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WO2008133493A1 - Prédiction de la réactivité à un traitement anti-œstrogénique dans le cancer du sein - Google Patents

Prédiction de la réactivité à un traitement anti-œstrogénique dans le cancer du sein Download PDF

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Publication number
WO2008133493A1
WO2008133493A1 PCT/NL2007/000112 NL2007000112W WO2008133493A1 WO 2008133493 A1 WO2008133493 A1 WO 2008133493A1 NL 2007000112 W NL2007000112 W NL 2007000112W WO 2008133493 A1 WO2008133493 A1 WO 2008133493A1
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embl
protein
group
antibody
proteins
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Arzu Umar
Theo Marten Luider
John A. Foekens
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Erasmus University Medical Center
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Erasmus University Medical Center
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Priority to PCT/NL2007/000112 priority Critical patent/WO2008133493A1/fr
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    • G01N33/57515—
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis

Definitions

  • the invention relates to the field of medical diagnostics, more specifically to the field of cancer diagnostics, especially breast cancer.
  • Faslodex Faslodex
  • this compound is approved as a second line therapy for patients who relapse while undergoing Tamoxifen treatment.
  • a significant percentage of patients with advanced breast cancer will likely develop resistance to all endocrine therapies, and additional approaches to treat these patients are needed. Therefore additional biomarkers are needed to identify patients who will not respond and to select patients for various tailored treatments.
  • On a gene level such a detection has already been described in WO 2005/054510 and Becker, M. et al, 2005, MoI. Cancer Ther. 4:151-168. There, it has been shown that using microarray expression profiling a prediction for the responsiveness or resistance to anti-estrogen therapy is feasible.
  • expression-based detection systems suffer from the disadvantages that, in general, not a single biomarker can be used for a proper classification, but that a so-called 'gene signature' needs to be defined consisting of several (up to several hundreds of genes) for which the expression values need to be determined, after which it should be assessed whether or not a patient is in possession of such a gene signature, based on an optimal cut-off in training and validation groups of patients.
  • WO 2005/054510 two nested signatures have been disclosed consisting of 81 and 44 genes respectively.
  • Another disadvantage is that often classification of a new patient is only possible by pooling the expression data with a standard group of responders and non- responders and then performing statistical analyses (clustering analyses) to evaluate whether the sample of the new patient ends up with pool of responders or with the pool of non-responders.
  • the invention comprises a method to predict responsiveness or resistance (non-responsiveness) to anti-estrogen therapy in breast cancer comprising a) isolating a tumor sample from a breast cancer patient; b) assaying said sample for the presence of one or more, preferably both of the proteins selected from the group A essentially consisting of: - nascent polypeptide associated complex alpha subunit (NACA, HSD48) (EMBL Q13765); - splice isoform 1 of epsin 4 (CLINTl, EPN4) (EMBL Q14677-1); c) assaying said sample for the presence of one or more, preferably two or more, preferably three or more, more preferably four or more, more preferably all of the proteins selected from the group B essentially consisting of:
  • AK3L1, AK3, AK4 mitochondrial
  • EPH receptor B2 EPH receptor B2 (EPHB2, ERK, EPTH3) (EMBL P29323-3);
  • said detection is performed by an immunoassay, comprising one or more antibodies specifically recognizing said protein(s).
  • the invention comprises a kit for performing a method as described above, comprising one or more antibodies capable of binding to one or more proteins of groups A and B, additional reagents, and optionally instructions for performing the assay.
  • the invention provides for the use of a protein, selected from the group consisting essentially of - nascent polypeptide associated complex alpha subunit (NACA, HSD48) (EMBL Q13765);
  • AK3L1, AK3, AK4 mitochondrial
  • ANXA8, ANX8 mitochondrial
  • COEOlB coronin-lB
  • EPH receptor B2 EPH receptor B2 (EPHB2, ERK, EPTH3) (EMBL P29323-3);
  • - splice isoform 2 of basigin precursor BSG, EMMPRIN, TCSF, CD 147) (EMBL P35613-2) in an assay for predicting the responsiveness of breast cancer patients to anti- estrogen therapy, more preferably tamoxifen therapy.
  • Fig. 1 Cluster analysis. Hierarchical clustering analysis was performed on the 8-protein profile, calculating z-scores for each protein across the different samples using average protein abundance levels. High protein abundance is shown in red and low abundance in blue.
  • Fig. 2 Principal Components Analysis.
  • PCA PCA on proteins. Proteins highly expressed in either OR (responsive, light blue) or PD (not responsive, dark blue) cluster together.
  • the group of 8 proteins that now has been found to be predictive for responsiveness to anti-estrogen, more specifically tamoxifen therapy is listed in Table 1 below.
  • the amino acid sequences of the proteins of Table 1 are represented herein below.
  • a biopsy of the tumor is a normal procedure for assessing the presence and nature of a tumor in patients suspected of breast cancer. This biopsy can thus further be used to test for the responsiveness to anti-estrogen treatment according to the present invention.
  • the biopsy or isolating the tumor cells from the biopsy tissue, it should be taken care that, at least for the assay of the present invention, sufficient tumor cells in relation to non- tumor cells are included. It is, however, submitted that even with an underrepresentation of tumor cells, it is believed that the level of one or more of the 8 proteins would be sufficient to perform the assay successfully.
  • the presence of a protein in the sample can be detected using various conventional methods, which will be well known to a person skilled in the art, such as mass spectrometry methods, e.g. MALDI-TOF, nanoLC-MALDI-TOF/TOF and/or MALDI-FTMS.
  • mass spectrometry methods e.g. MALDI-TOF, nanoLC-MALDI-TOF/TOF and/or MALDI-FTMS.
  • One of the preferred methods for the assay of the invention is a quantitative immunoassay, which can be an enzyme-immunoassay (EIA), and enzyme-linked immunosorbent assay (ELISA), or any other assay using immune reactions.
  • EIA enzyme-immunoassay
  • ELISA enzyme-linked immunosorbent assay
  • antibodies to the peptides should be generated.
  • a first option is formed by pooling the data with data from other breast cancer patients of whom the responsiveness to anti-estrogen treatment is known and performing an analysis as basically described in the Examples. If then a plot is made similar to the plot shown in Fig. 2B it can be easily determined whether the new patient falls in the category of responders or non-responders. For this option thus a pool of samples from patients with a known history of treatment responsiveness is needed.
  • the concentration of at least one of the proteins of Group A of Table 1 and at least one of the proteins of Group B of Table 1 should be determined (in any way as described above) and a hierarchical clustering and principal components analysis should be performed on these data, it is submitted that these statistical analysis methods are readily known to a person skilled in the art.
  • a second option is to regard one or a pool of samples from one known group (responders or non-responders) as a control for a new sample to be tested. Then, using the same method, the concentration of at least one protein of Group A and at least one of the proteins of Group B should be determined in both samples. Using the values obtained in the control sample, the test values of the test samples are corrected. This basically means that - if the control sample would e.g. consist of responders, the determined level of proteins of
  • the test sample is multiplied with (or divided by) a factor to equalize the values obtained in the control sample. This same factor is then used to multiply or divide the data obtained for the at least one protein of Group B, which is then indicated as the normalized concentration. If then the normalized level of said at least one protein of Group B is about similar to the level of said at least one protein of Group B in the control sample, then the test sample can be classified as responder. If the normalized level is much larger than the at least one protein of Group B in the control sample, then the test sample should be classified as non-responder.
  • a third possibility is to calculate the ration between the concentration of at least one of the proteins from Group A and at least one protein of Group B.
  • the ratio is calculated for more protein pairs selected from said groups of proteins of Table 1.
  • concentration is measured of a protein which is known to be present at a fairly constant level in tumor cells, whether they origin from responders or non-responders.
  • a protein can for instance be a housekeeping protein such as RNA polymerase or an enzyme from the Krebs cycle. It is then also possible to calculate the ratio of the concentration of at least one of the proteins from Group A and at least one of the proteins of Group B with the concentration of the housekeeping protein. If these ratios are calculated then the ratio of said ratios is determined. If this ratio is below or above a certain predetermined value (based on calculating the same ratio in known responders and non-responders) a classification of the new sample can be established.
  • synthetic antibody an antibody which is generated using recombinant DNA technology, such as, for example, an antibody expressed by a bacteriophage as described herein.
  • the term should also be construed to mean an antibody which has been generated by the synthesis of a DNA molecule encoding the antibody and which DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, wherein the DNA or amino acid sequence has been obtained using synthetic DNA or amino acid sequence technology which is available and well known in the art.
  • enzyme linked immuno- sandwich assay is an antibody based assay used for the identification of protein and for measurements of protein levels in cell or tissue preparations.
  • “Expression vector” refers to a vector comprising a recombinant polynucleotide comprising expression control sequences operatively linked to a nucleotide sequence to be expressed.
  • An expression vector comprises sufficient cis- acting elements for expression; other elements for expression can be supplied by the host cell or in an in vitro expression system.
  • Expression vectors include all those known in the art, such as cosmids, plasmids (e.g., naked or contained in liposomes) and viruses that incorporate the recombinant polynucleotide.
  • the instruction manual of the kit of the invention may, for example, be affixed to a container which contains the composition or be shipped together with a container which contains the composition. Alternatively, the instruction manual may be shipped separately from the container with the intention that the instructional material and the composition be used cooperatively by the recipient.
  • the "level" of a nucleic acid or polypeptide refers to a measurable amount of a nucleic acid or polypeptide.
  • the level of a protein of Table 1 can be ascertained by measuring the concentration of the protein in weight per unit volume or, as described above, it can be related to the level of a reference protein.
  • reference protein preferably a protein is used which is available at a nearly constant level in the tumor or surrounding cells, such as is the case for various housekeeping enzymes.
  • the complete absence of a protein of Table 1 from an assay sample can be referred to as a "zero level" of said protein in that assay.
  • peptide typically refers to short polypeptides. Conventional notation is used herein to portray polypeptide sequences: the left-hand end of a polypeptide sequence is the amino- terminus; the right-hand end of a polypeptide sequence is the carboxyl -terminus.
  • Antibodies against any protein of Table 1 are made by methods known to the skilled artisan, and summarized as follows. The generation of polyclonal antibodies is accomplished by inoculating the desired animal with the antigen and isolating antibodies, which specifically bind the antigen there of.
  • the proteins listed in Table 1, variants, and fragments thereof may be produced by transformation of a suitable host cell with all or part of a polypeptide-encoding nucleic acid molecule or fragment thereof in a suitable expression vehicle.
  • suitable host cell e.g., E.
  • coli or in a eukaryotic host (e.g., Saccharomyces cerevisiae, insect cells, e.g., S[pound]21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells).
  • a eukaryotic host e.g., Saccharomyces cerevisiae, insect cells, e.g., S[pound]21 cells, or mammalian cells, e.g., NIH 3T3, HeLa, or preferably COS cells.
  • Such cells are available from a wide range of sources (e.g., the American Type Culture Collection, Rockland, Md.; also, see, e.g., Ausubel et al., supra).
  • the method of transformation or transfection and the choice of expression vehicle will depend on the host system selected. Transformation and transfection methods are described, e.g., in Ausubel et al. (supra); expression vehicles may
  • Expression vectors useful for producing such proteins include, without limitation, chromosomal, episomal, and virus- derived vectors, e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof.
  • chromosomal, episomal, and virus- derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from transposons, from yeast episomes, from insertion elements, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as SV40, vaccinia viruses, adenoviruses, fowl po
  • coli pET expression system (Novagen, Inc., Madison, Wis). According to this expression system, DNA encoding a polypeptide is inserted into a pET vector in an orientation designed to allow expression. Since the gene encoding such a polypeptide is under the control of the T7 regulatory signals, expression of the polypeptide is achieved by inducing the expression of T7 ENA polymerase in the host cell. This is typically achieved using host strains that express T7 RNA polymerase in response to IPTG induction. Once produced, recombinant polypeptide is then isolated according to standard methods known in the art, for example, those described herein. Another bacterial expression system for polypeptide production is the pGEX expression system (Pharmacia).
  • This system employs a GST gene fusion system that is designed for high-level expression of genes or gene fragments as fusion proteins with rapid purification and recovery of functional gene products.
  • the protein of interest is fused to the carboxyl terminus of the glutathione S-transferase protein from Schistosoma japonicum and is readily purified from bacterial lysates by affinity chromatography using Glutathione Sepharose 4B. Fusion proteins can be recovered under mild conditions by elution with glutathione. Cleavage of the glutathione S- transferase domain from the fusion protein is facilitated by the presence of recognition sites for site-specific proteases upstream of this domain. For example, proteins expressed in pGEX-2T plasmids may be cleaved with thrombin; those expressed in pGEX-3X may be cleaved with factor Xa
  • the recombinant polypeptide of the invention is expressed, it is isolated, e.g., using affinity chromatography.
  • an antibody e.g., produced as described herein
  • a protein of the invention may be attached to a column and used to isolate the recombinant polypeptide. Lysis and fractionation of polypeptide-harboring cells prior to affinity chromatography may be performed by standard methods.
  • the recombinant protein can, if desired, be further purified, e.g., by high performance liquid chromatography (see, e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, 1980).
  • Polypeptides of the invention, particularly short peptide fragments can also be produced by chemical synthesis (e.g., by the methods described in Solid Phase Peptide Synthesis, 2nd ed., 1984 The Pierce Chemical Co., Rockford,
  • the invention further includes analogues of any naturally-occurring polypeptide of the invention. Analogues can differ from naturally-occurring polypeptides of the invention by amino acid sequence differences, by post- translational modifications, or by both. Analogues of the invention will generally exhibit at least 85%, more preferably 90%, and most preferably 95% or even 99% identity with all or part of a naturally-occurring amino acid sequence of the invention.
  • the length of sequence comparison is at least 10, 13, 15 amino acid residues, preferably at least 25 amino acid residues, and more preferably more than 35 amino acid residues.
  • a BLAST program may be used, with a probability score between e- 3 and e 10 ° indicating a closely related sequence.
  • Modifications include in vivo and in vitro chemical derivatization of polypeptides, e.g., acetylation, carboxylation, phosphorylation, or glycosylation; such modifications may occur during polypeptide synthesis or processing or following treatment with isolated modifying enzymes.
  • Analogues can also differ from the naturally-occurring polypeptides of the invention by alterations in primary sequence.
  • a fragment means at least 5, 10, 13, or 15 amino acids. In other embodiments a fragment is at least 20 contiguous amino acids, at least 30 contiguous amino acids, or at least 50 contiguous amino acids, and in other embodiments at least 60 to 80 or more contiguous amino acids. Fragments of the invention can be generated by methods known to those skilled in the art or may result from normal protein processing (e.g., removal of amino acids from the nascent polypeptide that are not required for biological activity or removal of amino acids by alternative mRNA splicing or alternative protein processing events).
  • Monoclonal antibodies directed against full length or peptide fragments of a protein or peptide may be prepared using any well known monoclonal antibody preparation procedures, such as those described, for example, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor, NY) and in Tuszynsld et al. (1988, Blood, 72:109-115). Human monoclonal antibodies may be prepared by the method described in U.S. patent publication 2003/0224490. Quantities of the desired peptide may also be synthesized using chemical synthesis technology. Alternatively, DNA encoding the desired peptide may be cloned and expressed from an appropriate promoter sequence in cells suitable for the generation of large quantities of peptide. Monoclonal antibodies directed against the peptide are generated from mice immunized with the peptide using standard procedures as referenced herein.
  • Nucleic acid encoding the monoclonal antibody obtained using the procedures described herein may be cloned and sequenced using technology which is available in the art, and is described, for example, in Wright et al. (1992, Critical Rev. in Immunol. 12(3,4): 125-168) and the references cited therein. Further, the antibody of the invention may be "humanized” using the technology described in Wright et al., (supra) and in the references cited therein, and in Gu et al. (1997, Thrombosis and Hematocyst 77(4): 755-759).
  • a cDNA library is first obtained from mRNA which is isolated from cells, e.g., the hybridoma, which express the desired protein to be expressed on the phage surface, e.g., the desired antibody. cDNA copies of the mRNA are produced using reverse transcriptase. cDNA which specifies immunoglobulin fragments are obtained by PCR and the resulting PNA is cloned into a suitable bacteriophage vector to generate a bacteriophage DNA library comprising DNA specifying immunoglobulin genes.
  • the procedures for making a bacteriophage library comprising heterologous DNA are well known in the art and are described, for example, in Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • Bacteriophages which encode the desired antibody, may be engineered such that the protein is displayed on the surface thereof in such a manner that it is available for binding to its corresponding binding protein, e.g., the antigen against which the antibody is directed.
  • the bacteriophage which express a specific antibody are incubated in the presence of a cell which expresses the corresponding antigen, the bacteriophage will bind to the cell.
  • Bacteriophages that do not express the antibody will not bind to the cell.
  • panning techniques are well known in the art and are described for example, in Wright et al., (supra).
  • a cDNA library is generated from mRNA obtained from a population of antibody-producing cells.
  • the mRNA encodes rearranged immunoglobulin genes and thus, the cDNA encodes the same.
  • Amplified cDNA is cloned into M 13 expression vectors creating a library of phage which express human Fab fragments on their surface.
  • Phage which display the antibody of interest are selected by antigen binding and are propagated in bacteria to produce soluble human Fab immunoglobulin.
  • this procedure immortalizes DNA encoding human immunoglobulin rather than cells, which express human immunoglobulin.
  • phages that encode the Fab portion of an antibody molecule.
  • the invention should not be construed to be limited solely to the generation of phage encoding Fab antibodies. Rather, phagesthat encode single chain antibodies (scFv/phage antibody libraries) are also included in the invention.
  • Fab molecules comprise the entire Ig light chain, that is, they comprise both the variable and constant region of the light chain, but include only the variable region and first constant region domain (CHl) of the heavy chain.
  • Single chain antibody molecules comprise a single chain of protein comprising the Ig Fv fragment.
  • An Ig Fv fragment includes only the variable regions of the heavy and light chains of the antibody, having no constant region contained therein.
  • Phage libraries comprising scFv DNA may be generated following the procedures described in Marks et al., 1991, J. MoI. Biol. 222:581-597. Panning of phage so generated for the isolation of a desired antibody is conducted in a manner similar to that described for phage libraries comprising Fab DNA.
  • the invention should also be construed to include synthetic phage display libraries in which the heavy and light chain variable regions may be synthesized such that they include nearly all possible specificities (Barbas, 1995, Nature Medicine 1 :837-839; de Kruif et al., 1995, J. MoI. Biol. 248:97- 105).
  • a preferred method of co-immunoprecipitation is described in the examples herein. See also Harlow et al., (1988, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY) and Harlow et ah, (1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  • a protein of Table 1 or a fragment thereof can be conjugated to an immunogenic carrier, e.g. bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH).
  • BSA bovine serum albumin
  • KLH keyhole limpet hemocyanin
  • adjuvants may be used to increase the immunological response, depending on the host species, including but not limited to Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Galmette-Guerin) and Corynebacterium parvum.
  • BCG Bacille Galmette-Guerin
  • Corynebacterium parvum bacille Galmette-Guerin
  • any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. These include but are not limited to the hybridoma technique originally developed by Kohler and Milstein (1975, Nature, 256:495- 497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., 1983, Immunology Today, 4:72; Cote et al., 1983, Proc. Natl.
  • monoclonal antibodies can be produced in germ-free animals (WO 89/12690).
  • techniques developed for the production of "chimeric antibodies" (Morrison et al., 1984, J. Bacteriol.
  • such fragments include but are not limited to: the F(ab') fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragments which can be generated by reducing the disulfide bridges of the F(ab')2 fragment, and the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
  • screening for the desired antibody can be accomplished by techniques known in the art, e.g., radioimmunoassay, ELISA (enzyme-linked immunosorbant assay), "sandwich” immunoassays (see Methods in Molecular Biology, Vol. 149; The ELISA
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labelled .
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention. For selection of an antibody specific to the protein of Table 1 from a particular species of animal, one can select on the basis of positive binding with said protein expressed by or isolated from cells of that species of animal.
  • the antibodies according to the invention also comprise antibody fragments obtained with the aid of phage libraries as described by Ridder et al., (1995, Biotechnology (NY), 13(3):255-260) or humanized antibodies as described by Reinmann et al. (1997, AIDS Res Hum Retroviruses, 13(ll):933-943) and Leger et al., (1997, Hum Antibodies, 8(1):3-16).
  • an antibody according to the invention is useful in immunological detection tests intended for the identification of the presence and/or of the quantity of antigens present in a sample. Therefore, an antibody according to the invention may comprise, in addition, a detectable marker that is isotopic or non-isotopic, for example fluorescent, or may be coupled to a molecule such as biotin, according to techniques well known to persons skilled in the art.
  • a detectable marker that is isotopic or non-isotopic
  • biotin for example fluorescent
  • yeast two-hybrid assay 1 can be identified using, for example, a yeast two-hybrid assay.
  • Yeast two hybrid assay methods are well-known in the art and can be performed using well documented techniques, for example those described in Bartel and Fields, (The Yeast Two-Hybrid System, Oxford University Press, Gary, N.C.). Therefore, once armed with the teachings provided herein, e.g., the full amino and derivable nucleic acid sequences of the protein(s) of Table 1, one skilled in the art can easily identify a protein that specifically binds with said protein(s).
  • detectable amino acid sequence or “detectable moiety” is meant a composition that when linked with the nucleic acid or protein molecule of interest renders the latter detectable, via any means, including spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include radioactive isotopes, magnetic beads, metallic beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (for example, as commonly used in an ELISA), biotin, digoxigenin, or haptens.
  • “Microarray” means a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support (for example, a chip, plate, or bead).
  • the diagnostic methods of the invention are used to assay the concentration of any protein of Table 1 in a biological sample relative to a reference (e.g., the level of said protein present in a corresponding control tissue).
  • the level of a protein is detected using an antibody that specifically binds said protein.
  • Methods for measuring an antibody- protein complex include, for example, detection of fluorescence, luminescence, chemiluminescence, absorbance, reflectance, transmittance, birefringence or refractive index.
  • Optical methods include microscopy (both confocal and non- confocal), imaging methods and non-imaging methods. Methods for performing these assays are readily known in the art.
  • Useful assays include, for example, an enzyme immune assay (EIA) such as enzyme-linked immunosorbent assay (ELISA), a radioimmune assay (RIA), a Western blot assay, or a slot blot assay.
  • EIA enzyme immune assay
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmune assay
  • Western blot assay or a slot blot assay.
  • kits for performing the diagnostic method of the invention in a biological sample obtained from a subject detects an increase in the concentration of any protein of Table 1 relative to a reference level.
  • the kit includes an antibody that binds to a protein of Table 1, more specifically the kit comprises a set of antibodies, wherein each antibody is able to bind to one of the proteins of Table 1. More preferably said kit comprises a set of 8 antibodies, wherein each antibody is capable to bind to a different protein of Table 1.
  • the kit also comprises an antibody which is able to specifically bind to a housekeeping protein.
  • the kit includes directions for monitoring the levels of the protein(s) of table 1 in a biological sample derived from a subject.
  • the kit comprises a sterile container, which contains the antibody or antibodies, or other detection regents; such containers can be boxes, ampules, bottles, vials, tubes, bags, pouches, blister-packs, or other suitable container form known in the art.
  • Such containers can be made of plastic, glass, laminated paper, metal foil, or other materials suitable for holding nucleic acids.
  • the instructions will generally include information about the use of diagnostic reagents described herein and their use in performing the diagnostic method of the invention.
  • the instructions include at least one of the following: description of the antibody or antibodies; methods for using the enclosed materials for the diagnostic method according to the invention; precautions; warnings; indications; clinical or research studies; and/or references.
  • the instructions may be printed directly on the container (when present), or as a label applied to the container, or as a separate sheet, pamphlet, card, or folder supplied in or with the container.
  • the diagnostic methods of the invention may also be used in microarray-based assays that provide for the high-throughput analysis of tissue samples.
  • the proteins of Table 1 or fragments or variants thereof are useful as hybridizable array elements in such a microarray.
  • the array elements are organized in an ordered fashion such that each element is present at a specified location on the substrate.
  • Useful substrate materials include membranes, composed of paper, nylon or other materials, filters, chips, glass slides, and other solid supports. The ordered arrangement of the array elements allows hybridization patterns and intensities to be interpreted as expression levels of particular genes or proteins. Methods for making polypeptide microarrays are described, for example, by Ge (Nucleic Acids Res.
  • protein microarrays feature a protein, or fragment thereof, bound to a solid support.
  • suitable solid supports include membranes (e.g., membranes composed of nitrocellulose, paper, or other material), polymer- based films (e.g., polystyrene), beads, or glass slides.
  • the proteins of Table 1, or fragments or variants thereof, or, alternatively, antibodies capable of binding said proteins, fragments or variants are spotted on a substrate using any convenient method known to the skilled artisan (e.g., by hand or by inkjet printer). Preferably, such methods retain the biological activity or function of the protein bound to the substrate (e.g., antibody binding).
  • the protein microarray is hybridized with a detectable probe.
  • Such probes can be polypeptide (e.g., an antibody) or small molecules. Binding conditions (e.g., temperature, pH, protein concentration, and ionic strength) are optimized to promote specific interactions. Such conditions are known to the skilled artisan and are described, for example, in Harlow, E. and Lane, D., Using Antibodies: A Laboratory Manual. 1998, New York: Cold
  • probes After removal of non-specific probes, specifically bound probes are detected, for example, by fluorescence, enzyme activity (e.g., an enzyme-linked calorimetric assay), direct immunoassay, radiometric assay, or any other suitable detectable method known to the skilled artisan.
  • enzyme activity e.g., an enzyme-linked calorimetric assay
  • direct immunoassay e.g., an enzyme-linked calorimetric assay
  • radiometric assay e.g., radiometric assay, or any other suitable detectable method known to the skilled artisan.
  • detection of one or more of the proteins of Table 1 may be combined with the detection of other biomarkers, where the presence or level of the biomarker is correlated with the responsiveness to anti-estrogen treatment.
  • the (detecting) reagent protein, peptide or antibody
  • the composition is generally a matter of simply adding the composition to the sample and incubating the mixture for a period of time long enough for the antibodies to form immune complexes with the antigen. Washing of the sample (i.e., tissue section, ELISA plate, dot blot or Western blot, microarray) is generally required to remove any non-specifically bound antibody species. The antigen-antibody complex (immunocomplex) is then detected using specific reagents.
  • the antigen detecting reagent is an antibody (a specific antibody)
  • this antibody may be (directly) labeled with a marker (fluorophore, chromophore, dye, enzyme, radioisotope, etc.) for enabling the detection of the complex.
  • a marker fluorophore, chromophore, dye, enzyme, radioisotope, etc.
  • a secondary binding ligand such as a secondary antibody or a biotin/avidin (streptavidin) (binding/ligand complex) arrangement, as is known in the art.
  • secondary antibodies may be labeled with a marker as described above or with an arrangement of biotin/avidin (i.e. avidin peroxidase) or biotin/streptavidin (i.e.
  • the secondary antibody will be an antibody directed to the specific antibody (primary antibody) of a defined isotype and species such as, for example, an anti-mouse IgG.
  • the detecting reagent may also be a polypeptide having affinity for a protein of Table 1, which forms a complex (i.e., polypeptide-polypeptide complex complex).
  • the polypeptide itself may be labeled using the markers described above, allowing direct detection.
  • the complex may be detected indirectly by adding a secondary (labeled) antibody or polypeptide.
  • Immunodetection methods such as enzyme-linked immunosorbent assays (ELISA), Western blots, etc. have utility in the diagnostic method of the present invention. However, these methods also have applications to nonclinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like.
  • ELISA enzyme-linked immunosorbent assays
  • Western blots etc.
  • these methods also have applications to nonclinical samples, such as in the titering of antibody samples, in the selection of hybridomas, and the like.
  • Immunoassays that may be performed using reagents of the present invention includes, for example, enzyme linked immunosorbent assays (ELISAs) and radioimmunoassays(RIA), which are known in the art lmmunohistochemical detection using tissue sections is also particularly useful However, it will be readily appreciated that detection is not limited to such techniques, and Western blotting, dot blotting, FACS analyses, and the like also may be used.
  • ELISAs enzyme linked immunosorbent assays
  • RIA radioimmunoassays
  • ELISA assays include the following, one or more antibodies binding to one or more proteins of Table 1 are immobilized onto a selected surface (i.e., a suitable substrate) exhibiting protein affinity, such as a well in a polystyrene microtiter plate (E.LISA plate) Then, a sample suspected of containing the polypeptide is added to the wells of the plate After binding and washing to remove non-specifically bound immunocomplexes, the bound protein may be detected Detection may be achieved by the addition of a second antibody specific for the target polypeptide, which is linked to a detectable label.
  • a selected surface i.e., a suitable substrate
  • E.LISA plate polystyrene microtiter plate
  • ELISA is a simple "sandwich ELISA "Detection also may be achieved by the addition of a second antibody, followed by the addition of a third antibody that has binding affinity for the second antibody, with the third antibody being linked to a detectable label (marker).
  • a detectable label marker
  • Another example of ELISA assay is the following, the samples suspected of containing the protein of interest are immobilized onto the surface of a suitable substrate and then contacted with the antibodies capable of binding said protein. After binding and washing to remove non-specifically bound immunocomplexes, the bound protein is detected The immunocomplexes may be detected directly or indirectly as described herein.
  • an ELISA assay is the following, again, proteins are immobilized to a substrate, however, in that case the assay involves a competition step
  • a known amount of the protein of interest is adsorbed to the plate
  • the amount of protein in an unknown sample is then determined by mixing the sample with a specific antibody before or during incubation with wells containing the immobilized protein.
  • a detection reagent is added (e g , 2 nd antibody, labeled) to quantify the antibody that is able to bind to the immobilized polypeptide.
  • the presence of the protein in the sample acts to reduce the amount of antibody available for binding to the protein contained in the well (immobilized polypeptide) and thus reduces the signal.
  • a control sample may be included during the assay.
  • known (predetermined) quantities of a polypeptide usually in a substantially pure form
  • the signal obtained for the unknown sample is then compared with the signal obtained for the control.
  • the intensity (level) of the signal is usually proportional to the amount of polypeptide (antibody bound to the polypeptide) in a sample.
  • the amount of control polypeptide and antibodies required to generate a quantitative assay needs to be evaluated first.
  • a plate with either an antigen (polypeptide) or antibody In coating a plate with either an antigen (polypeptide) or antibody, one will generally incubate the wells of the plate with a solution of the protein or antibody, either overnight or for a specified period of hours. The wells of the plate will then be washed to remove incompletely adsorbed material. Any remaining available surfaces of the wells are then "coated" with a non-specific protein that is antigenically neutral with regard to the test samples. These include bovine serum albumin (BSA), casein and solutions of milk powder.
  • BSA bovine serum albumin
  • the coating allows for blocking of non-specific adsorption sites on the immobilizing surface and thus reduces the background caused by non-specific binding of antisera onto the surface.
  • Conditions that may allow immunocomplex (antigen/antibody) formation include diluting the proteins and antibodies with solutions such as BSA, bovine gamma globulin (BGG) and phosphate buffered saline (PBS)/Tween. These added agents also tend to assist in the reduction of non-specific background. Suitable conditions involves that the incubation is at a temperature and for a period of time sufficient to allow effective binding. Incubation steps are typically from about 1 to 2 to 4 h, at temperatures preferably on the order of 20 0 C to 27°C, or may be overnight at about 4°C or so.
  • BSA bovine gamma globulin
  • PBS phosphate buffered saline
  • the detection of the immunocomplex is performed with a reagent that is linked to an enzyme. Detection usually requires the addition of the enzyme's substrate. Enzymes such as, for example, a phosphatase (e.g., alkaline phosphatase), a peroxidase, etc. when given an appropriate substrate will generate a reaction that may be quantified by measuring the intensity (degree) of color (radioactivity, fluorescence, etc.) produced. The reaction is usually linear over a wide range of concentrations and may be quantified using a visible spectra spectrophotometer.
  • a phosphatase e.g., alkaline phosphatase
  • peroxidase e.g., etc.
  • the reaction is usually linear over a wide range of concentrations and may be quantified using a visible spectra spectrophotometer.
  • the present invention also relates to immunodetection kits and reagents for use with the immunodetection methods described above.
  • the polypeptide of the present invention may be employed to detect antibodies and the corresponding antibodies may be employed to detect the polypeptide, either or both of such components may be provided in the kit.
  • the immunodetection kits may thus comprise, in suitable container means, one or more proteins selected from the proteins listed in Table 1, or one or more of a first antibody that binds to said one or more protein and/or an immunodetection reagent.
  • the kit may comprise also a suitable matrix, to which the antibody or protein of choice may already be bound. Suitable matrices include an ELISA plate. The plate provided with the kit may already be coated with the antibody or protein of choice.
  • the coated ELISA plate may also have been blocked using reagents described herein to prevent unspecific binding.
  • Detection reagents may also be provided and may include, for example, a secondary antibody or a ligand, which may carry the label or marker and/or an enzyme substrate.
  • Kits may further comprise an antibody or protein (usually of known titer or concentration) that may be used for control.
  • Reagents may be provided, for example, lyophilized or in liquid form (of a defined concentration) and are provided in suitable containers (ensuring stability of reagents, safety etc.).
  • any specified range or group is to be understood as a shorthand way of referring to each and every member of a range or group individually as well as each and every possible sub-ranges or sub-groups encompassed therein; and similarly with respect to any sub- ranges or sub- groups therein.
  • a time of 1 minute or more is to be understood as specifically incorporating herein each and every individual time, as well as sub-range, above 1 minute, such as for example 1 minute, 3 to 15 minutes, 1 minute to 20 hours, 1 to 3 hours, 16 hours, 3 hours to 20 hours etc.; and similarly with respect to other parameters such as concentrations, temperature, etc...
  • Tryptic digests were prepared and analyzed in triplicate; ⁇ 550 ng of peptides per analysis were separated on a 50 ⁇ m x 80 cm reversed phase nanoLC column prior to FTICR MS analysis.
  • Peptide mass and elution time features were matched to information in previously generated accurate mass and time (AMT) tag reference databases to identify peptide sequences and proteins, and the MS peak intensities were used to determine relative peptide abundances. More than 20,000 unique peptides were identified that corresponded to a total of 2309 non-redundant proteins identified with two or more peptides. From this total, 1713 (74%) proteins that overlapped between the two datasets were used for further statistical analysis.
  • the two datasets were separated into a 'training' and Validation' set and analyzed for differential protein abundance between OR and PD groups, using a univariate t-test from BRB array-tools software package, followed by a Wilcoxon rank-sum test.
  • 100 differentially abundant proteins were identified (p ⁇ 0.05), of which 8 were present in both sets (p ⁇ 0.015). These 8 proteins were subjected to hierarchical clustering and principal components analysis. Based upon this 8-protein profile, PD and OR samples clustered into two separate groups that correctly predicted therapy-response. The sensitivity and specificity of this protein profile will be determined in an independent validation set.
  • an 8-protein profile observed to predict tamoxifen-resistance in breast cancer was revealed by ultra-sensitive nanoLC-FTICE, technology for comprehensive proteome analyses of LCM cells.
  • the 8 proteins are listed in Table 1 and their amino acid sequences are provided below.
  • NACA (SEQ ID NO: 1) 1 mpgeatetvp ateqelpqpq aetgsgtesd sdesvpelee qdstqattqq aqlaaaaeid
  • AK3L1 (SEQ ID NO:3) 1 maskllravi lgppgsgkgt vcqriaqnfg lqhlssghfl renikastev gemakqyiek

Landscapes

  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé de prédiction de la réactivité à un traitement anti-œstrogénique chez des patientes atteintes du cancer du sein en dosant une ou plusieurs des protéines suivantes qui se sont avérées être exprimées différemment chez les sujets répondeurs et les sujets non répondeurs : - une sous-unité alpha de complexe lié au polypeptide naissant (NACA, HSD48) (EMBL Q13765) ; - une isoforme d'épissage 1 de l'epsine 4 (CLINT1, EPN4) (EMBL Q14677-1) ; une isoenzyme de l'adénylate kinase 4 mitochondriale (AK3L1, AK3, AK4) (EMBL P27144) ; l'annexine A8 (ANXA8, ANX8) (EMBL P13928) ; la coronine 1B (CORO1B) (EMBL Q9BR76) ; le récepteur EPH B2 (EPHB2, ERK, EPTH3) (EMBL P29323-3) ; une NADH-cytochrome B5 réductase (CYB5R3, DIA1) (EMBL P00387-1) ; et une isoforme d'épissage 2 de précurseur de la basigine (BSG, EMMPRIN, TCSF, CD147) (EMBL P35613-2).
PCT/NL2007/000112 2007-04-27 2007-04-27 Prédiction de la réactivité à un traitement anti-œstrogénique dans le cancer du sein Ceased WO2008133493A1 (fr)

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WO2000034788A1 (fr) * 1998-12-08 2000-06-15 Board Of Regents, The University Of Texas System Procedes de detection du cancer du sein resistant aux anti-oestrogenes
EP1167975A1 (fr) * 2000-06-22 2002-01-02 Universite Rene Descartes (Paris V) Compositions et méthodes pour détecter, traiter ou prédire la réponse des cellules tumorales à une thérapie endocrine
WO2003089904A2 (fr) * 2002-04-17 2003-10-30 Baylor College Of Medicine Aib1 utilise en tant que marqueur pronostique et predicteur de la resistance a l'endocrinotherapie
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WO2003089904A2 (fr) * 2002-04-17 2003-10-30 Baylor College Of Medicine Aib1 utilise en tant que marqueur pronostique et predicteur de la resistance a l'endocrinotherapie
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WO2005054510A2 (fr) * 2003-12-05 2005-06-16 Erasmus Mc Prediction de la receptivite et des resultats du traitement anti-oestrogenique du cancer du sein metastatique
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101716053B1 (ko) 2015-11-10 2017-03-14 충북대학교 산학협력단 Ｎａｃａ 단백질에 특이적으로 결합하는 ｒｎａ 앱타머 및 이의 용도

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