WO2003056341A2 - Method for the diagnosis of breast cancer diseases, associated peptides and the uses thereof - Google Patents

Abstract

The invention relates to defined peptides and the quantitative determination thereof in body fluids or tissue samples of patients suffering from breast cancer disease. The inventive peptides are processed in a specific manner and are post-translationally or chemically modified. They are modified in a specific manner for each peptide in the concentration thereof in breast cancer patients. A specific and significant modification in the concentration of said peptides indicates breast cancer. The invention can be used to verify the course of an illness, dividing the illness up into various stages, in the stratification of patients and in the development of diagnostic agents.

Description

Methods for diagnosing breast cancer, associated peptides and their uses

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.

12% of all women develop breast cancer during their lifetime. With 43,000 new cases annually in Germany and 180,000 in the USA, 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]. However, 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. 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. In addition, it is not possible to predict which peptide fragments of these proteins, which are not even known in their identity, could occur in vivo in changed concentrations due to the cancer. It is consequently very difficult to select experimentally from thousands of possibly modified proteins and from millions of different peptide fragments that can be derived theoretically from them, which indicate a tumor disease of the breast as a marker peptide or which may also be causally related to the disease.

Probably some of the new or missing 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].

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 (mammography) 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. If breast cancer is suspected, physical examinations of the breast and lymph nodes, X-ray and ultrasound examinations as well as magnetic resonance imaging (MRI, magnetic resonance tomography) and histological examinations of biopsy specimens are currently being carried out [1]. At the moment, routine examinations for breast cancer are used exclusively and physical examinations are almost exclusively used in the more detailed examination in a specific suspected case.

The determination of 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]. Previous clinical studies have also shown that the combinations of CEA and CA 15-3 or other potential markers for breast cancer, such as MCA (“mucin -like carcinoma-associated antigen ") or CA 549 (" cancer antigen 549 ") does not allow an improved diagnosis of breast cancer [3, 4]. Tests are often carried out to determine whether it is an estrogen receptor negative or positive breast tumor is because the therapy strategy of estrogen receptor negative and positive breast tumors is due to their different degrees of differentiation and due to Differences in their aggressiveness differs [1]. However, estrogen receptors are not markers of breast cancer.

With more than 5 million new cases worldwide in 2000

Cancer is one of the most common human diseases. Breast cancer is the most common with more than 1 million new cases

Cancer and in 2000 approximately 370,000 people died

Breast cancer [5]. Despite the resulting health and economic importance of breast cancer, no reliable, clinically measurable markers for this disease have been established so far, and the screening tests for breast cancer that have been practiced so far, which are mainly limited to mammography and breast scanning by the doctor, have so far been found insufficient acceptance among the population. An alternative to these physical precautionary screening examinations, which may be more widely accepted, would therefore be of enormous economic and health policy interest.

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. Under certain circumstances, 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. Additionally or alternatively, therapy with estrogen-blocking drugs, such as tamoxifen (trade name: Noivadex ™), can be carried out for estrogen receptor-positive tumors which often require estrogen for growth. First of all, the strong side effects of chemotherapy, but also the fact that a large part of the treatments Not leading to cure suggest that new, more effective, and / or fewer side effects therapies as an alternative to

Chemotherapy is urgently needed. An example of this would be e.g. a

Breast cancer therapy with antibodies such as the anti-HER-2 antibody "trastuzumab", which selectively recognize antigens or markers of breast tumor cells and can thus be used to destroy the tumor tissue.

Preferably embodiments of the invention

We have identified various peptides in cell lysates of the MCF-7 and HMEC breast tumor cell lines that enable a distinction to be made between healthy and malignant breast tissue. These peptides are referred to below as "Breast Cancer Eeptides" or abbreviated as BCAP. The peptides BCAP-1 to BCAP-40 represent peptide fragments which have never been described in the literature and are therefore new as substances. None of the peptides BCAP-1 Up to BCAP-40 has so far been described in the literature in connection with breast cancer diseases. The proteins from which the peptides described here are derived for the first time due to defined processing processes have already been described in part in connection with tumor diseases, but not the novel protein fragments disclosed here Proteins It is not possible for a person skilled in the art to predict which of the numerous proteins described in connection with breast cancer diseases are also present as proteolytic processing products in breast cancer cells, and it is impossible to predict what type of peptide fragments will result from these complicated processing processes, and how peptide fragments are stable enough to occur in biological samples in detectable amounts.

Surprisingly, we have found that the majority of these novel markers for breast cancer are peptides that are structural proteins that are responsible for the spatial structure of the cell, especially intermediate filament and histone proteins. The proteins from which intermediate filaments are made up have a common structure, which consists of a central alpha-helical domain (“rod domain”), which is only interrupted by 3 short non-helical sequence sections. This central domain becomes N- and C-terminal 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. The central structural element of the cell nucleus is again histone proteins and 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. As additional markers, we identified peptides derived from the human anterior gradient 2 homolog (hAG-2) and transgelin 2 (TAG2) (BCAP-29 and 30, or BCAP-33 and -34).

Table I

Function of the subgroup proteins BCAP peptides

proteins Structure intermediate filament - CK19, CK18, BCAP-3 to -22 and protein proteins in the cytosol CK17, CK14, BCAP-37 to -40 CK5, Tb-10

Histones in the cell nucleus H2B, H3B, H2A BCAP-23 to -28

Enzymes Cathepsine Cat-A, Cat-D BCAP-3, 4, 35, 36

Dehydrogenases in UGDH, G3P2 BCAP-1, 2, 31, 32

glycogen

metabolism

More hum. Anterior hAG-2 BCAP-29, 30 gradient -2-homolog Transgelin TAG 2 BCAP-33, 34

Table II:

BCAP template sequence

Peptide bank position

No. (A.S.) mass (Da) peptide sequence

UGD 369 - 400 3541, 9 PKVPREQIVVDLSHPGVSEDDQVSRLVTISKD H

UGD 378 - 385 *> 822.4 X1-VDLSHPGV-X2 H

35 Cat-A 29-72 5090.5 APDQDEIQRLPGLAKQPSFRQYSGYLKGSGS HLH YWFVESQKD

36 Cat-A 47-54> 1032.5 x27-FRQYSGYL-x28

3 Cat-D 169-190 2360.3 LGGVKVERQVFGEATKQPGITF

4 Cat-D 179-186> 876.4 x3-FGEATKQP-x4

5 CK19 369-400 3703.9 IKSRLEQEIATYRSLLEGQEDHYNNLSASKVL

6 CK19 381 -388> 930.5 x5-RSLLEGQE-x6

CK18 387-430 4875.4 GEDFNLGDALDSSNSMQTIQKTTTRRIVDGKVVSE TNDTKVLRH

CK18 390-430 4574.4 FNLGDALDSSNSMQTIQKTTTRRIVDGKVVSETND TKVLRH

CK18 411 - 418> 941.6 x7-RRIVDGKV-x8

37 CK17 392-431 4674.5 AHLTQYK EPVTTRQVRTIVEEVQDGKVISSREQV HQTTR

38 CK17 409-416> 931.4 X31-TIVEEVQD-X32

10 CK14 424-472 5383.7 AHLSSSQFSSGSQSSRDVTSSSRQIRTKVMDVHD GKVVSTHEQVLRTKN

11 CK14 445-452> 986.6 x9-SRQIRTKV-x10

39 CK5 159-184 3118.7 PSIQRVRTEEREQIKTLNNKFASFID

40 CK5 168-175> 1015.6 x33-EREQIKTL-x34

12 VIME 430-466 4224.2 SLPLVDTHSKRTLLIKTVETRDGQVINETSQHHDDL

e

13 VIME 444-466 2663.3 IKTVETRDGQVINETSQHHDDLE

14 VIME 444-465 2534.3 IKTVETRDGQVINETSQHHDDL 15 VIME 450- -457> 929.5 x11-RDGQVINE-x12

16 Tb- 10 6- 43 4365.3 MGEIASFDKAKLKKTETQEKNTLPTKETIEQEKRSEI S

17 Tb- 10 27 -44 2102.1 NTLPTKETIEQEKRSEIS

18 Tb- 10 13- -42 3576.9 FDKAKLKKTETQEKNTLPTKETIEQEKRSE

19 Tb- 10 13 -44 3777.0 FDKAKLKKTETQEKNTLPTKETIEQEKRSEIS

20 Tb- 10 14- -44 3629.9 DKAKLKKTETQEKNTLPTKETIEQE RSEIS

21 Tb- 10 31 • -38 ≥ 976.5 x13-TKETIEQE-x14

22 Tb- 10 19 -26> 990.5 X15-KKTETQEK-X16

23 H2B 100- - 127 2982.7 VRLLLPGELAKHAVSEGTKAVTKYTSSK

(N)

24 H2B 110 .-- 117> 827.4 x17- HAVSEGT-x18

(N)

25 * * * H3B 104- - 135 3720.0 FEDTNLCAIHAKRVTIMPKDIQLARRIRGERA

26 H3B 116-123> 971.6 X19-KRVTIMPK-X20

27 H2A 63 - 122 7477.3 ILELAGNAARDNKKTRIIPRHLQLAIRNDEELNKLLG (SLE) KVTIAQGGVLPNIQAVLLPKKTESHHKAKGK

28 H2A 93- 100> 913.6 x21-ELNKLLG -x22 (SLE)

29 hAG- 133 ^■ - 175 4864.7 PSLTVRADITGRYSNRLYAYEPADTALLLDNMK A 2 LKLLKTEL

30 hAG- 151 • - 158> 836.4 x23-AYEPADTA-x24 2

31 G3P2 46-80 3960.0 FQYDSTHGKFHGTVKAENGKLVINGNPITIFQERD

32 G3P2 61-68> 842.5 x25-AENGKLVI-x26

33 DAY2 148-199 5789.9 PNWFP KSKENPRNFSDNQLQEGKNVIGLQMGTN RGASQAGMTGYGMPRQIL

34 DAY2 169-176> 828.5 x29-EGKNVIGL-x30

* x1- corresponds to the sequence or parts of the sequence of UGDH from

Amino acid 379 to 369, where x1-, starting from amino acid 368 of UGDH, can be between 0 and 9 amino acids long. 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. The abbreviations for precursor proteins used in Table I have the following meaning: 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.

* * The symbol (is larger or the same) is to be understood in such a way that masses of any size are not possible for the corresponding BCAP peptides, but only the masses which may result from the amino acids which may also be located at the ends of these peptides. In addition, any amino acids may not be additionally present at the ends of the peptides, but only those which may be located at this sequence position due to the sequence of the associated BCAP peptide (see *). , Y. _r y, this peptide occurs in two forms, one with a cysteinyl

Modification in position 1 10 and once without this modification.

The BCAP peptides listed in Table III below are sequence fragments of protein database entries from the “GeneBank” databases and from the EMBL database. table

BCAP accession no. Protein database

Peptide No. Protein DNA / RNA Name

1, 2 AAC36095 AF061016 UGDH GeneBank

35, 36 CAA15501 AL008726 Cat-A Embel

3, 4 AAA51922 AH002629 Cat-D GeneBank

5, 6 AAA36044 J03607 CK19 GeneBank

7, 8, 9 AAH00698 BC000698 CK18 GeneBank

37, 38 NP_000413 NM_000422 CK17 GeneBank

10, 1 1 AAB59562 J00124 CK14 GeneBank

39, 40 NP_000415 NM_000424 CK5 GeneBank

12, 13, 14, 15 AAH00163 BC000163 VIME GeneBank

16, 17, 18, 19, 20, 21, AAH 16731 BC016731 Tb-10 GeneBank

22

23, 24 AAK84040 AF397301 H2B GeneBank

25, 26 CAB02546 Z80784 H3B Embel

27, 28 AAH 16677 BC016677 H2A GeneBank

29, 30 AAH 15503 BC015503 HAG-2 GeneBank

31, 32 NP_002037 NM_002046 G3P2 GeneBank

33, 34 AAH02616 BC002616 TAG2 GeneBank

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.

In particular, the peptides can be present with a cysteinyl modification.

Naturally occurring, derived from the corresponding proteins

Polymorphisms or mutants and the BCAP peptides and BCAP peptide fragments corresponding to these are also referred to as BCAP peptides.

Such 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

Activity of the original sequence or at least very similar to it. 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.

Determination of the Homology of Sequences To determine the homology between sequences, computer programs such as the GCG program package (Genetics Computer Group, University of Wisconsin, Madison, Wl, USA), including GAP [6], BLASTP, BLASTN, FASTA [7] or the well-known Smith Waterman algorithm can be used to determine the homologies. 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. The 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. 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. For a positive detection of the disease, it is provided that 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

Examinations such as breast scanning to identify malignancies

Changes by a suitably trained person.

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.

Suitable samples 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.

Manufacture of tissue extracts

In a further embodiment of this invention it is provided that 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

Be cooked for 10 minutes. 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.

Use of the BCAP peptides 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.

Detection methods for BCAP peptides

Various methods for detecting the

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

One embodiment of the invention is the use of physical

Methods which can indicate the peptides according to the invention qualitatively or quantitatively. These methods include mass spectrometry, liquid chromatography,

Thin layer chromatography and MRI (Magnetic Resonance Tomography) etc.

Here quantitative measurement results are obtained from a sample to be examined. From these results, the existence of a

Breast cancer and / or the severity or stage of this disease can be derived, as well as stratification of patients.

According to a preferred embodiment of this invention, 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. Another

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. For this, 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.

Reverse phase chromatography A particularly preferred embodiment of this invention comprises

Use of reverse phase chromatography, especially a C18

Reverse phase chromatography column using eluents consisting of trifluoroacetic acid and acetonitrile for the fractionation of peptides from cell extracts. E.g. fractions collected, each

Include 1/100 of the volume of solvent used. The fractions obtained in this way are supported using a mass spectrometer, preferably using a MALDI mass spectrometer (matrix-supported

Laser desorption ionization) using a matrix solution consisting of e.g. from L (-) fucose and alpha-cyano-4-hydroxycinnamic acid, dissolved in a mixture of acetonitrile, water, trifluoroacetic acid and acetone, analyzed and the presence of certain masses determined and the signal intensity quantified. These masses correspond to the masses of the BCAP peptides according to the invention.

Mass spectrometry

According to a preferred embodiment of the invention, 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.

Slight deviations from the theoretical, monoisotopic mass can occur due to the experimental error and the natural isotope distribution. 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. In addition, 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-1 = 3541, 9 / BCAP-2> 822.4 / BCAP-35 = 5090.5 / BCAP-36> 1032.5 /

BCAP-3 = 2360.3 / BCAP-4> 876.4 / BCAP-5 = 3703.9 / BCAP-6> 930.5 /

BCAP-7 = 4875.4 / BCAP-8 = 4574.4 / BCAP-9> 941, 6 / BCAP-37 = 4674.5 /

BCAP-38> 931, 4 / BCAP-10 = 5383.7 / BCAP-1 1> 986.6 / BCAP-39 = 3118.7 / BCAP-40> 1015.6 / BCAP-1 2 = 4224.2 / BCAP-13 = 2663.3 / BCAP-14 = 2534.3 / BCAP-1 5> 929.5 / BCAP-1 6 = 4365.3 / BCAP-1 7 = 2102.1 / BCAP-1 8 = 3576, 9 / BCAP-1 9 = 3777.0 / BCAP-20 = 3629.9 / BCAP-21> 976.5 / BCAP-22> 990.5 / BCAP-23 = 2982.7 / BCAP-24> 827.4 / BCAP-25 = 3720.0 / BCAP-26> 971, 6 / BCAP-27 = 7477.3 / BCAP-28> 913.6 / BCAP-29 = 4864.7 / BCAP-30> 836.4 / BCAP -31 = 3960.0 / BCAP-32 ≥ 842.5 / BCAP-33 = 5789.9 and BCAP-34> 828.5 daltons. The BCAP-25 peptide is also suitable with a cysteinyl modification as a marker and then has an experimentally determined mass of 3840 daltons.

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.

Mass spectrometric determination of the sequence of the MAC3 peptides In the further practical application of this embodiment, a further validation of the detection result is possible and recommended that the identity of the peptides corresponding to the mass is determined, whereby only peptide signals are taken into account, those of the protein precursor molecules of those in the context of this invention

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].

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.

Molecular biological detection techniques

Finally, 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

Patient and the subsequent determination of the concentration of an RNA

Transcripts corresponding to a gene of the precursor molecules represented by the BCAP peptides or corresponding to homologous genes.

Here quantitative measurement results (intensities) are obtained from a sample to be examined. Methods such as e.g. Reverse transcriptase polymerease chain reaction (RT-PCR, “reverse transcriptase polymerase chain reaction”), quantitative PCR, PCR, Northern blots or 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.

Immunological Detection Methods According to a further preferred embodiment of the invention, 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. To increase the specificity, 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. 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. Other detection methods suitable for peptide detection include Western blotting, immunoprecipitation, dot blots, protein chip experiments, plasmon resonance spectroscopy (BIACORE ™), phage particles, affinity matrices etc. In general, all substances / molecules are suitable as detection agents that allow a specific one Set up detection system for BCAP peptides.

Obtaining 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. 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). For the recombinant expression of BCAP peptides, for example, 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. The BCAP encoding

Nucleic acid sequence is used in combination with suitable regulatory

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

(Mammalian cells, e.g. CHO ("Chinese Hamster Ovary") cells), pYES2

(Yeast cells, Saccaromyces cerevisiae), pVL1393 (insect cells, Sf9 cells) or pBAD (prokaryotes, Escherichia coli) from Invitrogen. Other commercial manufacturers such as Stratagene, Clontech, etc. also offer expression vectors. The 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. 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

Production and production of BCAP peptide-specific antibodies that

Recognize epitopes. These antibodies recognize epitopes that are only present in BCAP peptides and do not occur in the corresponding complete precursor molecules. Such neo-epitope-specific anti-BCAP peptide antibodies enable specific immunological detection of BCAP peptides without the presence of the respective precursor molecule having a disruptive influence on the detection of the BCAP peptide.

Therapy development and monitoring through BCAP peptide determinations

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. In addition, after surgical removal of breast cancer tumors, 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.

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

(Seq. ID 41-56) to promote BCAP peptides. Fusion peptides can also be used

Treatment of the cell lines, e.g. Fusion peptides with

Peptide sequences that promote transport of the fusion peptide into the interior of the cell. Examples of possible fusion partners are HIV-TAT, Antennapedia, Herpes

Simplex VP22 sequences etc. Cell lines can also be used

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. Alternatively, 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. In particular, 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

Laboratory animals, e.g. Mice, rats, rabbits, dogs, monkeys etc. the in vivo effects of e.g. BCAP peptides are examined. You can do this

Peptides may be galenically prepared in this way. As 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

Sequence that are connected can be used. Peptides and

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. The determination of body function (temperature, respiratory rate, heart rate, etc.), 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.

As a further possibility for examining the function of BCAP peptides, 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.

The invention is illustrated in more detail below with the aid of examples. Reference is also made to illustrations.

Figure 1 Western blot for the detection of the precursor molecules of vimentin, cytokeratin 14 and 18 in HMEC and MCF-7 cell extracts

Figure 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.

Figure 3: MALDI as a relatively quantifying mass spectroscopic method

Figure 4: MS / MS fragment spectrum for the identification of peptides using the example of BCAP-1

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

Cell extracts from MCF-7 and HMEC cell lines and in

Cell extracts obtained from primary, human breast tumors (Cystosarcroma phylloides and invasieves, ductal carcinoma)

Example 1: Carrying out Western blots

Production of cell extracts for Western blots

Cells in vitro are cultured twice with PBS (“Phosphate Buffered

Saline ", phosphate-buffered saline), then detached from the surface of the cell culture flasks with cell scrapers, transferred to a 1.5 ml tube and centrifuged at low speeds (1000-2000 revolutions per minute). The cell pellet is placed in 1 ml of ice-cooled lysis buffer of the following composition resuspended: 50 mM HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), pH 7.5 with 0.1% Tween-20, 150 mM sodium chloride, 1 mM sodium fluoride, 1 mM EDTA (ethylene ediaminetetraacetic acid), 2.5 mM EGTA

(Ethylene bis (oxyethylenenitrilo)] tetraacetic acid), 10% glycerin, 0.1 mM ortho-vanadate, 10 mM beta-glycerophosphate, 10 tg / ml aprotinin, 10 μg / ml leupeptin, 0.1 mM PMSF (phenyl-methyl- sulfonyl fluoride) and 1 mM dithiothreitol. The lysate is then treated with an ultrasonic pulse 15 times for one second each time and centrifuged for 15 minutes at 4 ° C. and 15,000 g to remove insoluble constituents. The supernatant represents the cell extract and is stored at -70 ° C. until used in Western blots.

Western blots The cell extracts are in SDS ("Sodium Dodecyl Sulfate", sodium Dodecy-

Treated sulfate) sample buffer [1 1] for 2 minutes at 95 ° C and then a sample amount corresponding to 20 μg 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

Antibodies detected:

Table IV

Antigen Antibody used Manufacturer of the antibody

Vimentin V9 DAKO Diagnostika GmbH,

Hamburg, Germany Cytokeratin 14 DC-10 RDI Research Diagnostics Inc.,

Flanders, NJ, USA Cytokeratin 18 LL002 RDI Research Diagnostics Inc.,

Flanders, NJ, USA

All antibodies were used in a dilution of 1: 200 in a solution of 10 mM tris (hydroxymethyl) aminoethane hydrochloride, 150 mM sodium chloride, pH 7.5 with 5% bovine serum albumin. Antibodies coupled to peroxidase were used as secondary antibodies to detect the primary antibodies and the chemiluminescent substrate Super Signal from Pierce Chemical Company, Rockford, IL, USA, was used to detect peroxidase activity. The result of a corresponding Western blot is shown in Figure 1. Example 2: Extraction of cell extracts

From cell lines cultivated in vitro To obtain cell extracts, the human breast tumor cell line MCF-7 (human breast adenocarcinoma cell line, estrogen receptor positive), obtained from the American cell culture collection (“American tissue culture collection, ATCC, Rockville, MD, USA, and normal, human breast epithelial cells (HMEC, "Normal Human Mammary Epithelial Cells"), obtained from Clonetics CellSystems, St. Katharinen, Germany. 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 μg / 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), 10μg / ml insulin, 1 μg / ml hydrocortisone, 0.1 mg / ml Gentamycin and 0.1 μg / 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. After rinsing twice with PBS (“Phosphate Buffered Saline”, phosphate-buffered saline), 1 ml of a high-molar (4 to 10 M) solution of a chaotropic salt, such as, for example, urea or guanidinium, whose pH value of 10 to 50 mM is suitable Substance such as formic acid, glycine, citric acid or lactic acid was adjusted to a value of pH 2 to 4. After the lysis, the cells are detached from the surface of the cell culture bottle with a cell scraper and the cell extract obtained in this way Transferred plastic container. The cell culture bottle is then rinsed again with 1 ml of the same solution and combined with the first ml of the cell extract. The cell extracts obtained in this way are used until further notice

Processing stored at -70 ° C.

From tissue samples from breast cancer patients

To obtain cell extracts, 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 For the mass spectrometric detection of MAC3 peptides in CSF, a separation of the peptide ingredients is necessary in this example. 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. The following 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

25,000 to 1,000,000 cells, according to Example 2, used. The

Cell extracts are in 750 μl of the chaotropic saline solution, pH 2 to 4, are heated for 5 minutes at 95 ° C, with 0.1% trifluoroacetic acid in water

Volume of 1750 μl filled up, centrifuged for 15 minutes at 18600 x g at 4 ° C. and finally 1500 μl of the sample prepared in this way onto the

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.

Example 4: Determination of the Masses of Peptides Using MALDI

Mass spectrometry

For mass analysis, typical positive ion spectra of peptides are created in a 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 Bremen (BIFLEX). To prepare the samples, they are mixed with a matrix substance, which 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. To measure the BCAP peptides according to the invention, 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 μl of a matrix solution. This matrix solution contains, for example, 10 g / 1 α-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 μl 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.

The measurement is carried out in "Linear Mode" with "Delayed Extraction ™". Figure shows an example of a measurement of one of the BCAP peptides according to the invention

2A) the spectrum of BCAP-1 and Figure 2 D) the spectrum of BCAP-

25. The peak which corresponds to the peptide BCAP-1 or the peptide BCAP-25 is marked by an arrow. Another example is the spectrum of BCAP-14, determined from an HMEC sample (Figure 2 B) and determined from an MCF-7 sample (Figure 2 C). It can be seen from Figures 2 B) and 2 C) that 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. There is a specific relationship between the measurement signal and the concentration for each peptide, which means that 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. Figure 3 shows an example of a MALDI measurement as a relatively quantifying MS method. All signal intensities of the standards were standardized for their signal intensity at a concentration of 0.64 μmol (= 1). Each peptide shows an individual, typical ratio of signal strength to

Concentration, which can be read from the slope of the curve.

Example 5: Mass spectrometric identification of the sequences of BCAP peptides

To quantify the BCAP peptides according to the invention, it must be ensured that the mass signals to be analyzed of peptides in the fractions obtained by reverse phase chromatography according to Example 3 are actually the BCAP peptides according to the invention. The peptides according to the invention are identified in these fractions using, for example, nanoSpray-MS / MS [10]. 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. argon or nitrogen, and the resulting BCAP peptide fragments are detected in the mass spectrometer in an integrated analysis unit and corresponding m / z values are determined (principle of tandem mass spectrometry) [14 ]. 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. The same applies of course to other modifications of the peptides, such as phosphorylations, which increase the peptide mass by about 80 daltons per phosphate group. Other chemically or post-translationally modified peptides can also be identified in this way. In this special case, the mass spectrometric analysis was carried out using a quadrupole TOF instrument, model "QStar-Pulsar" from the company Applied Biosystems-Sciex, USA. An exemplary MS / MS fragment spectra of BCAP-1 is shown in Figure 4.

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. For this purpose, 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. 5B), BCAP-5 (Fig. 5C), BCAP-8 (Fig. 5D), BCAP -10 (Fig. 5E), BCAP-12 (Fig. 5F), BCAP-16 (Fig. 5G), BCAP-23 (Fig. 5H), BCAP-25 (Fig. 51), BCAP-28 (Fig. 5J) and BCAP-29 (Fig. 5K). Cell extracts were produced with 25,000, 50,000, 100,000, 25,000 and 500,000 cells, chromatographed and analyzed in MALDI. The MALDI signal intensity is plotted against the number of cells used. Figure 6 shows an example of a comparison of the MALDI signal intensities obtained with cell extracts from primary, human tumor tissue extracts, obtained from individual patients, with measured values from MCF-7 and HMEC cell extracts. 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. For the marker 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.

literature

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Basic local alignment search tool. J Mol Bio /. 215: 403-10. 8. Needleman, S.B., and CD. Wish. 1970. A general method applicable to the search for similarities in the amino acid sequence of two proteins. J Mo /

Bio /. 48: 443-53. 9. Henikoff, S., and JG Henikoff. 1992. Amino acid substitution matrices from protein blocks. Proc Natl Acad Be US A. 89: 10915-9. l O. Wilm, M., and M. Mann. 1996. Analytical properties of the nanoelectrospray ion source. Anal Chem. 68: 1-8. 1 1. Sambrook, J., and D. Proboscis. 2001. Molecular Cloning: A Laboratory

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Claims

claims:

1. A method for the detection of breast cancer diseases by determining at least one marker in a biological sample, characterized in that at least one BCAP peptide or a substance corresponding to SEQ ID 1 to 72 is used as the marker.

2. The method according to claim 1, characterized in that the marker is a BCAP peptide that a) is derived from a structural protein, in particular i) from an intermediate filament protein such as cytokeratin-19, -18, -17, -14, -5 or thymosin-beta 10, or ii) from a histone protein such as H2B, H3B or H2A, or b) derived from an enzyme, in particular i) from a cathepsin such as Cathepsin-A or Cathepsin-D, or ii) from one directly dehydrogenase involved in glycogen metabolism such as UDP-glucose 6-dehydrogenase or glyceraldehyde 3-phosphate dehydrogenase, or c) derived from human anterior gradient 2 homolog or transgelin 2.

3. The method according to claim 1 or 2, characterized in that a) a specific increase or decrease in concentration of the marker in the sample is determined for the respective marker, and b) a significant change in concentration of the marker in the manner mentioned under a) as a positive detection result is evaluated for a disease of breast cancer.

4. The method according to claim 1 to 3, characterized in that the measurement of at least two BCAP peptides or substances according to SEQ ID 1 to 72 is used in combination for a clear diagnosis.

5. The method according to claim 1 to 4, characterized in that the diagnosis a) enables a distinction between breast cancer and other cancers, or b) enables a distinction between gutartingen and malignant breast cancer diseases, or c) enables a distinction between non-metastatic and metastatic breast cancer diseases , or d) to increase the sensitivity and / or specificity of the diagnosis in combination with other diagnostic methods, or e) to observe the course of the disease of breast cancer patients, before, during or after therapy is used, in particular for the detection of recurrences and metastases or f) is used to stratify patients.

6. The method according to claim 1 to 5, characterized in that at least one marker a) a BCAP peptide or a substance according to Seq. ID 1 to Seq. ID 72, or b) is a descendant of a naturally occurring alley or a naturally occurring mutant of these peptides or nucleic acids, or c) is a BCAP peptide mutant with preferably no more than 2 different amino acids compared to the corresponding section of the non-mutated sequence, or d) a substance with at least 70% homology with Seq. ID 1 to 56 or

60% homology with Seq. ID 57 to 72, or e) is present in the sample in post-translational modifications or in a chemically modified form and is detected in this form, preferably with a cysteinyl modification.

7. The method according to any one of claims 1 to 6, characterized in that the biological sample blood, serum, plasma, urine, lymphatic fluid, milk, a

Sample obtained from a smear, a cell extract from blood cells

Tissue extract, preferably obtained from breast or lymph node tissue or obtained therefrom by processing.

8. The method according to any one of claims 1 to 7, characterized in that the marker is determined by its mass, preferably by mass spectrometry, further preferably by determining at least one of the following masses of a BCAP peptide of 3541, 9 /> 822.4 / 5090.5 /> 1032.5 / 2360.3 /> 876.4 / 3703.9 /> 930.5 / 4875.4 / 4574.4 /> 941.6 / 4674.5 /> 931.4 / 5383.7 /> 986.6 / 3118.7 /> 1015.6 / 4224.2 / 2663.3 / 2534.3 /> 929.5 / 4365.3 / 2102.1 / 3576.9 / 3777.0 / 3629.9 /> 976.5 /> 990.5 / 2982.7 /> 827.4 / 3720.0 /> 971.6 / 7477.3 /> 913.6 / 4864.7 /> 836.4 / 3960.0 /> 842.5 / 5789.9 I ≥ 828.5 daltons.

9. The method according to any one of claims 1 to 7, characterized in that the determination of the BCAP peptide or the peptide according to Seq. ID 1 to 56 using an immunological test using specific binding substances, such as Antibodies, antibody fragments, phage particles or affinity matrices are carried out, in particular using an ELISA test, an RIA, a protein chip assay or a Western blot.

10. Use of the peptides according to Seq. ID 1 to 56 corresponding nucleic acids or of nucleic acids corresponding to Seq. ID 57 to 72 for

Diagnosis of breast cancer, especially using PCR, quantitative PCR, Northerblots or nucleic acid chip assays.

11. The method according to any one of claims 1 to 10, characterized in that the sample is fractionated prior to the determination chromatographically or by electrophoresis and / or subjected to a precipitation reaction and / or a liquid phase separation, the fractions obtained are subsequently examined separately.

12. Substance of a) a peptide according to Seq. ID 1 to Seq. ID 40, or b) is a descendant of naturally occurring alleles or homologous mutants of a BCAP peptide, in particular with point mutated BCAP peptides is preferably not more than 2 amino acids which differ from the corresponding non-mutated original BCAP sequence, or c) is a peptide which has at least 70% homology with a sequence according to Seq. ID 1 to 56, or d) a chemically modified, enzymatically or post-translationally modified

Peptide according to Seq. ID 1 to Seq. ID 56 or according to claim 12 b) to c), or e) is a peptidomimetic of a peptide according to claim 12 a) to d).

13. Antibody, “single chain antibody” or antibody fragment, in particular Fab or F (ab) 2 fragment or antibody fusion protein, which binds to a substance according to claim 12.

14. Nucleic acid which a) codes for one of the substances according to claim 12 or 13 or a corresponding complementary nucleic acid b) is suitable for modulating the concentration of the substances according to claim 12 or claim 14 a), in particular an antisense nucleic acid, a triplex Nucleic acid, an RNAi nucleic acid, a PNA (peptide nucleic acid) or a ribozyme c) is suitable for the detection of nucleic acids according to claim 14 a), in particular as a PCR primer or as a probe for hybridization reactions d) for producing substances according to claim 12 , 13 or 14 a) to c) is suitable, in particular in the form of a vector, or a nucleic acid suitable for translation or transcription.

15. A method for obtaining a substance according to claim 12 to 14.

16. Use of a substance according to claims 12 to 14 for the manufacture or development of a diagnostic reagent or a therapeutic agent, in particular for the production of antibodies, PCR primers or nucleic acid probes for the diagnosis of breast cancer diseases.

17. Medicament for the therapy, diagnosis or prophylaxis of breast cancer diseases containing a) at least one substance according to claim 12 to 14, or b) at least one substance according to claim 12 to 14 and additionally at least one further pharmacologically acceptable substance, preferably a preservative, a filler, a solvent, color, flavor or fragrance.

18. Test kit for carrying out a method according to claim 1 to 11, characterized in that at least a) a substance is contained according to claim 12 to 14, and the substance is in immobilized or labeled form, or in a form which the immobilization or labeling enables, and / or b) a substance according to SEQ ID 1 to 72 or according to claim 12 or 14 for use as a standard is included.