVETRDGQVINETSQHHDDL 15 VIME 450- -457> 929.5 x11-RDGQVINE-x12
  • * x1- corresponds to the sequence or parts of the sequence of UGDH from
  • x2- corresponds to the Sequence or part of the sequence of UGDH from amino acid 386 to 400, where x2-, starting from amino acid 385 of UGDH between 0 and 15
  • Amino acids can be long. All other placeholders x3- to x32- represent sequences or partial sequences of the following proteins: x27- and x28- refer to the sequence of cathepsin-A, or BCAP-36, x3- and x4- refer to the sequence of Cathepsin-D, or on BCAP-3, x5- and x6- refer to the sequence of cytokeratin 19, or BCAP-5, etc.
  • UGDH UDP-Glucose 6-dehydrogenase
  • Cat-A Cathepsin-A
  • Cat-D Cathepsin-D
  • CK19 Cytokeratin-19
  • CK18 Cytokeratin-18
  • CK17 Cytokeratin-7
  • CK14 Cytokeratin-1
  • CK5 Cytokeratin -5
  • VIME vimentin
  • Tb-10 thymosin-beta 10
  • H2B histone H2B
  • H3B histone H3B
  • H2A histone H2A
  • hAG-2 human anterior gradient 2 homologue
  • G3P2 glyceraldehyde 3-phosphate dehydrogenase
  • TAG2 transgelin 2.
  • BCAP peptides listed in Table III below are sequence fragments of protein database entries from the “GeneBank” databases and from the EMBL database. table
  • the BCAP peptides according to the invention can be present in post-translational or chemical modification forms, such as, for example, as glycosylated, phosphorylated, sulfated, formylated, oxidized, amidated and acetylated peptides, etc. This affects, among other things, their masses and thus the mass spectrometric identification and also their elution behavior the chromatography, such as in reverse phase chromatography.
  • the peptides can be present with a cysteinyl modification.
  • BCAP peptides Polymorphisms or mutants and the BCAP peptides and BCAP peptide fragments corresponding to these are also referred to as BCAP peptides.
  • polymorphisms often represent almost identical proteins that differ from the original sequence by only one or a few amino acids. Their biological activity is usually identical to that
  • the peptides are also regarded as BCAP peptides in particular if a maximum of 2 of their amino acids deviate from the corresponding sequence of your precursor molecule. Point mutations, deletions, insertion of amino acids and N-terminal and / or C-terminal extensions are permitted as long as the peptide sequence is at least 8 amino acids long, at least 8 amino acids are conserved relative to the sequence of the precursor molecule, and at the same time a maximum of 2 amino acids from the precursor sequence differ.
  • GCG program package Genetics Computer Group, University of Wisconsin, Madison, Wl, USA
  • GAP [6] BLASTP, BLASTN, FASTA [7] or the well-known Smith Waterman algorithm
  • Preferred parameters for the amino acid sequence comparison include the algorithm by Needleman and Wunsch [8], the comparison matrix BLOSUM 62 [9], a gap value (gap penalty) of 12, a gap length value (gap length penalty) of 4 and a Homology threshold of 0.
  • GAP program is also suitable for use with the above parameters.
  • the above parameters are the error parameters (default parameters) for amino acid sequence comparisons, with gaps at the ends indicating the homology Do not decrease the value. In the case of very short sequences compared to the reference sequence, it may also be necessary to increase the expectation value to up to 100,000 (expectation value) and, if necessary, to reduce the word size to up to 2. Further exemplary algorithms, gap opening penalties, gap extension penalties, comparison matrices including those mentioned in the program manual, Wisconsin package, version 9, September 1997, can be used. The selection will depend on the comparison to be performed and also on whether the comparison is carried out between pairs of sequences, with GAP or the best fit being preferred, or between a sequence and an extensive sequence database, with FASTA or BLAST being preferred.
  • 70% homology A 70% agreement determined with the above-mentioned algorithm is referred to as 70% homology in the context of this application. The same applies to higher degrees of homology. Due to the degenerate genetic code, ie several different codons code for the same amino acid, a 70% homology at the amino acid level corresponds to a lower homology at the nucleic acid level. Therefore, in this application, a homology of 70% to specifically named peptides and only a homology of 60% for nucleic acids is set as the minimum value in order to describe peptides or nucleic acids as belonging to the present invention.
  • the concentration of the BCAP peptide (s) identified is significantly changed for each of these peptides in a defined concentration that is either always specifically higher or always specifically lower for the respective peptide.
  • the ratio of the concentrations of the respective peptides to the concentration in the control sample can be used to determine the severity or stage of breast cancer and to estimate the risk of developing metastases, in particular to supplement the test results with imaging methods for breast cancer diagnosis, such as mammography, ultrasound examinations or Computed tomography and to supplement physical
  • the control sample can be a pool sample from various controls.
  • the sample to be examined can also be a pool sample, with individual examinations being carried out if the result is positive.
  • the biological sample can preferably be a blood sample, plasma, serum or a tissue homogenate obtained from biopsy samples, from tissue sections or from tissue removed during surgery, preferably breast or lymph node tissue or tissue from metastases that can occur in the context of breast cancer diseases. his.
  • Samples of another biological material such as e.g. Breast secretions, milk, lymph fluid, cell homogenates, cell extracts from blood cells or other tissues, or a smear, urine, lavage fluid from the lungs, sputum or cerebrospinal fluid, etc. can be used.
  • the type and origin of the tissue to be examined depends, among other things. on the sensitivity of the chosen detection method (mass spectrometry, ELISA, histological examination etc.), the suspected stage of breast cancer, the position where the cancerous tissue is suspected, etc.
  • tissue extracts are prepared for the preparation of the sample to be examined, preferably from tissue samples obtained in the context of biopsies or from surgical interventions.
  • These fabrics can be used, for example, with manual homogenizers, with ultrasonic homogenizers or with electrically operated homogenizers such as Ultraturrax Cooling at temperatures below 4 ° C, preferably below 0 ° C, and then in a manner known to those skilled in the art in acidic, aqueous solutions with, for example, 0.1 to 0.2 M acetic acid for
  • the extracts are then subjected to the respective detection method, e.g. subjected to a mass spectrometric examination.
  • the samples can be prepared in the usual way, e.g. if necessary, diluted or concentrated, and stored.
  • the invention further comprises the use of at least one of the BCAP peptides according to the invention and the corresponding unprocessed proteins from which these BCAP peptides are derived for the diagnosis of breast cancer diseases, and the use of BCAP peptides for the production of antibodies or of other agents which, because of their BCAP-peptide-specific binding properties, are suitable for the development of diagnostic reagents for the detection of this disease.
  • the invention also encompasses the use of BCAP peptides to obtain phage particles which specifically bind these peptides or of phages which present BCAP peptides on their surface and thus enable the identification of binding partners of BCAP peptides.
  • BCAP peptides can be used. All methods that make it possible to specifically detect BCAP peptides in a patient's sample are suitable for this. Suitable methods include physical methods such as mass spectrometry or liquid chromatography, molecular biological methods such as reverse transcriptase polymerase chain reaction (RT-PCR) or immunological detection techniques such as "enzyme linked immunosorbent assays” (ELISA). Physical detection methods
  • Methods which can indicate the peptides according to the invention qualitatively or quantitatively include mass spectrometry, liquid chromatography,
  • Breast cancer and / or the severity or stage of this disease can be derived, as well as stratification of patients.
  • the peptides in the sample are separated chromatographically prior to identification, preferably with reverse phase chromatography, and separation of the peptides in the sample with high-resolution reverse phase high-performance liquid chromatography (RP-HPLC) is particularly preferred.
  • RP-HPLC reverse phase high-performance liquid chromatography
  • Embodiment of this invention is the implementation of precipitation reactions for fractionation of the sample using precipitation agents such as e.g. Ammonium sulfate, polyethylene glycol, trichloroacetic acid, acetone, ethanol etc.
  • the fractions thus obtained are then subjected to the respective detection method, e.g. the mass spectrometric examination.
  • Another embodiment of the invention is the use of liquid phase extraction.
  • the sample is e.g. mixed with a mixture of an organic solvent such as polyethylene glycol (PEG) and an aqueous saline solution. Due to their physical properties, certain constituents of the sample accumulate in the organic phase and others in the aqueous phase and can thus be separated from one another.
  • PEG polyethylene glycol
  • fractions obtained in this way are supported using a mass spectrometer, preferably using a MALDI mass spectrometer (matrix-supported
  • the BCAP peptides can be identified with the aid of a mass spectrometric determination, preferably MALDI (matrix-assisted laser desorption and ionization) mass spectrometry.
  • the mass spectrometric determination further preferably comprises at least one of the following mass signals, each calculated on the basis of the theoretical, monoisotopic mass of the corresponding peptide.
  • the measurement error is 500 ppm for peptides in the mass range from 1000 to 4000 daltons, the error of the measurement gradually increasing for peptides with a mass that is greater than 4000 daltons.
  • a proton is added to the peptides in MALDI mass determinations due to the measurement methodology, which causes the mass to decrease by 1 dalton elevated.
  • the following masses correspond to the theoretical, monoisotopic ones
  • Masses of peptides we identified; calculated with suitable software, here GPMAW 4.02. The calculated masses were rounded up to one decimal place after the decimal point. These theoretical, monoisotopic masses can occur individually or in combinations in a sample:
  • BCAP peptides Chemical or post-translational modifications possibly present on BCAP peptides increase the mass of these peptides accordingly, e.g. by an oxide group by approx. 16 daltons by a phosphate group by approx. 80 daltons, or by a cysteinyl modification by approx. 120 daltons etc.
  • the symbol> (is larger or the same) is to be understood such that not arbitrarily larger masses for the BCAP peptides concerned are possible, but only the masses which may result from the amino acids possibly present at the ends of these peptides.
  • Peptides have been identified, can be derived. This protection is carried out by identifying the peptide signals, preferably using mass spectrometric methods, e.g. an MS / MS analysis [10].
  • BCAP-1 to BCAP-40 Specific peptide fragments were identified for the first time by the method according to the invention and their significance was recognized. These peptides and their descendants are referred to here as BCAP-1 to BCAP-40. Their sequences are in the sequence listing under Seq. ID 1 to Seq. ID 40 specified.
  • the invention also encompasses the recombinantly, enzymatically (e.g. by in vitro translation) or synthetically produced BCAP peptides isolated from biological samples in unmodified, chemically modified or post-translationally modified form. Two point mutations as well as further deviations are possible as long as the BCAP peptide has at least 8 amino acids which correspond in identity and position within the peptide sequence to the precursor molecule.
  • the invention also encompasses nucleic acids which correspond to BCAP peptides, in particular those which correspond to the peptides BCAP-1 to BCAP-40 according to the invention, and which correspond to their use for the indirect determination and quantification of the associated protein precursor molecules (Seq. ID 41 to 56) the protein sequences, Seq. ID 57 to 72 the nucleic acid sequences of these precursor molecules).
  • This also includes nucleic acids which, for example, represent non-coding sequences, such as 5'- or 3'- untranslated regions of the mRNA, or nucleic acids which are one for specific hybridization experiments have sufficient sequence agreement with the nucleic acid sequence of complement C3, and therefore for indirect detection of the associated peptides, in particular the MAC3-
  • Peptides are suitable.
  • An exemplary embodiment of this includes the collection of tissue samples from
  • RT-PCR Reverse transcriptase polymerease chain reaction
  • PCR reverse transcriptase polymerase chain reaction
  • PCR quantitative PCR
  • PCR Northern blots
  • nucleic acid chip experiments can be used in a manner known to the person skilled in the art.
  • the results can be used to determine the likelihood of a disease Breast cancer and / or the stage of the disease are derived, as well as stratification of patients.
  • the BCAP peptides can be identified using an immunological detection system, preferably an ELISA (enzyme linked immunosorbent assay).
  • This immunological detection detects at least one BCAP peptide.
  • a so-called “sandwich ELISA” should also preferably be used, in which the detection of the BCAP peptide depends on the specificity of two antibodies which recognize different epitopes within the same molecule.
  • BCAP peptides For the detection of BCAP peptides, however, other ELISA systems, such as direct or competitive ELISA, are also used, and other ELISA-like detection techniques, such as RIAs (radio immunoassays), ELI spot, etc., are also suitable as immunological detection systems for BCAP peptides.
  • BCAP peptides isolated, recombinantly produced or chemically synthesized can be used as the standard for the quantification.
  • the BCAP peptide (s) can be identified, for example, generally with the aid of an antibody directed at the peptide or peptide fragment.
  • detection methods suitable for peptide detection include Western blotting, immunoprecipitation, dot blots, protein chip experiments, plasmon resonance spectroscopy (BIACORE TM), phage particles, affinity matrices etc.
  • BIACORE TM plasmon resonance spectroscopy
  • all substances / molecules are suitable as detection agents that allow a specific one Set up detection system for BCAP peptides.
  • BCAP Peptides and Anti-BCAP Peptide Antibodies Another embodiment of the invention is the extraction of BCAP peptides using recombinant expression systems, chromatography methods and chemical synthesis protocols which are known to the person skilled in the art.
  • the BCAP peptides obtained in this way can be used, inter alia, as standards for quantifying the respective BCAP peptides or as an antigen for producing BCAP peptide-specific antibodies.
  • Recombinant expression of pepids is one of the methods known and suitable for the person skilled in the art for isolating and obtaining BCAP peptides.
  • BCAP peptides For the expression of the BCAP peptides, cell systems such as bacteria such as Escherichia coli, yeast cells such as Saccharomyces cerevisiae, insect cells such as Spodoptera frugiperda (Sf-9) cells, or mammalian cells such as "Chinese Hamster Ovary” (CHO) cells can be used Cells are available from the American Tissue Culture Collection (ATCC).
  • ATCC American Tissue Culture Collection
  • nucleic acid sequences which code for BCAP peptides are inserted into an expression vector.
  • the sequence of the respective BCAP peptides can be encoded by a large number of different nucleic acid sequences, since several nucleic acid codes are possible for most amino acids and therefore different nucleic acid sequences are used can, as long as they encode a BCAP peptide sequence.
  • Nucleic acid sequences such as e.g. Promoters, antibiotic selection markers etc. inserted into an expression vector using molecular biological methods [1 1].
  • Suitable vectors for this are e.g. pcDNA3.1
  • BCAP peptide expression vectors obtained in this way can then be inserted into suitable cells, for example by electroporation.
  • the isolation of the BCAP peptides produced in this way can be obtained from cell extracts which are produced, for example, as described in Example 2, and the BCAP peptides can be isolated by reverse phase chromatography, for example as described in Example 3.
  • the BCAP peptides can be prepared by chemical synthesis, for example according to the Merrifield solid-phase synthesis protocol (tBOC synthesis protocol) [12] or the FMOC synthesis protocol using automatic synthesizers, which are available from various manufacturers, such as Applied Biosystems Inc., Foster City , CA (ABI 433A), Advanced ChemTech Inc., Louisville, KY (Apex 396), Gilson Medical Electronics Inc., Middleton, Wl, Rainin Instruments Co. Inc., Woburn, MA. (Symphony model), etc. are available.
  • tBOC synthesis protocol tBOC synthesis protocol
  • FMOC synthesis protocol automatic synthesizers
  • Another embodiment of this invention is the isolation of BCAP peptides from biological samples or from cell culture media or cell lysates of recombinant expression systems using, for example, reverse phase chromatography, affinity chromatography, ion exchange chromatography, gel filtration, isoelectric focusing, etc. or using other methods such as preparative immunoprecipitation, ammonium sulfate precipitation , Extraction with organic solvents, etc.
  • Another embodiment of the invention is the production of monoclonal or polyclonal antibodies using BCAP peptides. The antibodies are obtained in the usual way Expert familiar way. A preferred embodiment is
  • Another application example is the quantitative determination of the above-mentioned BCAP peptides to estimate the effectiveness of a therapy in development against breast cancer diseases.
  • the quantitative measurement results from a sample to be examined are compared with the measurement values obtained from a control group and a group of patients.
  • the effectiveness of a therapeutic agent can be derived from these results.
  • the efficacy test is of outstanding importance for the successful development of new therapeutics and new treatment concepts and so far there are no established clinically measurable parameters available for breast cancer that reliably enable this.
  • regular checks are required at intervals to determine whether tumors and metastases are recurring as early as possible.
  • BCAP peptides could also be used as markers for breast cancer tumors in these controls.
  • BCAP peptides Examination of the therapeutic efficacy of BCAP peptides and of agents which modulate the expression and the bioavailability of these substances.
  • An embodiment of this includes the cultivation of cell lines and their treatment with BCAP peptides or with substances which express the expression of Promote BCAP peptides, or those that process precursors
  • Peptide sequences that promote transport of the fusion peptide into the interior of the cell are HIV-TAT, Antennapedia, Herpes
  • Expression vectors are transfected which directly or indirectly influence the expression of BCAP peptides or the corresponding precursor molecules by the transfected cells, e.g. by coding directly for BCAP peptides, or by coding for molecules which are involved in the processing or expression of BCAP peptides or the corresponding precursor molecules.
  • the simultaneous transfection with several different expression vectors can also be carried out.
  • suitable cell lines can be treated with anti-BCAP peptide antibodies or antibodies against the corresponding precursor molecules or with corresponding nucleic acids which suppress the expression of BCAP peptides, e.g. Antisense, triplex, RNAi nucleic acids or ribozymes.
  • cell lines that are considered models for breast tumor cells such as MCF-7 cells can be used for such studies.
  • Tests which measure the rate of proliferation of the treated cells, their metabolic activity, the rate of apoptosis of the cells, changes in cell morphology, changes in the expression of cell-specific proteins or reporter genes added by the cells or which can be used as read-out systems for these studies determine the release of cytosolic cell components as markers for cell death.
  • Suitable strains of experimental animals for example mice or rats, which serve as models for tumor diseases, for example p53 deficient mice, can be used as further test systems. These test animals can be used to investigate the effectiveness of therapy strategies by modulating the concentration of BCAP peptides or 0 of the corresponding precursor molecules aim to be used. It can also be used in suitable
  • Peptides may be galenically prepared in this way.
  • a galenical preparation method e.g. Liposome-packaged peptides, or peptides that are covalent or non-covalent with transport peptides, e.g. the HIV-TAT
  • Proteins are chemically modified in such a way that they acquire lipophilic properties and can therefore pass through Zeil and Organell membranes more easily.
  • Peptides that are only sparingly soluble in aqueous solutions can be chemically modified in such a way that they become more hydrophilic and thus e.g. can be used as an intravenous injectable therapeutic agent.
  • Acid-resistant capsules can be used to protect orally administered sensitive substances in the stomach from being destroyed by gastric acid.
  • Measurement parameters in experiments with experimental animals can be the survival time of the animals, the tumor mass, the tumor diameter or the occurrence and the number of metastases.
  • body function temperature, respiratory rate, heart rate, etc.
  • determination of e.g. immunological mediators and antibodies from blood, urine or tissue samples e.g., the determination of e.g. immunological mediators and antibodies from blood, urine or tissue samples, metabolic tests, the expression of BCAP peptides and other peptides related to the disease, as well as morphological and histological tests on tissues, e.g. the tumor tissue or the tissue of lymph nodes and vessels.
  • experimental animals can be obtained using molecular biological methods, in whose organism the precursor molecules of the BCAP peptides are not produced or are produced in reduced or increased amounts. Thereby the expression can be changed both locally and in the entire organism of the test animal in a targeted manner.
  • Caenorhabditis elegans, Drosophila, zebra fish, mice, rats, etc. are suitable as experimental animals.
  • Figure 1 Western blot for the detection of the precursor molecules of vimentin, cytokeratin 14 and 18 in HMEC and MCF-7 cell extracts
  • FIG. 2 Mass spectrometric measurement (MALDI) using the example of A) BCAP-1 in MCF-7 cells, B) BCAP-14 in HMEC cells (signal present), C) BCAP-14 in MCF-7 cells (signals not available) and D) BCAP-25 in MCF-7 cells.
  • MALDI Mass spectrometric measurement
  • Figure 5 Diagram showing the MALDI signal intensities of A) BCAP-1, B) BCAP-35, C) BCAP-3, D) BCAP-5, E) BCAP-8, F) BCAP-37, G) BCAP -10, H) BCAP-39, I) BCAP-12, J) BCAP-16, K) BCAP-23, L) BCAP-25 with (BCAP-25 *) and without (BCAP-25) cysteinyl modification, M) BCAP-27, N) BCAP-29, O) BCAP-31 and P) BCAP-33 in cell extracts obtained from HMEC and MCF-7 cells.
  • Figure 6 Diagram showing the MALDI signal intensities of BCAP-29 (Fig. 6A) and BCAP-16 (Fig. 6B) in
  • Treated sulfate sample buffer [1 1] for 2 minutes at 95 ° C and then a sample amount corresponding to 20 ⁇ g protein of each cell extract in 12%
  • SDS-polyacrylamide gels separated electrophoretically in a manner known to those skilled in the art [1 1].
  • the proteins thus separated in the gels are transferred in a manner known to the person skilled in the art by applying an electric field to nitrocellulose membranes (0.2 m pore size) and the successful transfer of the proteins to the membrane is checked by reversibly staining the proteins with Ponceau S.
  • the presence of various proteins in the electrophoretically separated cell extracts is determined in a manner known to the person skilled in the art [13]. checked with Western blots. The following proteins were identified with the following
  • the human breast tumor cell line MCF-7 human breast adenocarcinoma cell line, estrogen receptor positive
  • American cell culture collection American tissue culture collection, ATCC, Rockville, MD, USA
  • normal, human breast epithelial cells HMEC, "Normal Human Mammary Epithelial Cells"
  • the HMEC cells had passed 7 passages on delivery and were used for a maximum of 5 further passages in culture before use
  • the cell culture medium used for the MCF-7 cell line RPMI 1640 was cell culture medium with 2 mM glutathione, 20 mM HEPES (4- (2-hydroxyethyl) piperazine-1-ethanesulfonic acid), pH 7.5 and 10% fetal calf serum, 0.2 units / ml insulin, 10 units / ml penicillin and 10 ⁇ g / ml streptomycin.
  • the HMEC cells were in MEBM ("Mammary Epithelial Cell Basal Medium”) cell ku ltur medium with 0.52 mg / ml bovine pituitary extract, 10 ng / ml human, recombinant EGF ("Epidermal Growth Factor", Epidermis growth factor), 10 ⁇ g / ml insulin, 1 ⁇ g / ml hydrocortisone, 0.1 mg / ml Gentamycin and 0.1 ⁇ g / ml amphotericin B cultivated.
  • the cells were cultivated in sterile 75 cm 2 plastic cell culture bottles at 37 ° C. in a C0 2 cell culture incubator until they had grown into a cell lawn that was not yet completely closed.
  • biopsy samples or tissue removed during surgical interventions are used.
  • Removal from the patient were stored at -70 ° C.
  • a tissue sample corresponding to approximately 25,000 to 1,000,000 cells is used to obtain cell extracts. Larger pieces of tissue are crushed in a frozen state with a scalpel and in the presence of a high-molar solution of a chaotropic salt, e.g. Urea or guanidinium and ground with ice in a mortar.
  • the cell extracts are then stored at -70 ° C until further processing.
  • Example 3 Chromatographic separation of peptides from cell extracts for mass spectrometric measurement of BCAP peptides
  • This sample pretreatment serves to enrich the peptides according to the invention and to separate components which can interfere with the measurement.
  • Reverse phase chromatography is used as the separation process.
  • Various RP chromatography resins and eluents are equally suitable.
  • the example below shows the separation of BCAP peptides using a C18 reverse phase chromatography column with the size 4 mm ⁇ 250 mm from Vydac.
  • compositions were used: solvent A: 0.06% (v / v) trifluoroacetic acid, solvent B: 0.05% (v / v) trifluoroacetic acid, 80% (v / v) acetonitrile. Chromatography was carried out at 33 ° C using a HP-ChemStation 1 100 from Agilent Technologies with a flow cell Micro from Agilent Technologies. Cell extracts obtained from were obtained as a sample
  • Cell extracts are in 750 ⁇ l of the chaotropic saline solution, pH 2 to 4, are heated for 5 minutes at 95 ° C, with 0.1% trifluoroacetic acid in water
  • Chromatography column applied.
  • the chromatography conditions were as follows: 5% solvent B at the time 0 min, from time 1 to 45 min continuous increase in the solvent B concentration to 50%, from time 45 to 49 min continuous increase in the solvent B concentration to 100% and then until Time 53 min constant 100% buffer B. 10 minutes after the start of the chromatography, 96 fractions of 0.25 ml each are started.
  • MALDI-TOF mass spectrometer matrix-assisted laser desorption ionization
  • Suitable MALDI-TOF mass spectrometers are manufactured by PerSeptive Biosystems Framingham (Voyager-DE, Voyager-DE PRO or Voyager-DE STR) or by Bruker Daltonik GmbH (BIFLEX).
  • a matrix substance typically consists of an organic acid.
  • Typical matrix substances which are suitable for peptides are 3,5-dimethoxy-4-hydroxycinnamic acid, alpha-cyano-4-hydroxycinnamic acid and 2,5-dihydroxybenzoic acid.
  • a dried equivalent of a cell extract according to Example 2 corresponding to 25,000 to 500,000 cells, obtained by reverse phase chromatography, is used.
  • the chromatographed sample is dissolved in 15 ⁇ l of a matrix solution.
  • This matrix solution contains, for example, 10 g / 1 ⁇ -cyano-4- hydroxycinnamic acid and 10 g / 1 L (-) fucose dissolved in one
  • Solvent mixture consisting of acetonitrile, water, trifluoroacetic acid and acetone in a volume ratio of 49:49: 1: 1. 0.3 ⁇ l of this solution are transferred to a MALDI carrier plate and the dried sample is analyzed in the MALDI mass spectrometer Voyager-DE STR from PerSeptive Biosystems.
  • BCAP-14 gives a clear signal only in HMEC samples, but not in MCF-7 samples, i.e. BCAP-14 is a good marker for differentiating between HMEC cells and MCF-7 cells, or between healthy breast cells and breast cancer cells.
  • MALDI-TOF mass spectrometry can be used to quantify peptides such as the BCAP peptides according to the invention if these peptides are present in a concentration that is in the dynamic measuring range of the mass spectrometer, thereby avoiding detector saturation.
  • concentration concentration for each peptide
  • MALDI mass spectrometry can preferably be used for the relative quantification of peptides. This is shown in Figure 3. If different amounts of different standard peptides are added to a sample, the intensity of both these standard signals and the sample signals can be determined.
  • a BCAP peptide ion is selected in the mass spectrometer on the basis of its specific m / z (mass / charge) value in a manner known to the person skilled in the art in the mass spectrometer. This selected ion is then fragmented by supplying collision energy with a collision gas, e.g.
  • the fragmentation behavior of peptides enables the BCAP peptides according to the invention to be uniquely identified with the mass accuracy of the devices used of 50 ppm using computer-assisted search methods [15] and sequence databases into which the sequences of the precursor molecules have been entered. Taking into account the positive shift of the molecular mass by approx. 16 daltons, which corresponds to the mass of an oxygen atom, oxidized peptides can also be identified with this method.
  • Example 6 Mass spectrometric quantification of BCAP peptides to compare their relative concentrations in the breast tumor line MCF-7, the control cell line HMEC and in tissue samples from breast cancer patients.
  • the concentration of BCAP peptides in the breast tumor cell line MCF-7 and in normal, human breast epithelial cells ( HMEC cells) compared.
  • cell extracts which were prepared in accordance with Example 2 were separated by chromatography in accordance with Example 3 and quantified in the MALDI spectrometrically.
  • Figure 3 shows the MALDI signal intensity relative to the peptide concentration using the example of standard peptides.
  • Figures 5A to 5K show the corresponding results for the peptides BCAP-1 (Fig. 5 A), BCAP-3 (Fig.
  • the primary breast tumors were a cystosarcroma phylloides tumor, which was negative for the estrogen receptor, and an invasive, ductal breast cancer, which was positive for the estrogen receptor.
  • the MCF-7 breast tumor cell line is also positive for the estrogen receptor.
  • Both the breast tumor cell line and the cells of the invasive, ductal breast cancer show a clear signal for BCAP-29, while the breast epithelial cell line HMEC and the cystosarcrom phylloides show no BCAP-29 signal.
  • BCAP-16 it can be seen that the two samples obtained from estrogen receptor negative cells (HMEC cells and Cystosarcroma phylloides) have a clear signal, which has a significantly lower signal intensity in the other two samples.

Abstract

La présente invention concerne des peptides définis et leur analyse quantitative dans des fluides corporels ou des échantillons tissulaires prélevés chez des patientes atteintes d'un cancer du sein. Ces peptides sont traités selon une procédure spécifique et subissent éventuellement une modification post-traductionnelle ou chimique. Ils varient de façon spécifique pour chaque peptide du point de vue de leur concentration chez les patientes atteintes d'un cancer du sein. Une modification spécifique et significative de la concentration de ces peptides est caractéristique d'un cancer du sein. Cette invention peut être mise en application pour contrôler l'évolution d'une maladie, décomposer cette évolution en différents stades pathologiques, effectuer une stratification des patientes ou développer des agents diagnostiques et thérapeutiques.
PCT/DE2002/004708 2001-12-21 2002-12-20 Methode de diagnostic de cancers du sein, peptides associes et leurs utilisations WO2003056341A2 (fr)

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WO2006013013A2 (fr) * 2004-08-04 2006-02-09 Bayer Healthcare Ag Agents pour le diagnostic et le traitement de maladies associees a la proteine protectrice pour la beta-galactosidase (ppgb)
EP2548888A1 (fr) * 2010-03-17 2013-01-23 Kagoshima University Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant
EP2727935A3 (fr) * 2009-07-14 2014-07-30 Temple University Of The Commonwealth System Of Higher Education Marqueurs sériques associés à des stades précoces et autres du cancer du sein
US8980269B2 (en) 2009-07-14 2015-03-17 Temple University Of The Commonwealth System Of Higher Education G-protein coupled receptor-associated sorting protein 1 as a cancer biomarker

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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2006013013A2 (fr) * 2004-08-04 2006-02-09 Bayer Healthcare Ag Agents pour le diagnostic et le traitement de maladies associees a la proteine protectrice pour la beta-galactosidase (ppgb)
WO2006013013A3 (fr) * 2004-08-04 2006-06-15 Bayer Healthcare Ag Agents pour le diagnostic et le traitement de maladies associees a la proteine protectrice pour la beta-galactosidase (ppgb)
US9140704B2 (en) 2007-07-26 2015-09-22 Temple University Of The Commonwealth System Of Higher Education Serum markers associated with early and other stages of breast cancer
EP2727935A3 (fr) * 2009-07-14 2014-07-30 Temple University Of The Commonwealth System Of Higher Education Marqueurs sériques associés à des stades précoces et autres du cancer du sein
US8980269B2 (en) 2009-07-14 2015-03-17 Temple University Of The Commonwealth System Of Higher Education G-protein coupled receptor-associated sorting protein 1 as a cancer biomarker
EP2548888A1 (fr) * 2010-03-17 2013-01-23 Kagoshima University Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant
JPWO2011115225A1 (ja) * 2010-03-17 2013-07-04 国立大学法人 鹿児島大学 歯周病特異的ペプチド、並びにそれを用いた歯周病の治療および診断
EP2548888A4 (fr) * 2010-03-17 2013-07-31 Univ Kagoshima Peptide spécifique d'une pathologie parodontale et traitement et diagnostic d'une pathologie parodontale l'incluant
JP5961809B2 (ja) * 2010-03-17 2016-08-02 国立大学法人 鹿児島大学 歯周病特異的ペプチド、並びにそれを用いた歯周病の治療および診断
US10627412B2 (en) 2010-03-17 2020-04-21 Kagoshima University Periodontal-disease-specific peptide, and treatment and diagnosis of periodontal disease using same

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