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WO2005040811A1 - Utilisation de la proteine tip47 comme marqueur du cancer du sein - Google Patents

Utilisation de la proteine tip47 comme marqueur du cancer du sein Download PDF

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Publication number
WO2005040811A1
WO2005040811A1 PCT/EP2004/011659 EP2004011659W WO2005040811A1 WO 2005040811 A1 WO2005040811 A1 WO 2005040811A1 EP 2004011659 W EP2004011659 W EP 2004011659W WO 2005040811 A1 WO2005040811 A1 WO 2005040811A1
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WIPO (PCT)
Prior art keywords
tip47
breast cancer
diagnosis
protein
sample
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Inventor
Gabriele Pestlin
Peter Berndt
Marie-Luise Hagmann
Johann Karl
Hanno Langen
Norbert Wild
Werner Zolg
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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F Hoffmann La Roche AG
Roche Diagnostics GmbH
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    • G01N33/57515—

Definitions

  • BC breast cancer
  • the prognosis in advanced stages of tumor is poor.
  • More than one third of the patients will die from progressive disease within five years after diagnosis, corresponding to a survival rate of about 40% for five years.
  • Current treatment is only curing a fraction of the patients and clearly has the best effect on those patients diagnosed in an early stage of disease.
  • BC as a public health problem, it is essential that more effective screening and preventative measures for breast cancer will be developed.
  • the earliest detection procedures available at present for breast cancer involve using clinical breast examination and mammography.
  • significant tumor size must typically exist before a tumor is palpable or can be detected by a mammogram.
  • the densitiy of the breast tissue and the age are important predictors of the accuracy of screening mammography.
  • the sensitivity ranges from 63% in women with extremely dense breasts to 87% in women with almost entirely fatty breasts.
  • the sensitivity increases with age from 69% in women of about 40 years of age to 83% in women 80 years and older (Carney, P.A., et al., Ann. Intern. Med. 138 (3) (2003) 168-175). Only 20 - 25 % of mammographically detected abnormalities that are biopsied prove to be malignant.
  • WO 00/60076 shall be mentioned and discussed.
  • This application describes and claims more than two hundred isolated polynucleotides and the corresponding polypeptides as such, as well as their use in the detection of BC.
  • differences on the level of mRNA are not mirrored by the level of the corresponding proteins.
  • a protein encoded by a rare mRNA may be found in very high amounts and a protein encoded by an abundant mRNA may nonetheless be hard to detect and find at all (Chen, G., et al., Molecular and Cellular Proteomics, 1.4 (2002) 304- 313).
  • This lack of correlation between mRNA-level and protein level is due to reasons like mRNA stability, efficiency of translation, stability of the protein, etc.
  • WO 02/23200 reports about twelve breast cancer-associated spots as found by surface-enhanced laser desorption and ionization (SELDI). These spots are seen more frequently in sera obtained from patients with BC as compared to sera obtained from healthy controls. However, the identity of the molecule(s) comprised in such spot, e.g their sequence, is not known.
  • Nipple aspirate fluid has been used for many years as a potential non- invasive method to identify breast cancer-specific markers.
  • Kuerer et al. compared bilateral matched pair nipple aspirate fluids from women with unilateral invasive breast carcinoma by 2D gel electrophoresis (Kuerer, H.M., et al., Cancer 95 (2002) 2276-2282).
  • 30 to 202 different protein spots were detected in the NAF of breasts suffering from breast carcinoma and not in the matched NAF of the healthy breasts. These spots were detected by a gel image analysis. But the identity of the protein spots is not known.
  • a new diagnostic marker as a single marker should be at least as good as the best single marker known in the art. Or, a new marker should lead to a progress in diagnostic sensitivity and/or specificity either if used alone or in combination with one or more other markers, respectively.
  • the diagnostic sensitivity and/or specificity of a test is best assessed by its receiver-operating characteristics, which will be described in detail below.
  • CA 15-3 cancer antigen 15- 3
  • CEA carcinoembryonic antigen
  • CA 15-3 is usually increased in patients with advanced breast cancer.
  • CA 15-3 levels are rarely elevated in women with early stage breast cancer (Duffy, M.J., Critical Reviews in Clinical Laboratory Sciences 38 (2001) 225-262). Cancers of the ovary, lung and prostate may also raise CA 15-3 levels. Elevated levels of CA 15-3 may be associated with non-cancerous conditions, such as benign breast or ovary disease, endometriosis, pelvic inflammatory disease, and hepatitis.
  • Pregnancy and lactation can also cause CA 15-3 levels to raise (National Cancer Institute, Cancer Facts, Fact Sheet 5.18 (1998) 1-5).
  • the primary use of CEA is in monitoring colon cancer, especially when the disease has metastasized.
  • a variety of cancers can produce elevated levels of CEA, including breast cancer.
  • the present invention therefore relates to a method for the diagnosis of breast cancer comprising the steps of a) providing a liquid sample obtained from an individual, b) contacting said sample with a specific binding agent for TIP47 under conditions appropriate for formation of a complex between said binding agent and TIP47, and c) correlating the amount of complex formed in (b) to the diagnosis of breast cancer
  • Another preferred embodiment of the invention is a method for the diagnosis of breast cancer comprising the steps of a) contacting a liquid sample obtained from an individual with a specific binding agent for TIP47 under conditions appropriate for formation of a complex between said binding agent and TIP47, and b) correlating the amount of complex formed in (a) to the diagnosis of breast cancer.
  • any such diagnosis is made in vitro.
  • the patient sample is discarded afterwards.
  • the patient sample is merely used for the in vitro diagnostic method of the invention and the material of the patient sample is not transferred back into the patient's body.
  • the sample is a liquid sample.
  • the cargo selection protein (TIP47) (Swiss-PROT: O60664) is characterized by the sequence given in SEQ ID NO:l. This sequence translates to a theoretical molecular weight of 47033 Da and to an isoelectric point at pH 5.21.
  • TIP47 that binds selectively to the cytoplasmic domains of mannose 6-phosphate receptors (MPRs) and is considered to be a protein required for MPR transport from endosome to trans-Golgi. TIP47 is present in cytosol and on endosomes (Diaz, E., Pfeffer, S.R., Cell 93 (1998) 433-443).
  • T47D non-metastatic cancer cell lines
  • MDA-MB-435 fully metastatic cell lines
  • the present invention shall not be construed to be limited to the full-length protein TIP47 of SEQ ID NO:l.
  • Physiological or artificial fragments of TIP47, secondary modifications of TIP47, as well as allelic variants of TIP47 are also encompassed by the present invention.
  • Artificial fragments preferably encompass a peptide produced synthetically or by recombinant techniques, which at least comprises one epitope of diagnostic interest consisting of at least 6 contiguous amino acids as derived from the sequence disclosed in SEQ ID NO:l. Such fragment may advantageously be used for generation of antibodies or as a standard in an immunoassay. More preferred the artificial fragment comprises at least two epitopes of interest appropriate for setting up a sandwich immunoassay.
  • novel marker TIP47 may be used for monitoring as well as for screening purposes.
  • the diagnostic method according to the present invention may help to assess tumor load, efficacy of treatment and tumor recurrence in the follow-up of patients.
  • Increased levels of TIP47 are directly correlated to tumor burden. After chemotherapy a short term (few hours to 14 days) increase in TIP47 may serve as an indicator of tumor cell death. In the follow- up of patients (from 3 months to 10 years) an increase of TIP47 can be used as an indicator for tumor recurrence.
  • the diagnostic method according to the present invention is used for screening purposes. I.e., it is used to assess subjects without a prior diagnosis of BC by measuring the level of TIP47 and correlating the level measured to the presence or absence of BC.
  • the staging of cancer is the classification of the disease in terms of extent, progression, and severity. It groups cancer patients so that generalizations can be made about prognosis and the choice of therapy.
  • TNM the most widely used classification of the anatomical extent of cancer. It represents an internationally accepted, uniform staging system. There are three basic variables: T (the extent of the primary tumor), N (the status of regional lymph nodes) and M (the presence or absence of distant metastases).
  • TNM criteria are published by the UICC (International Union against Cancer) (Sobin, L.H., Wittekind, Ch. (eds): TNM Classification of Malignant Tumours, fifth edition, 1997).
  • UICC International Union against Cancer
  • the staging system for breast cancer has recently been revised (Singletary, S.E., et al., Journal of Clinical Oncology 20 (2002) 3628-3636).
  • early diagnosis of BC refers to a diagnosis at a pre-cancerous state (DCIS) or at a tumor stage where no metastases at all (neither proximal nor distal), i.e., T is , NO, M0 or Tl-4; NO; M0 are present.
  • T ⁇ s denotes carcinoma in situ.
  • the diagnostic method according to the present invention is based on a liquid sample which is derived from an individual. Unlike to methods known from the art TIP47 is specifically measured from this liquid sample by use of a specific binding agent.
  • a specific binding agent is, e.g., a receptor for TIP47, a lectin binding to TIP47 or an antibody to TIP47.
  • a specific binding agent has at least an affinity of 10 7 1/mol for its corresponding target molecule.
  • the specific binding agent preferably has an affinity of 10 1/mol or even more preferred of 10 1/mol for its target molecule.
  • specific is used to indicate that other biomolecules present in the sample do not significantly bind to with the binding agent specific for TIP47.
  • the level of binding to a biomolecule other than the target molecule results in a binding affinity which is only 10%, more preferably only 5% of the affinity of the target molecule or less.
  • a most preferred specific binding agent will fulfill both the above minimum criteria for affinity as well as for specificity.
  • a specific binding agent preferably is an antibody reactive with TIP47.
  • the term antibody refers to a polyclonal antibody, a monoclonal antibody, fragments of such antibodies, as well as to genetic constructs comprising the binding domain of an antibody. Any antibody fragment retaining the above criteria of a specific binding agent can also be used.
  • Antibodies are generated by state of the art procedures, e.g., as described in Tijssen (Tijssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam).
  • Tijssen Tejssen, P., Practice and theory of enzyme immunoassays 11 (1990) the whole book, especially pages 43-78; Elsevier, Amsterdam.
  • the skilled artisan is well aware of methods based on immunosorbents that can be used for the specific isolation of antibodies. By these means the quality of polyclonal antibodies and hence their performance in immunoassays can be enhanced. (Tijssen, P., supra, pages 108-115).
  • monoclonal and polyclonal antibodies have been used.
  • Polyclonal antibodies have been raised in rabbits.
  • polyclonal antibodies from different species e.g. rats or guinea pigs can also be used.
  • Monoclonal antibodies have been produced using spleen cells from immunized mice. Since monoclonal antibodies can be produced in any amount required with constant properties, they represent ideal tools in development of an assay for clinical routine.
  • the generation and use of monoclonal antibodies to TIP47 in a method according to the present invention is yet another preferred embodiment.
  • a synthetic peptide comprises a subsequence of SEQ ID NO:l which is specific for TIP47, i.e., which has a comparatively low homology to other/related polypeptides. It is preferred that the synthetic peptide comprises a contiguous subsequence consisting of 5 to 25 amino acid residues of SEQ ID NO:l. More preferred, the peptide comprises a contiguous subsequence consisting of 10 to 15 amino acid residues of SEQ ID NO:l.
  • DNA immunization also known as DNA vaccination may be used.
  • the liquid sample obtained from an individual is incubated with the specific binding agent for TIP47 under conditions appropriate for formation of a binding agent TIP47-complex.
  • Such conditions need not be specified, since the skilled artisan without any inventive effort can easily identify such appropriate incubation conditions.
  • the amount of complex is measured and correlated to the diagnosis of BC.
  • the skilled artisan will appreciate there are numerous methods to measure the amount of specific binding agent TIP47-complex all described in detail in relevant textbooks (cf., e.g., Tijssen P., supra, or Diamandis et al., eds. (1996) Immunoassay, Academic Press, Boston).
  • TIP47 is detected in a sandwich type assay format.
  • a first specific binding agent is used to capture TIP47 on the one side and a second specific binding agent, which is labeled to be directly or indirectly detectable is used on the other side.
  • TIP47 can be measured from a liquid sample obtained from an individual sample. No tissue and no biopsy sample is required to apply the marker TIP47 in the diagnosis of BC.
  • the method according to the present invention is practiced with serum as liquid sample material.
  • the method according to the present invention is practiced with plasma as liquid sample material.
  • the method according to the present invention is practiced with whole blood as liquid sample material.
  • the method according to the present invention is practiced with nipple aspirate fluid as liquid sample material.
  • Antibodies to TIP47 with great advantage can be used in established procedures, e.g., to detect breast cancer cells in situ, in biopsies, or in immunohistological procedures.
  • an antibody to TIP47 is used in a qualitative (TIP47 present or absent) or quantitative (TIP47 amount is determined) immunoassay.
  • the present invention relates to use of protein TIP47 as a marker molecule in the diagnosis of breast cancer from a liquid sample obtained from an individual.
  • marker molecule is used to indicate that an increased level of the analyte TIP47 as measured from a bodily fluid of an individual marks the presence of BC.
  • the novel marker TIP47 in the early diagnosis of breast cancer.
  • the use of protein TIP47 itself represents a significant progress to the challenging field of BC diagnosis.
  • the present invention relates to the use of TIP47 as a marker molecule for breast cancer in combination with one or more marker molecules for breast cancer in the diagnosis of breast cancer from a liquid sample obtained from an individual.
  • the expression "one or more” denotes 1 to 10, preferably 1 to 5, more preferred 3.
  • Preferred selected other BC markers with which the measurement of TIP47 may be combined are CEA and CA 15-3.
  • TIP47 is used as part of a marker panel at least comprising TIP47 and CA 15-3.
  • a further preferred embodiment of the present invention is the use of the protein TIP47 as a marker molecule for breast cancer in combination with one or more marker molecules for breast cancer in the diagnosis of breast cancer from a liquid sample obtained from an individual, whereby the at least one other marker molecule is CA 15-3.
  • the inventive method is used with samples of patients suspected of suffering from breast cancer.
  • An individual suspected of suffering from breast cancer is an individual for which other types of cancers have been excluded.
  • Other cancers include but are not limited to cancers of the colon, lung, stomach, ovary, and prostate.
  • a preferred embodiment of the invention is therefore a method for the diagnosis of breast cancer comprising the steps of a) providing a liquid sample obtained from an individual suspected of suffering from breast cancer, b) contacting said sample with a specific binding agent for TIP47 under conditions appropriate for formation of a complex between said binding agent and TIP47, and c) correlating the amount of complex formed in (b) to the diagnosis of breast cancer.
  • Diagnostic reagents in the field of specific binding assays usually are best provided in the form of a kit, which comprises the specific binding agent and the auxiliary reagents required to perform the assay.
  • the present invention therefore also relates to an immunological kit comprising at least one specific binding agent for TIP47 and auxiliary reagents for measurement of TIP47.
  • Accuracy of a test is best described by its receiver-operating characteristics (ROC) (see especially Zweig, M. H., and Campbell, G., Clin. Chem. 39 (1993) 561-577).
  • ROC receiver-operating characteristics
  • the ROC graph is a plot of all of the sensitivity/specificity pairs resulting from continuously varying the decision thresh-hold over the entire range of data observed.
  • the clinical performance of a laboratory test depends on its diagnostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Diagnostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • the ROC plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 - specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true-positive test results) (number of true-positive + number of false- negative test results)].
  • This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the false-positive fraction or 1 - specificity [defined as (number of false- positive results) / (number of true-negative + number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • the area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area - 1.0).
  • FIG. 1 The figure shows a typical example of a 2D-gel, loaded with a tumor sample (left side), and a gel, loaded with a matched control sample (right side).
  • the circle in the enlarged section of these gels indicates the position for the protein TIP47. Using the same method this protein has not been detected in healthy tissue. TIP47 migrates in the 2D gel corresponding to an isoelectric point of about (above) pH 5 and an apparent molecular weight of about 40 to 45 kDa.
  • tissue specimen from 14 patients suffering from breast cancer are analyzed. From each patient two different tissue types are collected from therapeutic resections: Tumor tissue (> 80% tumor) (T), and adjacent healthy tissue (N). The latter tissue type serves as matched healthy control sample. Tissues are immediately snap frozen after resection and stored at -80°C before processing. Tumors are diagnosed by histopathological criteria.
  • lysis buffer 40 mM Na-citrate, 5 mM MgCl 2 , 1% Genapol X-080, 0.02% Na-azide, Complete EDTA-free [Roche Diagnostics GmbH, Mannheim, Germany, Cat. No. 1 873 580]
  • a Wheaton® glass homogenizer 20 x loose fitting, 20 x tight fitting.
  • 3 ml of the homogenate are subjected to a sucrose-density centrifugation (10-60% sucrose) for 1 h at 4,500 x g.
  • fractions are obtained.
  • the fraction on top of the gradient contains the soluble proteins and is used for further analysis.
  • Freeze-dried CNBr-activated Sepharose 4B (Amersham Biosciences, 17-0430-01) is reswollen and washed according to the instructions of the manufacturer.
  • Monoclonal antibody directed against human albumin is dissolved in 0.1 M NaHCO 3 , pH 8.3, 0.5 M NaCl, 10 mg/ml.
  • 1 ml antibody solution is mixed with 1 ml reswollen CNBr-activated Sepharose 4B.
  • the reaction time is 1 h. Blocking of the remaining acitve groups and washing of the gel is carried out according to the instructions of the manufacturer.
  • sample buffer 7 M urea, 2 M fhiourea, 2% CHAPS, 0.4% IPG buffer pH 4-7, 0.5% DTT
  • sample buffer 7 M urea, 2 M fhiourea, 2% CHAPS, 0.4% IPG buffer pH 4-7, 0.5% DTT
  • the samples are concentrated in an Amicon ® Ultra- 15 device (Millipore GmbH, Schwalbach, Germany) and the protein concentration is determined using the Bio-Rad ® protein assay (Cat.No. 500-0006; Bio-Rad Laboratories GmbH, M ⁇ nchen, Germany) following the instructions of the supplier's manual.
  • Bio-Rad ® protein assay Cat.No. 500-0006; Bio-Rad Laboratories GmbH, M ⁇ nchen, Germany
  • IPG strips pH 4-7 (Amersham Biosciences, Freiburg, Germany) overnight.
  • the IEF is performed using the following gradient protocol: (1.) 1 minute to 500 V; (2.) 2 h to 3500 V; (3.) 22 h at constant 3500 V giving rise to 82 kVh. After IEF, strips are stored at -80°C or directly used for SDS-PAGE.
  • the strips Prior to SDS-PAGE the strips are incubated in equilibration buffer (6 M urea, 50 mM Tris/HCl, pH 8.8, 30% glycerol, 2 % SDS), for reduction DTT (15 min, + 50 mg DTT/10 ml), and for alkylation IAA (15 min, + 235 mg iodacetamide/10 ml) is added.
  • the strips are put on 12.5% polyacrylamide gels and subjected to electrophoresis at 1 W/gel and thereafter 1 h at 17 W/gel. Subsequently, the gels are fixed (50% methanol, 10% acetate) and stained overnight with Novex Colloidal Blue Staining Kit (Invitrogen, Düsseldorf, Germany, Cat No. LC6025, 45-7101) Detection of TIP47 as a potential marker for breast cancer
  • protein TIP47 is found to be specifically expressed or strongly overexpressed in tumor tissue and not detectable in healthy control tissue. It therefore - amongst many other proteins - qualifies as a candidate marker for use in the diagnosis of breast cancer.
  • Polyclonal antibody to the breast cancer marker protein TIP47 is generated for further use of the antibody in the measurement of serum and plasma and blood levels of TIP47 by immunodetection assays, e.g. Western Blotting and ELISA
  • recombinant expression of the protein is performed for obtaining immunogens.
  • the expression is done applying a combination of the RTS 100 expression system and E. coli.
  • the DNA sequence is analyzed and recommendations for high yield cDNA silent mutational variants and respective PCR-primer sequences are obtained using the "ProteoExpert RTS E.coli HY” system. This is a commercial web-based service (www.proteoexpert.com).
  • the "RTS 100 E. coli Linear Template Generation Set, His-tag” (Roche Diagnostics GmbH,
  • the construct is transformed into E. coli BL 21 (DE 3) (Studier, F.W., et al., Methods Enzymol. 185 (1990) 60-89) and the transformed bacteria are cultivated in a 1 1 batch for protein expression.
  • His-TIP47 fusion protein Purification of His-TIP47 fusion protein is done following standard procedures on a Ni-chelate column. Briefly, 1 1 of bacteria culture containing the expression vector for the His-TIP47 fusion protein is pelleted by centrifugation. The cell pellet is resuspended in lysis buffer, containing phosphate, pH 8.0, 7 M guanidium chloride, imidazole and thioglycerole, followed by homogenization using a Ultra-Turrax ® . Insoluble material is pelleted by high speed centrifugation and the supernatant is applied to a Ni-chelate chromatographic column. The column is washed with several bed volumes of lysis buffer followed by washes with buffer, containing phosphate, pH 8.0 and urea. Finally, bound antigen is eluted using a phosphate buffer containing SDS under acid conditions.
  • Hemocyanin is brought to 10 mg/ml in 100 mM NaH 2 PO 4 /NaOH, pH 7.2. Per ml hemocyanin 100 ⁇ l MHS (12.3 mg in DMSO) are added and incubated for 1 h. The sample is dialyzed over night against 100 mM NaH 2 PO 4 /NaOH, pH 6.5 and adjusted to 6 mg/ml with dialysis buffer. A selected cysteine containing TIP47- peptide was dissolved in DMSO (5 mg/ml for a peptide of 1500 Dalton).
  • mice 12 week old A/J mice are initially immunized intraperitoneally with 100 ⁇ g TIP47 or hemocyanin-peptide-conjugate (see above). This is followed after 6 weeks by two further intraperitoneal immunizations at monthly intervals. In this process each mouse is administered 100 ⁇ g TIP47 or hemocyanin-peptide-conjugate adsorbed to aluminium hydroxide and 10 germs of Bordetella pertussis. Subsequently the last two immunizations are carried out intravenously on the 3rd and 2nd day before fusion using 100 ⁇ g TIP47 or hemocyanin-peptide-conjugate in PBS buffer for each.
  • Spleen cells of the mice immunized according to a) are fused with myeloma cells according to Galfre, G., and Milstein, C, Methods in Enzymology 73 (1981) 3-46. In this process ca. lxlO 8 spleen cells of the immunized mouse are mixed with 2xl0 7 myeloma cells (P3X63-Ag8-653, ATCC CRL1580) and centrifuged (10 min at
  • the cells are then washed once with RPMI 1640 medium without foetal calf serum (FCS) and centrifuged again at 400 x g in a 50 ml conical tube. The supernatant is discarded, the cell sediment is gently loosened by tapping, 1 ml PEG (molecular weight 4000, Merck, Darmstadt) is added and mixed by pipetting. After 1 min in a water-bath at 37°C, 5 ml RPMI 1640 without FCS is added drop-wise at room temperature within a period of 4-5 min. Afterwards 5 ml RPMI 1640 containing 10% FCS is added drop-wise within ca.
  • FCS foetal calf serum
  • TIP47- positive primary cultures are cloned in 96-well cell culture plates by means of a fluorescence activated cell sorter. In this process again interleukin 6 at 100 U/ml is added to the medium as a growth additive. c) Immunoglobulin isolation from the cell culture supernatants
  • the hybridoma cells obtained are sown at a density of lxlO 5 cells per ml in RPMI 1640 medium containing 10% FCS and proliferated for 7 days in a fermenter (Thermodux Co., Wertheim/Main, Model MCS-104XL, Order No. 144-050). On average concentrations of 100 ⁇ g monoclonal antibody per ml are obtained in the culture supernatant. Purification of this antibody from the culture supernatant is carried out by conventional methods in protein chemistry (e.g. according to Bruck, C, et al., Methods in Enzymology 121 (1986) 587-695).
  • a fresh emulsion of the protein solution (lOO ⁇ g/ml TIP47 or hemocyanin-peptide-conjugate) and complete Freund's adjuvant at the ratio of 1:1 is prepared.
  • Each rabbit is immunized with 1 ml of the emulsion at days 1, 7, 14 and 30, 60 and 90. Blood is drawn and resulting anti-TIP47 serum used for further experiments as described in Examples 3 and 4.
  • IgG immunoglobulin G
  • rabbit serum is diluted with 4 volumes of acetate buffer (60 mM, pH 4.0). The pH is adjusted to 4.5 with 2 M Tris-base. Caprylic acid (25 ⁇ l/ml of diluted sample) is added drop-wise under vigorous stirring. After 30 min the sample is centrifuged (13,000 x g, 30 min, 4°C), the pellet discarded and the supernatant collected. The pH of the supernatant is adjusted to 7.5 by the addition of 2 M Tris-base and filtered (0.2 ⁇ m).
  • the immunoglobulin in the supernatant is precipitated under vigorous stirring by the drop-wise addition of a 4 M ammonium sulfate solution to a final concentration of 2 M.
  • the precipitated immunoglobulins are collected by centrifugation (8,000 x g, 15 min, 4°C). The supernatant is discarded.
  • the pellet is dissolved in 10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl and exhaustively dialyzed.
  • the dialysate is centrifuged (13,000 x g, 15 min, 4°C) and filtered (0.2 ⁇ m).
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, pH 7.5,
  • Polyclonal rabbit IgG is brought to 10 mg/ml in 10 mM NaH 2 PO 4 /NaOH, 30 mM NaCl, pH 7.5.
  • Per ml IgG solution 50 ⁇ l digoxigenin-3-O-methylcarbonyl- ⁇ - aminocaproic acid-N-hydroxysuccinimide ester (Roche Diagnostics, Mannheim, Germany, Cat. No. 1 333 054) (3.8 mg/ml in DMSO) are added. After 30 min at room temperature, the sample is chromatographed on Superdex® 200 (10 mM NaH 2 PO 4 /NaOH, pH 7.5, 30 mM NaCl). The fractions containing digoxigenylated IgG are collected. Monoclonal antibodies are labeled with digoxigenin according to the same procedure.
  • the membranes are washed 3 times in PBS/0.05 % Tween-20 and blocked with SuperBlock Blocking Buffer (Pierce Biotechnology, Inc., Rockford, IL, USA).
  • the biotinylated primary antibody is diluted in SuperBlock Blocking Buffer (0.01-0.2 ⁇ g/ml) and incubated with the membrane for lh.
  • the membranes are washed 3 times in PBS/0.05 % Tween-20.
  • the specifically bound biotinylated primary antibody is labeled with a streptavidin- HRP-conjugate (20 mU ABT s/ml in SuperBlock Blocking Buffer). After incubation for 1 h > the membranes are washed 3 times in PBS/0.05 % Tween-20.
  • the bound streptavidin-HRP-conjugate is detected using a chemiluminescent substrate (SuperSignal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, IL, USA) and autoradiographic film. Exposure times varies from 10 min to over night.
  • chemiluminescent substrate SuperSignal West Femto Substrate, Pierce Biotechnology, Inc., Rockford, IL, USA
  • Example 4 ELISA for the measurement of TIP47 in human serum and plasma samples.
  • a sandwich ELISA For detection of TIP47 in human serum or plasma, a sandwich ELISA is developed. For capture and detection of the antigen, aliquots of the anti-TIP47 polyclonal antibody (see Example 2) are conjugated with biotin and digoxygenin, respectively.
  • Streptavidin-coated 96-well microtiter plates are incubated with 100 ⁇ l biotinylated anti-TIP47 polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. After incubation, plates are washed three times with 0.9% NaCl , 0.1% Tween-20. Wells are then incubated for 2 h with either a serial dilution of the recombinant protein (see Example 2) as standard antigen or with diluted plasma samples from patients. After binding of TIP47, plates are washed three times with 0.9% NaCl , 0.1% Tween-20.
  • wells are incubated with 100 ⁇ l of digoxygenylated anti- TIP47 polyclonal antibody for 60 min at 10 ⁇ g/ml in 10 mM phosphate, pH 7.4, 1% BSA, 0.9% NaCl and 0.1% Tween-20. Thereafter, plates are washed three times to remove unbound antibody. In a next step, wells are incubated with 20 mU/ml anti- digoxigenin-POD conjugates (Roche Diagnostics GmbH, Mannheim, Germany,
  • Accuracy is assessed by analyzing individual liquid samples obtained from well- characterized patient cohorts, i.e., 50 patients having undergone mammography and found to be free of BC, 50 patients each diagnosed and staged as invasive ductal and invasive lobular Tl-3, NO, M0 of BC, 50 patients diagnosed with progressed BC, having at least tumor infiltration in at least one proximal lymph node or more severe forms of metastasis, 50 patients each diagnosed with medullary, mucinous, tubular, or papillary breast carcinoma, and 50 patients diagnosed with DCIS, respectively.
  • CA 15-3 as measured by a commercially available assay (Roche)
  • TIP47 with the established marker CA 15-3 is calculated by regularized discriminant analysis (Friedman, J. H., Regularized Discriminant Analysis, Journal of the American Statistical Association 84 (1989) 165-175).

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  • Peptides Or Proteins (AREA)

Abstract

La présente invention concerne le diagnostic du cancer du sein. L'invention concerne également l'utilisation de la protéine de sélection de cargo (TIP47) dans le diagnostic du cancer du sein. L'invention concerne en outre une méthode de diagnostic du cancer du sein à partir d'un échantillon de liquide prélevé chez un individu, par mesure de la protéine TIP47 dans ledit échantillon. La mesure de la protéine TIP47 peut, par exemple, être utilisée dans le dépistage ou le diagnostic précoce du cancer du sein.
PCT/EP2004/011659 2003-10-15 2004-10-15 Utilisation de la proteine tip47 comme marqueur du cancer du sein Ceased WO2005040811A1 (fr)

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EP03023510 2003-10-15
EP03023510.5 2003-10-15

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WO2005040811A1 true WO2005040811A1 (fr) 2005-05-06

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Publication number Priority date Publication date Assignee Title
WO2009144702A1 (fr) * 2008-04-17 2009-12-03 Diagnostic Technologies Ltd. Nouveau procédé pour diagnostiquer des complications liées à la grossesse

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US5989820A (en) * 1997-12-12 1999-11-23 Incyte Pharmaceuticals, Inc. Polynucleotides encoding a human adipophilin protein homolog
WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein
WO2004063355A2 (fr) * 2003-01-10 2004-07-29 Protein Design Labs, Inc. Nouveaux procedes de diagnostic d'un cancer metastatique, compositions et procedes de depister des modulateurs du cancer metastatique
WO2004094648A2 (fr) * 2003-04-18 2004-11-04 Cytovia, Inc. Et Al. Methodes de traitement de maladies sensibles a une induction de l'apoptose et analyses de criblage

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Publication number Priority date Publication date Assignee Title
US5989820A (en) * 1997-12-12 1999-11-23 Incyte Pharmaceuticals, Inc. Polynucleotides encoding a human adipophilin protein homolog
WO2002059377A2 (fr) * 2001-01-24 2002-08-01 Protein Design Labs Procedes de diagnostic du cancer du sein, compositions et procedes de criblage de modulateurs du cancer du sein
WO2004063355A2 (fr) * 2003-01-10 2004-07-29 Protein Design Labs, Inc. Nouveaux procedes de diagnostic d'un cancer metastatique, compositions et procedes de depister des modulateurs du cancer metastatique
WO2004094648A2 (fr) * 2003-04-18 2004-11-04 Cytovia, Inc. Et Al. Methodes de traitement de maladies sensibles a une induction de l'apoptose et analyses de criblage

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Title
BARBERO PIERRE ET AL: "TIP47 is not a component of lipid droplets", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 276, no. 26, 29 June 2001 (2001-06-29), pages 24348 - 24351, XP002317522, ISSN: 0021-9258 *
MELLICK ALBERT S ET AL: "Differential gene expression in breast cancer cell lines and stroma-tumor differences in microdissected breast cancer biopsies revealed by display array analysis.", INTERNATIONAL JOURNAL OF CANCER. JOURNAL INTERNATIONAL DU CANCER. 10 JUL 2002, vol. 100, no. 2, 10 July 2002 (2002-07-10), pages 172 - 180, XP002317523, ISSN: 0020-7136 *
MIURA SHINJI ET AL: "Functional conservation for lipid storage droplet association among perilipin, ADRP, and TIP47 (PAT)-related proteins in mammals, Drosophila, and Dictyostelium", JOURNAL OF BIOLOGICAL CHEMISTRY, vol. 277, no. 35, 30 August 2002 (2002-08-30), pages 32253 - 32257, XP002317521, ISSN: 0021-9258 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009144702A1 (fr) * 2008-04-17 2009-12-03 Diagnostic Technologies Ltd. Nouveau procédé pour diagnostiquer des complications liées à la grossesse

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