use of a histone variant or an isoform associated with a cell-free nucleosome, a kit for the detecti

专利摘要:
The invention relates to a method for detecting and measuring the presence of mono-nucleosomes and oligo-nucleosomes and nucleosomes containing particular histone variants and the use of such measurements for the detection and diagnosis of illness. The invention also relates to a method of identifying histone variant biomarkers for the detection and diagnosis of disease and to the biomarkers identified by said method.
公开号:BR112014004584B1
申请号:R112014004584-4
申请日:2012-08-31
公开日:2021-07-06
发明作者:Jacob Vicent Micallef
申请人:Singapore Volition Pte Limited；
IPC主号:

专利说明:

FIELD OF THE INVENTION
[001] The invention relates to a method for detecting and measuring the presence of mono-nucleosomes and oligo-nucleosomes and nucleosomes that contain particular histone variants and the use of such measurements for the detection and diagnosis of the disease. The invention also relates to a method of identifying histone variant biomarkers for the detection and diagnosis of disease and to the biomarkers identified by said method. FUNDAMENTALS OF THE INVENTION
[002]The human body comprises several hundred types of cells. All of these cell types contain the same genome, but they have very different phenotypes and different functions in the body. This phenotypic diversity is due to differential expression of the genome in different cell types. Differential gene expression control is not fully understood, but basic mechanisms include gene regulation by various interconnected epigenetic signals associated with the gene, including control of chromatin packaging such as euchromatin or heterochromatin, control of nucleosome positioning and accessible nuclease sites , DNA methylation and variation in the structure of the nucleosomes around which the DNA is wrapped.
[003] The nucleosome is the basic unit of chromatin structure and consists of a protein complex of eight highly conserved nuclear histones (comprising one pair each of histones H2A, H2B, H3 and H4). Around this complex are wrapped approximately 146 base pairs of DNA. Another histone, H1 or H5, acts as a linker and is involved in chromatin compaction. DNA wraps around consecutive nucleosomes in a structure, often said to resemble "beads on a necklace" and this forms the basic opening or euchromatin structure. In compacted or heterochromatin, this necklace is rolled and super-coiled into a closed and complex structure (Herranz and Esteller, 2007).
[004] The structure of nucleosomes can vary by Post-Transcriptional Modification (PTM) of histone proteins and by the inclusion of variant histone proteins. Histone protein MTPs typically occur in the histone tails of the nucleus and common modifications include acetylation, methylation or ubiquitination of lysine residues, as well as methylation of arginine residues and phosphorylation of serine residues, and many others. Histone modifications are known to be involved in epigenetic regulation of gene expression (Herranz and Esteller, 2007). The nucleosome structure can also be varied by including alternative histone isoforms or variants that are different gene or splice products and have different amino acid sequences. Histone variants can be classified into several families that are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and publicly available, for example, in the National Human Genome Research Institute NHGRI Histone Database (Marino-Ramírez, L., Levine, KM, Morales, M., Zhang, S., Moreland, RT, Baxevanis, AD, and Landsman, D. The Histone Database: An Integrated Resource for Histone and Fold-Containing Histone Proteins Database Vol, 2011. (Presented) and http://genome.nhgri .nih.gov/histones/complete.shtml), GenBank Database (NIH Genetic Sequence), EMBL Nucleotide Sequence Database, and Japan DNA Database (DDBJ).
[005] Normal cell renewal in adult humans involves the creation by cell division of a few cells daily and the death of a similar number, mainly by apoptosis. During the process of apoptosis, chromatin is broken down into mononucleosomes and oligonucleosomes that are released from cells. Under normal conditions, the level of circulating nucleosomes found in healthy individuals has been reported to be low. Elevated levels are found in individuals with a variety of conditions including many cancers, autoimmune diseases, inflammatory conditions, stroke, and myocardial infarction (Holdenreider & Stieber, 2009).
Mononucleosomes and oligonucleosomes can be detected by Enzyme Linked Immunosorbent Assay (ELISA) and several methods have been reported (Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003). These assays typically use an anti-histone antibody (e.g. anti-H2B, anti-H3 or anti-H1, H2A, H2B, H3 and H4) as the capture antibody and an anti-DNA or anti-complex antibody. H2A-H2B-DNA as detection antibody. Using these assays, field workers report that the level of nucleosomes in the serum is higher (by up to an order of magnitude) than in plasma samples taken from the same patients. This is also true for serum and plasma DNA measurements made by PCR (Holdenrieder et al, 2005). The reason for this is not known, but the authors speculate that it may be due to the additional release of DNA during the clotting process. However, we have found that results from current technique nucleosome ELISA assays do not compete with each other. Furthermore, although the most circulating DNA in serum or plasma is reported to exist as mono-nucleosomes and oligo-nucleosomes (Holdenrieder et al, 2001), measured levels of nucleosomes and DNA in serum or plasma do not compete as well. The correlation coefficient between ELISA results for circulating cell free nucleosome levels and circulating DNA levels as measured by real-time PCR (Polymerase Chain Reaction) was reported to be r = 0.531 in serum and r = 0.350 in plasma (Holdenrieder et al, 2005).
[007] Current nucleosome ELISA methods are used in cell culture, primarily as a method to detect apoptosis (Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003), and are also used for the measurement of circulating cell-free nucleosomes in serum and plasma (Holdenrieder et al, 2001). The levels of cell-free nucleosomes in serum and plasma released into the circulation by dying cells have been measured by ELISA methods in studies of several different cancers to assess their use as a potential biomarker (Holdenrieder et al, 2001). Average circulating nucleosome levels were reported to be high in most, but not all, cancers studied. The highest circulating nucleosome levels have been observed in individuals with lung cancer. The lowest levels were seen in prostate cancer, which were within the normal range for healthy individuals. However, patients with malignant tumors were reported to have serum nucleosome concentrations that varied considerably, and some patients with advanced tumor disease were found to have low circulating nucleosome levels within the range measured for healthy individuals (Holdenrieder et al, 2001 ). Because of this and the variety of non-cancerous causes of elevated nucleosome levels, circulating nucleosome levels are not used clinically as a cancer biomarker (Holdenrieder and Stieber, 2009). Surprisingly, we have shown that many individuals with cancer whose circulating nucleosome levels are low or undetectable as measured by these current-technological nucleosome ELISA methods do indeed have elevated levels of free circulating cell nucleosomes. We have designed and demonstrated new nucleosome ELISA methods that detect nucleosomes not detected by current technical ELISA methods.
[008] ELISA methods for the detection of histone MTPs are also known in the art. ELISA methods for the detection of MTPs in free histone proteins (not bound to other histones and DNA in a nucleosome complex) are used for the detection of MTPs in extracted histones, usually by acid extraction, from of cell lysates. Immunoassay for the detection of MTPs in free circulating cell nucleosomes has been reported (Bawden et al, 2005). A method for the ELISA detection of histone MTPs in purified nucleosomes directly coated on microtiter wells has recently been reported (Dai et al, 2011). In this method, nucleosomes obtained by digesting chromatin extracts from cultured cells are coated directly onto microtiter wells and reacted with anti-PTM antibodies. It will be clear to those skilled in the art that this method requires relatively pure nucleosome samples and is not suitable for the direct measurement of histone MTPs in complex biological media such as blood or serum.
[009] A modified chromatin immunoprecipitation (ChIP) method for the detection of a histone MTP (H3K9Me, monomethylated histone H3 in lysine residue K9) in cell-free nucleosomes associated with a particular DNA sequence has been reported in plasma. The level of sequence-specific histone methylation has been reported to be independent of the concentration of circulating nucleosomes (Deligezer et al, 2008).
[0010] In addition to epigenetic signaling mediated by nucleosome position and nucleosome structure (in terms of both constituent histone protein variant and PTM structures), control of gene expression in cells is also mediated by DNA nucleotide modifications including the methylation state of DNA cytosine. It has been known in the art for some time that DNA can be methylated at the 5-position of cytosine nucleotides to form 5-methylcytosine. Methylated DNA in the form of 5-methylcytosine has been reported to occur at positions in the DNA Sequence where a cytosine nucleotide occurs next to a guanine nucleotide. These positions are called “CpG” for the short form. It has been reported that more than 70% of CpG positions are methylated in vertebrates (Pennings et al, 2005). Regions of the genome that contain a high proportion of CpG sites are often termed “CpG islands”, and approximately 60% of human gene promoter sequences are associated with such CpG islands (Rodriguez- Paredes and Esteller, 2011). In active genes, these CpG islands are usually hypomethylated. Gene methylation promoter sequences are associated with stable gene inactivation. DNA methylation also commonly occurs in repetitive elements including Alu repetitive elements and long intercalated nucleotide elements (Herranz and Estellar, 2007; Allen et al, 2004).
[0011] The involvement of DNA methylation in cancer was reported as far back as 1983 (Feinberg and Vogelstein, 1983). The DNA methylation patterns observed in cancer cells differ from those in healthy cells. Repetitive elements, particularly around pericentromeric areas, have been reported to be hypomethylated in cancer relative to healthy cells, but specific gene promoters have been reported to be hypermethylated in cancer. The balance of these two effects has been reported to result in general DNA hypomethylation in cancer cells (Rodriguez-Paredes; Esteller, 2007).
[0012] Hypermethylation of certain specific genes can be used as a diagnostic biomarker for cancers. For example, a reported method for detecting Septin 9 gene hypermethylation by PCR amplification of DNA extracted from plasma has been reported to detect 72% of colon cancers with a 10% false positive rate (Grutzmann et al, 2008 ). The DNA methylation status of specific genes or locus is usually detected by selective bisulfite deamination from cytosine, but not 5-methylcytosine, to uracil, leading to a primary DNA sequence change that can be detected by sequencing or others. means (Allen et al, 2004).
[0013] General DNA hypomethylation is a peculiarity of cancer cells (Estellar 2007 and Hervouet et al, 2010). General DNA methylation can be studied in cells using immunohistochemical (IHC) techniques. Alternatively, DNA is extracted from cells for analysis. Several methods have been reported for the detection of general methylation in DNA extracted from cells including restriction digestion and nearest neighbor analysis, fluorescent assays using chloracetaldehyde, inverse determination by methylation of all CpG sites using DNA methyltransferase in conjunction with S -tritium-labeled adenosyl methionine to calculate the amount of unmethylated CpG and single nucleotide DNA digestion for analysis by high-performance liquid chromatography, thin layer chromatography, or liquid chromatography followed by mass spectroscopy. The disadvantages of these methods are that they are labor intensive and/or require large amounts of good quality extracted DNA (Allen et al 2004). PCR-based methods involving bisulfite deamination overcome the need for large amounts of DNA, but must amplify specific genome regions, typically repetitive sequences, as indicative of the total genome content of 5-methylcytosine (Allen et al 2004). These methods for general DNA measurement methylation were used to study DNA extracted from a variety of cells and tissues. Some workers have studied DNA extracted from white blood cells in whole blood as this is easier to obtain in a minimally invasive way (Moore et al, 2008; Ting Hsiung et al, 2007; Mansour et al, 2010). Liquid chromatography with mass spectrometry is considered the gold standard for general DNA measurement methylation, but it is expensive, and DNA must be digested at the single nucleotide level prior to analysis (Vasser et al, 2009).
[0014] Recent methods for estimating general DNA methylation include ultra-high pressure liquid chromatography with tissue hydrolyzate extracted DNA mass spectrometry (Zhang et al, 2011) and a specific methylation digital sequencing (MSDS) method (Ogoshi et al 2011). A classic competitive immunoassay for general DNA methylation (as well as a similar assay for general 5-hydroxymethylcytosine methylation) has been described. In this method, DNA extracted from cells or tissues is added to a microtiter well coated with a 5-methylated cytidine conjugate, an anti-5-methylcytidine antibody is added and the antibody distribution that binds between the conjugate of 5 -methylcytidine coated and the methylated DNA in the extracted sample is compared to that of known standards to estimate the level of overall DNA methylation present in the sample (Cell Biolabs, 2011). In another immunoassay similar to the method DNA extracted from tissue samples or plasma or serum is coated to a microtiter well and methylated DNA is detected using an anti-5-methylcytosine antibody (Vasser, et al, 2009; Epigentek, 2009) . A disadvantage of these methods is that they require DNA extraction involving the denaturation and removal of all nucleosome and chromatin structure from the DNA. They are not suitable, for example; for direct measurement of general DNA methylation in biological fluids such as tissue lysate, blood, plasma or serum without a DNA extraction step.
The 5-hydroxymethyl modification of cytosine bases in DNA has also been reported. The role of 5-hydroxymethylation is not yet well understood, but it appears to be involved in gene regulation (Stroud et al, 2011).
[0016] Current methods for detecting general DNA methylation involve DNA extraction or purification and are not suitable for rapid, high-throughput, low-cost, minimally invasive diagnostic methods. Similarly, DNA analysis for other modified or unusual bases (eg, uracil, inosine, xanthine and hypoxanthine) can only be investigated by analyzing substantially pure or extracted DNA. Such analysis cannot be performed directly in complex biological media such as tissue lysate, blood, plasma or serum.
[0017] Histone variants (also known as histone isoforms) are also known to be epigenetic regulators of gene expression (Herranz and Esteller, 2007). Histone variants have been studied in vivo and in vitro using a variety of techniques including knock-down studies of the gene encoding a particular variant (eg using RNAi knock-down), chromatin immunoprecipitation, stable isotope labeling amino acid and proteomics from quantitative mass spectrometry, immunohistochemistry, and Western Blotting (Whittle et al, 2008; Boulard et al, 2010; Sporn et al, 2009; Kapoor et al, 2010; Zee et al, 2010; Hua et al, 2008).
[0018] Immunohistochemistry studies of histone variant expression in tissue samples removed surgically or by biopsy from individuals diagnosed with lung cancer, breast cancer, and melanoma have been reported. These immunohistochemical studies report that staining of histone macroH2A (mH2A) and H2AZ variants in resected cancer tissue samples may have prognostic application in these cancers (Sporn et al, 2009, Hua et al, 2008, Kapoor et al, 2010 ). A disadvantage of immunohistochemical methods for clinical use is that tissue sample collection is invasive involving surgery or biopsy. Another disadvantage of immunohistochemical methods is that they are inadequate for early diagnosis or screening diagnoses as a reasonable expectation of the disease must usually already exist before a biopsy or tissue resection is performed. Minimally invasive blood ELISA tests are suitable for a wider range of applications and would overcome these disadvantages and would be preferable to the patient, as well as faster, less costly and higher throughput for the healthcare provider.
[0019] However, cell-free nucleosomes containing particular nucleotides, modified nucleotides or histone variants have not been measured in blood or any other medium and no such measurements have been suggested or considered. No studies on the presence or absence of nucleotides, modified nucleotides or histone variants in blood cell-free nucleosomes have been reported, nor whether they have value as blood biomarkers of disease. There are currently no methods for detecting or measuring nucleotides, modified nucleotides or histone variants in intact cell-free nucleosomes. We will now report the methods for such tests and their use in plasma and serum samples taken from healthy and sick individuals. Surprisingly, we have shown that high levels of intact nucleosomes comprising specific histone variants can be detected in plasma and serum samples so that no nucleosomes, or low levels, are detected by current art nucleosome ELISA methods. SUMMARY OF THE INVENTION
[0020] According to a first aspect of the invention there is provided a cell-free nucleosome comprising a histone variant or histone isoform for use as a biomarker for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis , chronic obstructive pulmonary disorder, Crohn's disease, and rheumatoid arthritis.
[0021] According to a second aspect of the invention there is provided a method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample comprising the steps of: contacting the sample with a binding agent which binds to histone variant or histone isoform; detecting or quantifying the binding of said binding agent to the histone variant or histone isoform in the sample; and using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.
[0022] According to a third aspect of the invention there is provided a method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample comprising the steps of: contacting the sample with a first binding agent that binds to nucleosomes; contacting the nucleosomes or sample with a second binding agent that binds to the histone variant or histone isoform; detecting or quantifying the binding of said second binding agent to the histone variant or histone isoform in the sample; and using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.
[0023] According to a fourth aspect of the invention there is provided a method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a sample comprising the steps of: contacting the sample with a first binding agent that binds to histone variant or histone isoform; contacting the nucleosomes or sample with a second binding agent that binds to the nucleosomes; detecting or quantifying the binding of said second binding agent to nucleosomes in the sample; and using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.
[0024] According to another aspect of the invention there is provided a method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a blood, serum or plasma sample comprising the steps of: removing, releasing or extracting the histone variant or isoform from the nucleosome complex to produce a portion of free histone variant or isoform detecting or quantifying the free histone variant or isoform in the sample; and using the presence or amount of free histone variant or isoform as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the sample.
[0025] According to another aspect of the invention there is provided a method for detecting the presence of a nucleosome containing a histone variant or histone isoform in a cell comprising the steps of: isolating chromatin from a cell; digest, sonicate or otherwise break down chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and detecting or measuring the presence of the histone variant or histone isoform in said nucleosomes according to a method of the invention.
[0026] According to another aspect of the invention there is provided a method for detecting or diagnosing a disease state in an animal or a human subject comprising the steps of: detecting or measuring nucleosomes containing a histone variant or histone isoform in an individual's fluid body; and using the level of histone variant or nucleosome-associated histone isoform detected to identify the individual's disease state.
[0027] According to another aspect of the invention there is provided a method for evaluating an animal or human subject of suitability for medical treatment comprising the steps of: detecting or measuring nucleosomes containing a histone variant or histone isoform in a fluid body of the individual; and using the level of histone variant or nucleosome-associated histone isoform detected as a parameter for selecting an appropriate treatment for the individual.
[0028] According to another aspect of the invention there is provided a method for monitoring a treatment of an animal or a human subject comprising the steps of: detecting or measuring nucleosomes containing a histone variant or histone isoform in a body fluid of the individual; repeat detection or measurement of nucleosomes containing a histone variant or histone isoform in a subject's body fluid on one or more occasions; and using any changes in the level of histone variant or nucleosome-associated histone isoform detected as a parameter for any changes in the individual's condition.
[0029] According to another aspect of the invention there is provided a method for identifying a histone variant or histone isoform biomarker for detecting or diagnosing a disease state in an animal or a human subject comprising the steps of: detecting or measuring nucleosomes containing the histone variant or histone isoform in an individual's body fluid; detecting or measuring nucleosomes containing the histone variant or histone isoform in a body fluid of a healthy subject or a control subject; and using the difference between levels detected in diseased and control subjects to identify whether a histone variant or histone isoform is useful as a biomarker for disease state.
[0030] According to another aspect of the invention there is provided a biomarker identified by said method of the invention.
According to another aspect of the invention there is provided a kit for detecting a histone variant or histone isoform associated with the nucleosome comprising a specific ligand or ligand for the histone variant or histone isoform or its component, or a structural mimicry/shape of the histone variant or histone isoform or its component, along with instructions for using the kit. BRIEF DESCRIPTION OF THE FIGURES
[0032] Figure 1. ELISA dose-response curve for the detection of human cell-free nucleosomes prepared by a published method (*Holdenrieder et al, 2001) containing histone variant macroH2A1.1 diluted in serum of veal.
Figure 2. ELISA dose-response curve for detection of histone variant macroH2A2 in cell-free nucleosomes in crosslink-digested chromatin extracted from MCF7 cells diluted in calf serum.
[0034] Figure 3. ELISA dose-response curve for the detection of histone H2AZ variant in cell-free nucleosomes in crosslink-digested chromatin extracted from MCF7 cells diluted in calf serum.
Figure 4. Nucleosome levels detected for EDTA serum and plasma samples taken from 20 healthy volunteers using current technique nucleosome ELISA methods.
Figure 5. Nucleosome-associated levels of histone variant mH2A1.1 cells detected for EDTA serum and plasma samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 6. Nucleosome-associated levels of histone variant mH2A2 cells detected for EDTA serum and plasma samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 7. Nucleosome-associated levels of histone H2AZ variant cells detected for EDTA serum and plasma samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 8. Cell-free nucleosome-associated levels of PH2AX(Ser139) detected for EDTA serum and plasma samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 9. Cell-free nucleosome-associated levels of 5-methylcytosine methylated DNA detected for EDTA serum and plasma samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 10. Cell-free nucleosome-associated levels of methylated 5-hydroxymethylcytosine DNA detected for serum samples taken from 20 healthy volunteers using the ELISA of the invention.
Figure 11. Cell-free nucleosome-associated levels of histone and nucleotide types detected for EDTA plasma samples taken from 3 individuals with colon cancer detected using the ELISA methods of the invention.
[0043] Figure 12. Cell-free nucleosome-associated levels of the histone and nucleotide types detected for the EDTA plasma samples taken from 13 individuals with lung cancer detected using the ELISA methods of the invention.
[0044] Figure 13. Cell-free nucleosome-associated levels of histone and nucleotide types detected for EDTA plasma samples taken from 2 individuals with pancreatic cancer detected using the ELISA methods of the invention.
Figure 14. Cell-free nucleosome-associated levels of histone and nucleotide types detected for an EDTA plasma sample taken from 1 individual with oral cancer detected using the ELISA methods of the invention.
[0046] Figure 15. Cell-free nucleosome-associated levels of the histone and nucleotide types detected for EDTA plasma samples taken from 4 different cancer diseases normalized as a proportion of nucleosome-associated 5-methylcytosine methylated DNA levels detected using the ELISA methods of the invention. Normalized levels for a sample containing nucleosomes from healthy volunteers produced by the method of *Holdenrieder et al 2001 are shown for comparison.
Figure 16. Nucleosome-associated histone H2AZ levels of cells from human EDTA plasma samples taken from cancer patients measured using a biotinylated anti-H2AZ detection antibody with two different monoclonal anti-histone capture antibodies .
[0048] Figure 17. Cell-free nucleosome-associated levels of mH2A1.1, H2AZ, P-H2AX(Ser139) and 5-methylcytosine (5mc) detected in EDTA plasma, citrate plasma, and heparin plasma taken from healthy volunteers using the ELISA method of the invention.
Figure 18. Cell-free nucleosome-associated 5-methylcytosine levels detected for serum samples taken from 3 healthy volunteers and 10 individuals with colon cancer detected using the ELISA method of the invention.
Figure 19. Cell-free nucleosome-associated histone variant H2AZ levels detected for EDTA plasma samples taken from 13 healthy volunteers and 55 cancer patients. Cutoff points defined as the mean value of the healthy samples plus one or two standard deviations from the mean are shown.
Figure 20. Cell-free nucleosome-associated histone variant H2AZ levels detected for EDTA plasma samples taken from 10 healthy volunteers and 61 cancer patients. The cutoff point defined as the mean value of the healthy samples plus two standard deviations of the mean is shown.
Figure 21. Cell-free nucleosome-associated histone variant H2AZ levels detected for serum samples taken from 4 healthy volunteers and 20 patients with pancreatic cancer. The cutoff shown is defined as the mean value of the healthy samples plus two standard deviations of the mean.
Figure 22. Cell-free nucleosome-associated histone variant H2AZ levels detected for EDTA plasma samples taken from lung cancer patients with increased tumor size, stage, and nodal development of disease.
[0054]N1 Metastasis in ipsilateral peribronchial and/or ipsilateral hilar lymph nodes and intrapulmonary nodules, including involvement by direct extension
[0055]N2 Metastasis in ipsilateral and/or subcarinal mediastinal lymph node(s)
[0056]N3 Metastasis in contralateral mediastinal, contralateral hilar, ipsilateral or contralateral scalene, or supraclavicular lymph node(s)
[0057]T1: tumor < 3 cm
[0058]T2: 3 cm < tumor < 7 cm
[0059]T3: tumor > 7 cm
[0060]T4: tumor of any size that invades another organ, tissue
[0061]M0: no spread of disease beyond regional nymph nodules
[0062]M1: disease spread in distant metastases.
[0063] Figure 23. Cell-free nucleosome-associated levels mean nucleotides and histone types detected using the ELISA methods of the invention for EDTA plasma samples taken from 10 different cancer diseases normalized as a proportion of DNA levels 5-methylcytosine methylated acid (5mc) associated with the nucleosome and expressed in relation to the mean proportions found in 11 healthy individuals.
[0064] Figure 24. Nucleosome-associated levels of mean cell nucleotides and histone types detected using the ELISA methods of the invention for EDTA plasma samples taken from 2 patients with cardiomyopathy, 10 patients with systemic lupus erythematosus (lupus ), 12 patients with ulcerative colitis, 10 patients with chronic obstructive pulmonary disease (COPD), 8 patients with Crohn's disease, and 10 patients with rheumatoid arthritis (RA) normalized as a proportion of 5-methylcytosine methylated DNA levels (5mc) associated with the nucleosome and expressed in relation to the mean proportions found in 11 healthy individuals. DETAILED DESCRIPTION OF THE INVENTION
[0065] According to a first aspect of the invention there is provided a cell-free nucleosome comprising a histone variant or histone isoform for use as a biomarker for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis , chronic obstructive pulmonary disorder, Crohn's disease, and rheumatoid arthritis.
[0066] In one embodiment, the nucleosome is a mononucleosome or oligonucleosome.
[0067] According to a particular aspect of the invention which may be mentioned, the use of a histone variant or histone isoform as a biomarker for the diagnosis of cancer is provided.
[0068] In one embodiment, the cancer is a cancer of the bladder, breast, colon, cervix, esophagus, kidney, large intestine, lung, oral cavity, ovary, pancreas, prostate, rectum, skin or stomach. In a particular embodiment which may be mentioned the cancer is a cancer of the colon, lung, oral cavity or pancreas.
[0069] We developed ELISA tests for the detection and measurement of nucleosomes containing the histone variants macroH2A1.1 (mH2A1.1), macroH2A2 (mH2A2) and H2AZ. We use an anti-histone antibody as the capture antibody for these assays in combination with an appropriate anti-histone-specific variant antibody as the detection antibody. We demonstrate that these ELISA methods work with alternative anti-nucleosome capture antibodies. We also use the assays to show that nucleosomes containing specific histone variants can be measured in blood samples taken from sick individuals and are discriminating for use as non-invasive or minimally invasive biomarkers. Histone variant levels detected in nucleosomes in serum and plasma samples taken from diseased individuals, relative to levels of other nucleosome epitopes, differ from those detected in samples from healthy individuals. Furthermore, the pattern of levels of nucleosomes containing different histone variants detected in nucleosomes in different disease samples was found to differ for different diseases, particularly when the nucleosome-associated histone variant patterns were examined in combination with the patterns determined for the nucleosomes containing different nucleotides and PTMs, such that a differential diagnosis of the disease was possible. It will be clear to those skilled in the art that the inclusion of tests for nucleosomes containing histone or histone variant modifications or different or additional nucleotides would be likely to improve differential diagnosis discrimination using such standards.
[0070]At the investigated levels of nucleosomes found in healthy individuals using the methods of the current technique, we measured the nucleosomes in serum and plasma samples, taken from 20 healthy individuals. Both methods of the current technique produced higher signals in serum samples taken from healthy individuals than in plasma samples. The results are shown in Figure 4. This is consistent with published data, in which nucleosome levels are higher in serum than in plasma (*Holdenrieder et al, 2001).
[0071] At the investigated levels of nucleosomes found in healthy subjects using the methods of the invention, we measured the nucleosomes containing the three histone variants in the serum of 20 healthy subjects and in healthy bovine serum. Serum results for all three ELISA tests were all low or undetectable for all 20 healthy subjects. We also conducted a similar test on EDTA plasma samples taken from 20 healthy subjects for the three ELISA methods of the invention and surprisingly the highest signals were observed. High levels of cell-free nucleosomes containing the histone variants mH2A1.1, mH2A2, H2AZ as well as histone P-H2AX(Ser139) were detectable by methods of the present invention in healthy human EDTA plasma, lower levels were detected in serum healthy human as shown in Figures 5 to 8. Figures 9 and 10 show that similar results were obtained for other nucleosome structures. This finding is unexpected and different from both the published results (*Holdenrieder et al, 2001) and the results we found for the nucleosome ELISA methods of the current technique. Thus, surprisingly, the methods of the invention produce results opposite to methods of the current art for the relative levels of nucleosomes that occur in serum and plasma samples with EDTA.
[0072] We investigated whether nucleosome structures are detectable in all of the many common types of plasma that can be collected. We found that high levels of H2AZ, mH2A1.1 and P-H2AX(Ser139) associated with the cell-free nucleosome were detectable by the method of the invention in EDTA plasma and, to a lesser extent, in citrate plasma taken from healthy but these nucleosome-associated H2AZ, mH2A1.1 and P-H2AX(Ser139) were undetectable in background signals of buffer or horse serum in heparin plasma taken from healthy subjects. Some heparin plasma samples (2 of 5) were found to contain detectable levels of nucleosome-associated 5-methylcytosine. The results are shown in Figure 17. To summarize, cell-free nucleosomes are found at relatively high concentrations in most or all EDTA plasma and citrate plasma samples taken from healthy individuals using the method of the invention, but are low or absent in heparin plasma or serum samples taken from healthy individuals. Therefore, it is clear that the precise choice of sample type will be critical for different applications.
[0073] We demonstrate that sample selection for the detection of free nucleosomes from cells containing particular histone structures involves several parameters. These include the low levels of cell-free nucleosomes generally present in serum and heparin plasma samples taken from healthy individuals, the highest levels generally present in EDTA and citrate plasma samples taken from healthy individuals, the recommendation that serum samples containing cell-free nucleosomes should be stabilized by the addition of EDTA after separation of the serum from the clot (*Holdenreider et al, 2001), and the serum sampling protocol. Other stabilizing agents (eg, protease inhibitors) can also be used. Where possible, we used serum samples centrifuged within 1 hour of venipuncture after 10mM EDTA was added and the sample frozen.
[0074] The choice of blood sample type for clinical samples should be made on the basis of optimal clinical discrimination for the particular test. Following our finding of consistently low nucleosome levels by the method of the invention in the serum of healthy individuals, we measured nucleosomes containing the histone variants mH2A1.1 and H2AZ in serum samples taken from individuals with a variety of cancer diseases. Clinical sensitivities of up to 75% for lung cancer and 80% for pancreatic cancer (Figure 21) were observed. We also tested cancer patient serum samples for nucleosomes containing 5-methylcytosine and observed clinical sensitivities of up to 100% as shown in Figure 18 for colon cancer samples.
We also measured the relative levels of cell-free nucleosomes containing various histone variants and other nucleosome structures in EDTA plasma samples taken from individuals with a variety of diseases. Cell-free nucleosome levels are high in EDTA plasma samples taken from both healthy and diseased individuals and EDTA plasma samples would therefore likely be the best sample choice for a sensitive discriminator of sick individuals and healthy. However, we have demonstrated that the levels and composition of free circulating cell nucleosomes, in terms of the relative levels of nucleosomes containing different histone variants (as well as other nucleosome structures), vary between sick and healthy individuals and also between different diseases. . We are thus the first to report that both (i) high levels of circulating nucleosomes are present in all or most EDTA plasma samples taken from both healthy and sick individuals, but this is not true for everyone the types of blood samples; and also that (ii) surprisingly, disease detection and disease type discrimination can, however, be done by analyzing these plasma nucleosomes with EDTA on the basis of the levels and structural profile of one or more of the relative types of nucleosome structures present in the plasma of sick and healthy individuals.
We measured EDTA plasma cell-free nucleosomes taken from healthy subjects and 117 subjects with a variety of cancers in two experiments consisting of 55 and 62 subjects with cancer, respectively. In total, 90% (105 of 117) of cancer samples were correctly identified as cancer positive using the method of the invention for the nucleosome-associated histone variant H2AZ using a cut-off level of the mean score for healthy subjects + 2 standard deviations of the average.
[0077] In the first of these 2 experiments, we measured EDTA plasma cell-free nucleosomes taken from 13 healthy subjects and 55 subjects with cancer of the stomach, large intestine, rectum, lung (small cell carcinoma and various non-cell carcinomas). small), breast, ovary, prostate, kidney and various oral cancers (oral cavity, palate, pharynx and larynx). All samples from healthy individuals and cancer patients were positive for cell-free nucleosomes. However, the levels detected in samples taken from individuals with cancer were higher than those found in samples from healthy individuals and the results showed that healthy individuals and those with cancer can be discriminated against. For example, the normal range calculated in terms of OD as the mean of ± 2 standard deviations from the mean for the nucleosome H2AZ assay was 0 to 0.95. Using this cutoff level of 0.95; all 13 healthy subjects were negative for elevated nucleosome H2AZ levels. In contrast, a positive result for elevated nucleosome H2AZ levels was found for 46 out of 55 cancer samples (an overall clinical sensitivity of 84%) including the cancer samples being 100% (8 of 8) from 100% stomach (5 of 5) large intestine, 67% (2 of 3) rectum, 83% (5 of 6) small cell lung, 79% non-small cell lung, 50% (3 of 6) of breast, 100% (1 of 1) ovary, 83% (5 of 6) prostate, 100% (1 of 1) kidney and 100% (5 of 5) mouth. The results are shown in Figure 19.
[0078] In an embodiment of the invention, a control sample is provided and the cut-off level for the assay to distinguish between positive or negative results is defined in relation to the result for the control sample. This can be any proportion equal to above or below the level of the control sample result. Patient results below this level are considered negative and patient results above this level are considered positive. There may also be a “grey area” range of patient results too close to the cutoff level for the decision to be considered indeterminate and/or the test must be repeated.
[0079] Similarly, for the nucleosome-associated mH2A1.1 assay the normal range was 0 to 0.91. Using this cut-off value, all 13 healthy specimens were negative and 64% (35 of 55) of the cancer specimens were positive. For the nucleosome-associated P-H2AX(Ser139) assay, the normal range was 0 to 1.08. Using this cut-off value, all 13 healthy specimens were negative and 60% (33 of 55) of the cancer specimens were positive. Nucleosome-associated 5-methylcytosine was also measured and the normal range was 0 to 1.41. Using this cut-off value all 13 healthy specimens were negative and 55% (30 of 55) of the cancer specimens were positive. Thus, some nucleosome assays exhibit better clinical sensitivity than others.
[0080] In addition, it is possible to use the pattern of nucleosome structures to improve the clinical utility of the invention. This can be done, for example, by lowering the nucleosome-associated H2AZ assay cut-off by the mean of + 1 standard deviation which gives a range of up to 0.69. In this case, the number of false negatives is reduced to 3 providing an improved clinical sensitivity of 95% (52 of 55) at the expense of an increase in false positive results for samples taken from healthy individuals from 0% to 23% (3 of 13). The results are shown in Figure 19.
[0081] Samples found positive for H2AZ-associated nucleosomes, or any nucleosomes, can be interrogated for the profile of the nucleosome structure. The nucleosome profile can be used to distinguish between healthy and sick patients as illustrated in Figures 23 and 24 where the relative proportions of various nucleosome structures in sick patients are expressed relative to those found in healthy patients and patients with other non-healthy diseases. cancerous. This shows that investigation of multiple nucleosome structures in a test panel can facilitate better clinical discrimination.
[0082] Similarly, the diagnostic specificity and/or sensitivity of the invention can be increased by combining data from more than one test in the form of ratios. For example, by using the ratio of H2AZ:mH2A1.1 associated with the nucleosome.
[0083] Thus, the methods of the invention are capable of detecting cancer in both plasma and serum samples taken from cancer patients.
[0084] We measured the levels of free circulating cell nucleosomes containing three different histone variants in EDTA plasma samples taken from 3 colon cancer patients, 13 lung cancer patients, 2 pancreatic cancer patients, and 1 cancer patient mouth and compared these with levels present in blood samples from 20 healthy individuals, as well as with an artificially produced preparation of nucleosomes from healthy individuals prepared as described in the literature (*Holdenreider et al, 2001). We also express the observed levels in a normalized form as level proportions of nucleosomes containing different epitopes and show that such proportions or patterns of proportions are useful for the diagnosis of cancer in general and for the differential diagnosis of specific types of cancer. We also investigated whether the level of nucleosome-associated histone H2AZ varies with disease progression. We observe that the mean level of free nucleosomes of cells containing histone variants increases with disease severity and rises with increasing disease spread to lymph nodes and with increased tumor size and stage. This provides evidence that the detected nucleosomes are tumor associated.
[0085] We also measured the nucleosomes present in these 19 cancer samples using two nucleosome ELISA methods of the current technique. Of the 19 subjects with most cancers studied were found to have low EDTA plasma nucleosome levels as determined by current technique nucleosome ELISA 1 and 2 . This result illustrates one reason why current technique assays are not used for routine clinical purposes.
[0086] We used the ELISA methods of the present invention to measure the nucleosomes containing the histone variants mH2A1.1, mH2A2 and H2AZ in the same 19 samples. Nucleosomes containing histone variants, particularly mH2A2 and H2AZ, were detectable in 16 of 19 samples. Thus, in one embodiment, the invention provides a novel nucleosome ELISA method capable of detecting nucleosomes not detected by current art nucleosome assays.
[0087] We also measured the levels of nucleosomes containing 2 different nucleotides and a histone MTP in the same 19 samples taken from individuals with cancer, as well as a sample of nucleosomes generated from healthy individuals by a method described in the literature (*Holdenrieder et al, 2001). We use these measurements in conjunction with the nucleosome-associated histone variant measurements described here, as a panel of the variety of cell-free nucleosomes present in biological fluids taken from individuals with 4 different types of cancers and with nucleosomes generated from healthy individuals. Surprisingly, the nucleosome pattern found in the 4 types of cancer investigated (lung, colon, pancreatic and mouth) were all distinguishable from those found in a nucleosome sample generated from healthy individuals. Furthermore, the different types of cancer were also distinguishable from each other based on the pattern of cell-free nucleosomes detectable in the blood of individuals. Thus, in one embodiment of the invention, a method is provided for detecting or diagnosing the presence, type, occurrence or severity of a disease or evaluating the ideal drug or other treatment options by testing a sample for a panel of different nucleosome epitopes which consists of two or more measurements of nucleosomes containing different histone variants or a combination of one or more histone DNA variants and one or more histone DNA bases and/or one or more histone modifications and/or measurements of nucleosomes per se, or any combination or reason of any of these, as an indicator of an individual's health or disease state.
[0088] We similarly used the ELISA methods of the invention to detect variability in the histone and nucleotide structures of circulating cell-free nucleosomes in a variety of cancer and non-cancerous diseases and compared this to the nucleosome structure found in 11 healthy individuals . Nucleosomes were found to be present in all cancers and non-cancerous diseases investigated and were found to have profiles that differ from those of healthy individuals.
[0089] We studied EDTA plasma samples taken from 2 patients with cardiomyopathy, 10 patients with systemic lupus erythematosus (lupus), 12 patients with ulcerative colitis, 10 patients with chronic obstructive pulmonary disease (COPD), 8 patients with Crohn's disease and 10 patients with rheumatoid arthritis (RA) and we normalized the levels of various structures of the nucleosome as a proportion of the levels of 5-methylcytosine associated with the nucleosome and expressed these in relation to the levels found in healthy individuals. We found that the diseases were associated with nucleosome structure profiles that differed from those of healthy individuals or those with cancer. Thus, nucleosome structure profiles can be used as a diagnostic tool for detecting, predicting prognosis, monitoring and predicting therapeutic efficacy in a wide variety of non-cancer diseases. The results are shown in Figure 24.
[0090] We also studied the variability in structure of cell-free nucleosomes in terms of the types of histones and nucleotides detected using the ELISA methods of the invention for EDTA plasma samples taken from 55 patients with 10 different cancer diseases normalized as a proportion of methylated DNA levels of 5-methylcytosine (5mc) associated with the nucleosome and expressed in relation to the mean proportions found in 11 healthy individuals. We found nucleosomes present in all individuals and nucleosome structure profiles that varied between cancerous, non-cancerous and healthy individuals. Thus, nucleosome structure profiles can be used as a diagnostic tool for detection, prognosis prediction, monitoring and prediction of therapeutic efficacy in cancer and other diseases. The results are shown in Figures 23 and 24.
[0091] Multiple isoforms or variants have been reported for the histones H2A, H2B and H3. Histone H4, on the other hand, is reported to exist as a unique form (Tachiwana et al, 2011). It will be clear to those skilled in the art that an ELISA method of the invention utilizing an antibody or ligand directed to bind to histone H4 will bind to virtually all nucleosomes in a sample. Thus, in one embodiment, the invention provides a new method for detecting nucleosomes per se, wherein nucleosomes containing a common histone variant are measured as a way to ensure that all or most of the nucleotides are detected. . It will further be clear to those skilled in the art that suitable antibodies or ligands produced for this application can be targeted to regions of histone H4 that are not subject to PTM modification. This will further increase the universality of the selected epitope as an epitope common to all or most nucleosomes. Similarly, it will be clear to those skilled in the art that similar suitable antibodies can be directed to bind the regions of other selected histone moieties, such that the regions are common for all or most histone variants or isoforms of said histone moieties and that are not subject to PTM (eg, without limitation; common histone regions H2A, H2B, or H3). Thus, the present invention described provides a means to detect all or most nucleosomes in a sample despite variation in constituent histone isoforms and PTMs.
[0092] We conclude that the method of the present invention is a successful method for the detection and measurement of nucleosomes containing specific histone isoforms or variants, in which this method can also be used as a method for the detection of nucleosomes per se and that is a superior method for the detection of nucleosomes per se than the methods in the current art. The methods of the invention thus used have advantages over methods for measuring nucleosomes in the current art. It will be clear to those skilled in the art that the methods of the invention can be used to detect and measure nucleosomes directly in any samples where they occur, for example, in samples obtained by digestion of chromatin extracted from cells or in biological fluids such as such as blood, serum or plasma samples. It will also be clear that the methods described herein can be developed for any histone variant or histone variant modified so that an antibody or other ligand can be produced.
[0093] The invention has been tested on many cancerous and non-cancerous diseases and has been found to be effective in detecting all tested diseases. This includes the detection of prostate cancer, in cases that have been reported to be undetectable by current-tech nucleosome tests (Holdenrieder, 2001). It is clear that the invention is effective in detecting all or most cancers. It will be clear to those skilled in the art that the clinical performance of the invention can be further improved by including other nucleosome structure tests and by examining the proportions of different nucleosome structures present.
[0094] According to one aspect of the invention there is provided a method for detecting and measuring cell-free nucleosomes containing specific histone variants or isoforms in a sample by an immunoassay comprising the steps of: contacting the sample with an antibody or other ligand that binds to a histone variant; detecting and/or quantifying the binding of said antibody or other ligand to the histone variant species in the sample; and using the presence or degree of such binding as a measure of the presence of a histone variant-associated nucleosome in the sample.
[0095] According to another aspect of the invention there is provided a dual antibody, immunometric or sandwich immunoassay method for detecting and measuring the free nucleosomes of cells containing specific histone variants or isoforms in a sample. One embodiment of this aspect is an immunoassay which comprises the steps of: contacting the sample which may contain nucleosomes with a first antibody or other ligand that binds to the nucleosomes; contacting the nucleosomes or sample with a second antibody or other ligand that binds to a histone variant; detecting and/or quantifying the binding of said second antibody or other ligand to a histone variant species in the sample; and using the presence or degree of such binding as a measure of the presence of a nucleosome-associated histone variant in the sample.
[0096] According to another embodiment a method is provided for detecting and measuring cell-free nucleosomes containing specific histone variants or isoforms in a sample by an immunoassay comprising the steps of: contacting the sample which may contain nucleosomes with a first antibody or other ligand that binds to a histone variant; contacting the nucleosomes or sample with a second antibody or other ligand that binds to the nucleosomes; detecting and/or quantifying the binding of said second antibody or other ligand to nucleosomes in the sample; and using the presence or degree of such binding as a measure of the presence of a nucleosome-associated histone variant in the sample.
A variety of antibodies or other ligands can be used in the invention as a ligand that binds to nucleosomes. These include ligands aimed at binding to epitopes that occur on intact nucleosomes and not on free histones (eg; an epitope found at the junction between two histones in a nucleosome) and also ligands aimed at any component of the nucleosome including protein, histone or common nucleosome nucleic acid epitopes. We conducted samples with the method of the invention using two different capture antibodies and showed that the particular capture antibody used does not materially affect the results of the method of the invention. The results are shown in Figure 16.
[0098] It will be clear to those skilled in the art that the methods of the invention described include a variety of embodiments including classic competitive immunoassays, as well as biosensor type assays and label-free assays of the commercial type, e.g., by ForteBio Incorporated from the USA which may be immunometric in nature.
[0099] According to an embodiment of the invention, a method is provided for detecting and measuring an isoform or histone variant, or a nucleosome-associated histone isoform or variant, in a sample by a label-free immunometric immunoassay which comprises the steps of: contacting the sample with an antibody or other ligand that binds to a histone isoform or variant; detecting and/or quantifying the binding of said antibody or other ligand to a histone isoform or variant in the sample; and using the presence or degree of such binding as a measure of the presence of an isoform or histone variant or a nucleosome-associated histone isoform or variant in the sample.
[00100] According to another embodiment of the invention there is provided a method for detecting and measuring a cell-free histone isoform or variant, or a nucleosome-associated histone isoform or variant, in a sample by a competitive immunoassay that comprises the steps of: contacting the sample with an antibody or other ligand that binds to a histone isoform or variant; detecting and/or quantifying the binding of said antibody or other ligand to a histone isoform or variant in the sample; and using the presence or degree of such binding as a measure of the presence of a histone isoform or variant in the sample.
[00101] According to another aspect of the invention there is provided a method for detecting the proportion of nucleosomes comprising a histone isoform in a sample comprising the steps of: detecting or measuring the level of nucleosomes in a sample; detecting or measuring the level of a nucleosome-associated histone isoform in accordance with a method of the invention; and use the two measurements to determine the proportion of nucleosomes that contain the histone isoform.
[00102] According to an embodiment of this aspect of the invention; both the level of total nucleosome in the sample and the level of histone variant associated with the nucleosome of interest are measured using the method of the invention. In another embodiment, current art nucleosome ELISA methods are used to determine total nucleosome levels. In yet another embodiment a measure of total DNA is used as a surrogate for the total nucleosome level.
[00103] We demonstrate that the detection and measurement of nucleosomes containing histone variants in blood taken from individuals can be used as a diagnostic method to identify individuals with cancer and to differentiate them from healthy individuals. Furthermore, we demonstrate that nucleosome patterns containing a panel of different histone variants, histone nucleotides and MTPs can be used to distinguish between different cancers. It will be clear to those skilled in the art that this provides the basis for a cancer blood test that will detect cancer in individuals and can be used to distinguish between cancer types in cancer positive individuals. According to another aspect of the invention there is provided a method for detecting or diagnosing the presence of a disease by measuring or detecting the presence and/or level or concentration of free nucleosomes of cells containing a histone variant in a body fluid, and using the detected level as a biomarker of an individual's disease state including, without limitation, a clinical diagnosis of a disease, a differential diagnosis of disease type or subtype, or a disease prognosis, or a disease relapse, or a diagnosis of the individual's susceptibility to treatment regimens. It will be appreciated by those skilled in the art that the body fluids used for diagnostic testing include without limitation blood, serum, plasma, urine, cerebrospinal fluid and other fluids. In a preferred embodiment, the body fluid selected as the sample is blood, serum or plasma. The assay response level, concentration, or amount of a nucleosome-associated histone variant in a body fluid can be expressed in absolute terms or relative terms, for example, without limitation, as a proportion of the total nucleosome or total DNA level. present or as a proportion at the level of nucleosomes containing another histone or nucleotide variant or PTM.
[00104] According to another aspect of the invention there is provided a method for detecting or measuring the presence and/or level of nucleosomes containing a particular histone variant in a cell comprising the steps of: isolating chromatin from a cell; breaking down chromatin to form mono-nucleosomes and/or oligo-nucleosomes; and detecting or measuring the presence of a histone variant in the mono- and/or oligo-nucleosomes by means of an immunoassay method as described herein.
[00105] Methods to produce chromatin mono-nucleosomes and/or oligo-nucleosomes are well known in the art and include enzymatic digestion and sonication (Dai et al, 2011). We produce cell-free nucleosomes from MCF7 cells using standard procedures and use the method of the invention to show that these MCF7 nucleosomes include nucleosomes containing the histone variants mH2A1.1, H2AZ as well as P-H2AX(Ser139) .
[00106] In one embodiment, the histone variant selected for detection by the method is a commonly occurring isoform occurring in all or most intact nucleosomes, providing a method for detecting or measuring nucleosomes per se. In another embodiment, the epitope on a histone isoform selected for detection by the method is located in a region of the histone isoform that is common to, and occurs in, all or most of said histone isoforms and consequently in all or most of the nucleosomes are intact and not yet subject to MTP, providing a method for detecting or measuring nucleosomes per se.
[00107] It will be appreciated by those skilled in the art that the described method of detecting nucleosome-associated histone variants in cells or tissues is simpler, faster, cheaper, more quantitative and/or more reproducible than currently used methods including IHC , Western Blotting or FACS. The level, concentration, or amount of a particular nucleosome-associated histone variant can be expressed in absolute terms or relative terms, for example, as a proportion of the total nucleosomes or total DNA present or as a proportion to the level of nucleosomes containing a another histone variant or PTM or nucleotide.
[00108] It will be clear to those of skill in the art that the terms antibody, ligand or ligand in relation to any aspect of the invention are not limiting, but are intended to include any ligand capable of binding to specific molecules or entities and that any ligand suitable can be used in the method of the invention. It will also be clear that the term nucleosomes is intended to include mononucleosomes and oligonucleosomes and any fragments of chromatin that can be analyzed in fluid media.
[00109] According to another aspect of the invention there is provided a kit for detecting or measuring nucleosomes comprising a specific ligand or ligand for the histone variant or its component, or a structural mimicry/shape of the nucleosome or its component, together with instructions for using the kit according to any of the methods defined herein.
[00110] According to another aspect of the invention there is provided a kit for detecting or measuring nucleosomes containing a particular histone variant which comprises a specific ligand or ligand for the histone variant or component thereof, or a structural mimicry/shape of the nucleosome or its component, along with instructions for using the kit according to any of the methods defined herein.
[00111] According to another aspect of the invention there is provided a method for identifying a histone variant biomarker for detecting or diagnosing disease state in animals or humans comprising the steps of: detecting or measuring the level of free nucleosomes of cells containing a histone variant in a body fluid from sick individuals; detecting or measuring the level of free nucleosomes of cells containing a histone variant in a body fluid of control subjects; and using the difference between levels detected in diseased and control subjects to identify whether a particular histone variant is useful as a biomarker for this disease.
[00112] It will be clear to those skilled in the art that control individuals may be selected on a variety of bases which may include, for example, individuals known to be disease free or may be individuals with a different disease (eg; for investigation of differential diagnosis).
[00113] According to another aspect of the invention there is provided a method for identifying a histone variant biomarker for assessing the prognosis of a patient animal or human subject comprising the steps of: detecting or measuring the level of cell-free nucleosomes containing a histone variant in a body fluid from sick individuals; and to correlate the level of free nucleosomes of cells containing a histone variant detected in a body fluid of sick individuals with the outcome of the individuals' disease.
[00114] According to another aspect of the invention there is provided a method for identifying a histone variant biomarker to be used for selecting a treatment regimen for a patient animal or human subject in need of treatment comprising the steps of : detecting or measuring the level of free nucleosomes of cells containing a histone variant in a body fluid of sick individuals; and correlating the level of free nucleosomes of cells containing a histone variant detected in a body fluid from diseased individuals with the observed efficacy of a treatment regimen in those individuals.
[00115] According to another aspect of the invention there is provided a method for identifying a histone variant biomarker to be used to monitor the treatment of a sick animal or human subject comprising the steps of: detecting or measuring the level of nucleosomes free from cells containing a histone variant in a patient's body fluid; repeating said detection or measurement on one or more occasions during the individual's disease progression; and correlating the level of free nucleosomes of cells containing a histone variant detected in a patient's body fluid with the progression of the disease in the individual.
[00116] According to another aspect of the invention, a biomarker identified by the method as defined herein is provided.
[00117] It will be clear to those skilled in the art that cell-free nucleosomes containing a histone variant can also be detected in a biological fluid including blood, plasma, serum and urine by a procedure involving the extraction of the histone variant protein from the nucleosome complex followed by a method for detecting or quantifying the extracted free histone variant protein. Suitable extraction procedures include commonly used acid extraction procedures for histones that utilize the basic nature of histone proteins. Detection of the free histone variant can be carried out, for example, by an immunoassay for the free histone moiety. Thus, in one embodiment of the invention, a histone variant is extracted from a biological fluid including blood, plasma, serum and urine and the extract is tested for the presence of a histone variant.
[00118] It is known in the art that one can detect the presence of a protein that is comprised as part of a complex containing other portions by immunoassay methods. It will be clear to those skilled in the art that cell-free nucleosomes containing a histone variant can be detected in a biological fluid including blood, plasma, serum and urine by a procedure that involves the direct immunoassay of the histone variant itself. fluid. In this procedure, a single antibody immunoassay, using one antibody directed to an epitope present on a histone variant, or a 2-site immunoassay, using two antibodies directed to two epitopes present on a histone variant, is used to detect the presence of a histone variant within a nucleosome. Thus, in another embodiment of the invention, a histone variant contained within a nucleosome is directly detected in a biological fluid including blood, plasma, serum and urine by using an immunoassay method for a histone variant .
[00119] Thus, in an embodiment of the invention, a histone variant is extracted from a biological fluid including blood, plasma, serum and urine and the extract is tested for the presence of a histone variant.
[00120] Another aspect of the invention provides linkers, such as naturally occurring or chemically synthesized compounds, capable of binding to the specific biomarker. A linker or ligand according to the invention may comprise a peptide, an antibody or a fragment thereof, or a synthetic linker such as a plastic antibody, or an aptamer or oligonucleotide, capable of binding to the specific biomarker. The antibody can be a monoclonal antibody or a fragment thereof capable of binding the specific biomarker. A binder according to the invention may be labeled with a detectable label, such as a luminescent, fluorescent, enzyme or radioactive label; alternatively or additionally a binder according to the invention can be tagged with an affinity tag, for example a biotin, avidin, streptavidin or His (for example hexa-His) tag. Alternatively, the binding of the ligand can be determined using a label-free type technology, for example, from ForteBio Inc.
[00121] A biosensor, according to the invention, may comprise the biomarker or a structural mimicry/shape thereof capable of binding a specific antibody against the biomarker. An order comprising a linker or mimicry as described herein is also provided.
[00122] Also provided by the invention is the use of one or more linkers as described herein, which may be naturally occurring or chemically synthesized, and is suitably a peptide, antibody or fragment thereof, aptamer or oligonucleotide, or the use of a biosensor of the invention, or an order of the invention, or a kit of the invention to detect and/or quantify the biomarker. In these uses, detection and/or quantification can be performed on a biological sample as defined herein.
[00123] Diagnostic or monitoring kits are provided to carry out the methods of the invention. Such kits, suitably, will comprise a binder according to the invention, for detection and/or quantification of the biomarker, and/or a biosensor, and/or an order as described herein, optionally, together with instructions for using the kit. .
[00124] Another aspect of the invention is a kit for detecting the presence of a disease state, comprising a biosensor capable of detecting and/or quantifying one or more of the biomarkers as defined herein.
[00125]Biomarkers to detect the presence of a disease are essential targets for the discovery of new targets and drug molecules that slow or stop the progression of the disorder. As the level of the biomarker is indicative of the disorder and drug response, the biomarker is useful for identifying new therapeutic compounds in in vitro and/or in vivo assays. The biomarkers of the invention can be used in methods for screening for compounds that modulate biomarker activity.
[00126] Thus, in another aspect of the invention, there is provided the use of a ligand or linker, as described, which may be a peptide, antibody or fragment thereof or aptamer or oligonucleotide, according to the invention; or the use of a biosensor according to the invention, or an order according to the invention; or a kit according to the invention, to identify a substance capable of promoting and/or suppressing the generation of the biomarker.
[00127] A method of identifying a substance capable of promoting or suppressing the generation of the biomarker in an individual is also provided, comprising administering a test substance to an animal subject and detecting and/or quantifying the level of the biomarker present in a sample of test from the individual.
[00128]The term “biomarker” means a biologically or biologically distinctive indicator derived from a process, event or condition. Biomarkers can be used in diagnostic methods, eg, clinical screening, and prognostic assessment, and in monitoring therapy outcomes, identifying patients most likely to respond to a particular therapeutic treatment, drug screening and development. Biomarkers and their use are of great value for identifying new drug treatments and for discovering new targets for drug treatment.
[00129] The terms "detect" and "diagnose", as used herein, encompass the identification, confirmation, and/or characterization of a disease state. The methods of detecting, monitoring and diagnosing, in accordance with the invention, are useful to confirm the existence of a disease, to monitor the development of the disease by assessing the onset and progression, or to assess the improvement or regression of the disease. Methods of detecting, monitoring and diagnosing are also useful in methods for evaluating clinical screening, prognosis, choice of therapy, assessment of therapeutic benefit, ie, for drug screening and drug development.
[00130] Efficient diagnostic and monitoring methods provide very potent "patient solutions" with the potential for improved prognosis by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus decreasing unnecessary exposure to drug side effects harmful), and reducing recidivism rates.
[00131] In one embodiment, said biomarker is released from the cells of a tumor. Thus, according to another aspect of the invention there is provided a method for detecting a tumor growth which comprises the steps of (i) measuring a biomarker in a biological sample that is associated with or released from the cells of a tumor and (ii) demonstrate that the level of said biomarker is associated with the size, stage, aggressiveness or spread of the tumor.
[00132] It is known that increased cell removal, cell death and apoptosis lead to increased circulatory levels of cell-free nucleosomes (Holdenrieder et al, 2001). The level of free circulating cell nucleosomes is a non-specific indicator and occurs in a variety of conditions including inflammatory diseases, a wide range of benign and malignant conditions, autoimmune diseases, as well as after trauma or ischemia (Holdenrieder et al. al 2001). It will be clear to those skilled in the art that the invention will find application in a variety of disease areas where circulating nucleosomes have been found in individuals. These include, without limitation, trauma (eg, severe injury or surgery), extreme exercise (eg, running a marathon), stroke and heart attack, and sepsis or other serious infection. We use the immunoassay method of the invention to measure nucleosome levels and investigate their variability of histone and nucleotide structure in a variety of diseases including cardiomyopathy, systemic lupus erythematosus, ulcerative colitis, chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis and we compared these with the results of healthy individuals. We can detect nucleosomes and determine their relative structures (in terms of histone and nucleotide composition) in all these diseases. As the methods of the current invention are capable of detecting a wider range of nucleosomes than current nucleosome ELISA methods, the methods of the invention have applications in a wide range of areas of cancerous and non-cancerous disease.
[00133] The immunoassays of the invention include immunometric assays using enzyme detection methods (eg, ELISA), fluorescently labeled immunometric assays, time-resolved fluorescence labeled immunometric assays, chemiluminescent immunometric assays, immunoturbidimetric assays, assays particulate labeled immunometrics and immunoradiometric assays and competitive immunoassay methods including competitive labeled antigen and labeled antibody immunoassay methods with a variety of label types including radioactive, enzyme, fluorescent, time-resolved fluorescent and particulate labels . All of said immunoassay methods are well known in the art, see, for example, Salgame et al, 1997 and van Nieu-wenhuijze et al, 2003.
[00134] In one embodiment, said biological sample comprises a body fluid. For example, biological samples that can be tested in a method of the invention include cerebrospinal fluid (CSF), whole blood, blood serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), respiration, for example, as condensed respiration, or an extract or purification, or dilution thereof. Biological samples also include specimens from a live individual, or taken post-mortem. Samples can be prepared, for example, where appropriate diluted or concentrated, and stored in the usual way.
[00135] In one embodiment, the method of the invention is repeated on multiple occasions. This embodiment provides the advantage of allowing detection results to be monitored over a period of time. Such a provision will provide the benefit of monitoring or evaluating the effectiveness of the treatment of a disease state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilization, improvement, relapse and/or remission.
[00136] Thus, the invention also provides a method of effectiveness of monitoring a therapy for a disease state in an individual suspected of having such a disease, comprising detecting and/or quantifying the biomarker present in a biological sample from said individual . In monitoring methods, test samples can be taken on two or more occasions. The method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more controls and/or with one or more previous test samples taken earlier from the same test subject, by for example, before starting therapy, and/or from the same test subject at an earlier stage of therapy. The method may comprise detecting a change in the nature or amount of the biomarker(s) in test samples taken on different occasions.
[00137] Thus, according to another aspect of the invention, there is provided a method for monitoring efficacy of therapy for a disease state in a human or animal subject, comprising: quantifying the amount of the biomarker as defined herein; and comparing the amount of said biomarker in a test sample with the amount present in one or more controls and/or one or more earlier test samples taken at an earlier time from the same test subject.
[00138] A change in the level of the biomarker in the test sample from the level in a previous test sample taken earlier from the same test subject may be indicative of a beneficial effect, for example stabilization or improvement, of said therapy on the disorder or suspected disorder. Furthermore, once the treatment is completed, the method of the invention can be periodically repeated in order to monitor for the occurrence of a disease.
[00139] Methods to monitor the effectiveness of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in animals (eg, in animal models). These monitoring methods can be incorporated into screens for new drug substances and substance combinations.
[00140] In another embodiment, monitoring of faster changes due to fast acting therapies can be conducted in short intervals of hours or days.
According to another aspect of the invention, a method is provided for identifying a biomarker for detecting the presence of a disease state. The term "identify", as used herein, means confirming the presence of the biomarker present in the biological sample. Quantifying the amount of biomarker present in a sample may include determining the concentration of the biomarker present in the sample. Identification and/or quantification can be performed directly on the sample, or indirectly on an extract, or on a dilution thereof.
[00142] In alternative aspects of the invention, the presence of the biomarker is assessed by detecting and/or quantifying the antibody or fragments thereof capable of binding to the specific biomarker that are generated by the individual's body in response to the biomarker and thus are present in a biological sample from an individual having a disease state.
[00143] The identification and/or quantification can be performed by any suitable method to identify the presence and/or amount of a specific protein in a biological sample of a patient or a purification or extract of a biological sample or a dilution thereof. In methods of the invention, quantification can be performed by measuring the concentration of the biomarker in the sample or samples. Biological samples that can be tested in a method of the invention include those as defined above. Samples can be prepared, for example, where appropriate diluted or concentrated, and stored in the usual way.
[00144]The identification and/or quantification of biomarkers can be performed by detecting the biomarker or a fragment thereof, for example, a fragment with C-terminal truncation, or with N-terminal truncation. Fragments are suitably greater than 4 amino acids in length, for example 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. In particular, it has been observed that peptides of the same or related sequence as histone tails are particularly useful fragments of histone proteins.
[00145]The biomarker can be directly detected, for example, by SELDI or MALDI-TOF. Alternatively, the biomarker can be detected directly or indirectly through interaction with a ligand or ligands, such as an antibody or a fragment linked to the biomarker thereof, or another peptide, or ligand, for example, aptamer, or oligonucleotide, able to specifically bind to the biomarker. The linker or ligand may have a detectable tag, such as a luminescent, fluorescent or radioactive tag, and/or an affinity tag.
[00146]For example, detection and/or quantification can be performed by one or more methods selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis , a 2-D gel-based analysis, mass spectrometry (MS), reversed-phase LC (RP), size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other based techniques of LC or LC MS. Appropriate LC MS techniques include ICAT®(Applied Biosystems, CA, USA), or iTRAQ®(Applied Biosystems, CA, USA). Liquid chromatography (eg high pressure liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR (nuclear magnetic resonance) spectroscopy may also be used.
The methods of diagnosing or monitoring, according to the invention, may comprise analyzing a sample by SELDI TOF or MALDI TOF to detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring therapy outcomes, identifying the patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identifying new targets for drug treatment.
[00148]The identification and/or quantification of analyte biomarkers can be performed using an immunological method, involving an antibody, or a fragment thereof capable of binding to the specific biomarker. Suitable immunological methods include sandwich immunoassays, such as a sandwich ELISA, in which detection of analyte biomarkers is performed using two antibodies that recognize different epitopes on an analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme-linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), fluorescence immunoassays (FIA), western blotting, immunoprecipitation-based immunoassay and any particle (for example, using gold, silver, or latex particles, magnetic particles or Q-dots). Immunological methods can be carried out, for example, in a microtiter plate or strip format.
[00149] In one embodiment, one or more of the biomarkers may be replaced by a molecule, or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.
[00150]The identification of specific biomarkers important to a disease is central to the integration of diagnostic procedures and therapeutic regimens. Using diagnostic tools appropriate to predictive biomarkers such as biosensors can be developed; therefore, in methods and uses of the invention, identification and quantification can be performed using a biosensor, microanalytical system, microengineered system, microseparation system, immunochromatography system or other suitable analytical devices. The biosensor may incorporate an immunological method for detecting the biomarker(s), electrical, thermal, magnetic, optical (eg hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker(s) at the anticipated concentrations found in biological samples.
[00151] As used herein, the term "biosensor" means something capable of detecting the presence of the biomarker. Examples of biosensors are described here.
Biosensors according to the invention may comprise a ligand ligand or ligands, as described herein, capable of binding to the specific biomarker. Such biosensors are useful in detecting and/or quantifying a biomarker of the invention.
[00153] The biomarkers of the invention can be detected using a biosensor incorporating technologies based on "intelligent" holograms, or high frequency acoustic systems, such systems are particularly responsible for "barcode" or ordering configurations.
[00154] In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored in a thin polymeric film that is sensitized to specifically react with the biomarker. On exposure, the biomarker reacts with the polymer leading to a change in the image displayed by the hologram. A reading off the test result may be a change in optical brightness, image, color and/or image position. For qualitative and semi-quantitative applications, a hologram of the sensor can be read with the naked eye, thus removing the need for detection equipment. A single color sensor can be used to read the signal when quantitative measurements are needed. The opacity or color of the sample does not interfere with sensor operation. The sensor format allows multiplexing for simultaneous detection of multiple substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a biomarker of particular interest is feasible.
[00155] Suitably, the biosensors for the detection of one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert the detection of the presence, or quantification, of the biomarker in the sample into a signal. Biosensors can be adapted for “alternative site” diagnostic testing, for example, in the ward, outpatient department, operating room, home, field and workplace.
Biosensors for detecting one or more biomarkers of the invention include acoustic, plasma resonance, holographic, Bio-Layer Interferometry (BLI) and microengineered sensors. Printed recognition elements, thin-film transistor technology, magnetic acoustic resonator devices and other new acoustic-electrical systems can be used in biosensors for detecting one or more biomarkers of the invention.
[00157] Methods involving the identification and/or quantification of one or more biomarkers of the invention can be performed on benchtop instruments, or can be incorporated into disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, for example, in the doctor's office or at the patient's bedside. Biosensors suitable for carrying out the methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow collected data to be electronically transmitted to the physician for interpretation and thus can form the basis for e-medicine.
[00158]Diagnostic kits for diagnosing and monitoring the presence of a disease state are described here. In one embodiment, the kits additionally contain a biosensor capable of identifying and/or quantifying a biomarker. Suitably, a kit according to the invention may contain one or more components selected from the group: a specific ligand or ligands for the biomarker or a structural mimicry/form of the biomarker, one or more controls, one or more reagents and one or more consumables; optionally, along with instructions for using the kit, according to any of the methods defined herein.
[00159]The identification of biomarkers for a disease state allows the integration of diagnostic procedures and therapeutic regimens. Detection of a biomarker of the invention can be used to screen individuals prior to their participation in clinical trials. Biomarkers provide the means to indicate therapeutic response, inability to respond, unfavorable side-effect profile, degree of medication adherence, and achievement of adequate serum drug levels. Biomarkers can be used to provide warning of adverse drug response. Biomarkers are useful in the development of personalized therapies, as response assessment can be used to fine-tune the dosage, minimize the number of medications prescribed, reduce the delay in achieving effective therapy, and avoid adverse drug reactions. Thus, by monitoring a biomarker of the invention, patient care can be precisely adjusted to match the needs determined by the disorder and the patient's pharmacogenomic profile, the biomarker can thus be used to titrate the optimal dose, predict a therapeutic response positive and identify patients at high risk of severe side effects.
[00160] Biomarker-based tests provide a first line of assessment of 'new' patients, and provide objective measurements for an accurate and rapid diagnosis, not feasible using current measurements.
[00161] In addition, diagnostic biomarker tests are useful to identify family members or patients with mild or asymptomatic disease or who may be at high risk of developing symptomatic disease. This allows for initiation of appropriate therapy, or preventive measures, eg managing risk factors. These approaches are recognized to improve outcome and may prevent the onset of the disorder.
[00162] Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring instruments to allow the physician to determine whether the relapse is due to worsening of the disorder. If pharmacological treatment is judged to be inadequate, then therapy can be reinstated or augmented; a change in therapy can be provided if appropriate. As biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy.
The invention will now be illustrated with reference to the following non-limiting examples. EXAMPLE 1
[00164] A human blood sample containing cell-free nucleosomes from healthy individuals prepared according to the method described by Holdenrieder (*Holdenrieder et al, 2001) was tested using an ELISA for the nucleosome-associated histone variant macroH2A1.1 using a solid phase anti-histone capture antibody that binds to intact nucleosomes and a biotinylated affinity purified polyclonal macroH2A1.1 polyclonal anti-histone variant detection antibody. The human sample was serially diluted in fetal calf serum and tested in duplicate in the undiluted ELISA and at 1:2, 1:4 and 1:8 dilutions. Pure fetal calf serum was also conducted in the ELISA as a control sample not containing cell-free nucleosomes. The assay method was as follows: A solution of anti-histone antibody in 0.1M phosphate buffer pH 7.4 was added to microtiter wells (100 µL/well) and incubated overnight at 4°C to coat the wells with capture antibody. Excess anti-histone antibody was decanted. A bovine serum albumin solution (20 g/L) was added to the wells (150 µL/well) and incubated for 60 minutes at room temperature to block excess protein binding sites in the wells. Excess bovine serum albumin solution was decanted and the wells were washed twice with wash buffer (200 µL/well, 0.05M TRIS/HCl buffer pH 7.5 containing 1% Tween 20). Sample (10 μL/well) and assay buffer (50 μL/well, 0.05M TRIS/HCl pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and Nonidet P40 substitute a 1%) were added to the wells and incubated for 90 minutes at room temperature with gentle agitation. The sample and assay buffer mixture was decanted and the wells were washed three times with wash buffer (200 µL/well). A biotinylated affinity purified polyclonal histone macroH2A1.1 anti-variant detection antibody solution was added (50 µl/well) and incubated for 90 minutes at room temperature with gentle agitation. Excess detection antibody was decanted and the wells were again washed three times with wash buffer (200 µL/well). A solution containing a streptavidin-horseradish peroxidase conjugate was added (50 µL/well) and incubated for 30 minutes at room temperature with gentle agitation. Excess conjugate was decanted and the wells were again washed three times with wash buffer (200 µL/well). A colored substrate solution (100 μL/well, 2,2’-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added and incubated for 30 minutes at room temperature with gentle agitation. A STOP solution (100 µL/well) containing 1% sodium dodecyl sulfate was added and the optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtiter plate reader. A color reproducible dose-response curve increased with increased nucleosome-associated histone variant macroH2A1.1 concentration was observed with a low background signal seen in the absence of nucleosome-associated histone variant macroH2A1.1 (fetal calf serum). The positive ELISA signal indicates that the histone macroH2A1.1 variant detected by the ELISA is incorporated within a nucleosome as the capture antibody used binds to histones within intact nucleosomes and does not bind to histone H2. The results are shown in Figure 1. EXAMPLE 2
[00165] A commercially available nucleosome preparation produced by digestion of chromatin extracted from MCF7 cells, in which the DNA and proteins in the nucleosome are cross-linked for stability (ensuring that all histones present in the preparation are incorporated into the intact nucleosomes) was evaluated using an ELISA method for nucleosome-associated histone macroH2A2 variant using a solid phase anti-histone capture antibody that binds to intact nucleosomes and a biotinylated polyclonal affinity-purified histone macroH2A2 anti-histone variant detection antibody. The nucleosome sample was serially diluted in fetal calf serum and was tested in duplicate in the ELISA. Pure fetal calf serum was also conducted in the ELISA as a control sample not containing cell-free nucleosomes. The assay method was as follows: A solution of anti-histone antibody in 0.1M phosphate buffer pH 7.4 was added to the microtiter wells (100 µL/well) and incubated overnight at 4°C to coat the wells with capture antibody. Excess anti-histone antibody was decanted. A bovine serum albumin solution (20 g/L) was added to the wells (200 µL/well) and incubated for 30 minutes at room temperature to block excess protein binding sites in the wells. Excess bovine serum albumin solution was decanted and the wells were washed three times with wash buffer (200 µL/well, 0.05M TRIS/HCl buffer pH 7.5 containing 1% Tween 20). Sample (10 μL/well) and assay buffer (50 μL/well, 0.05M TRIS/HCl pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and 1 Nonidet P40 substitute %) were added to the wells and incubated overnight at 4°C. The sample and assay buffer mixture was decanted and the wells were washed three times with wash buffer (200 µL/well). A biotinylated affinity purified polyclonal histone macroH2A1.1 anti-variant detection antibody solution was added (50 µL/well) and incubated for 90 minutes at room temperature with gentle agitation. Excess detection antibody was decanted and the wells were again washed three times with wash buffer (200 µL/well). A solution containing a streptavidin-horseradish peroxidase conjugate was added (50 μL/well) and incubated for 30 minutes at room temperature with gentle agitation. Excess conjugate was decanted and the wells were again washed three times with wash buffer (200 µL/well). A colored substrate solution (100 μL/well, 2,2’-azinobis[3-ethylbenzothiazoline-6-sulfonic acid]-diammonium salt) was added and incubated 20 minutes at room temperature with gentle agitation. The optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtiter plate reader. An increased color dose-response curve with increased nucleosome-associated histone variant macroH2A2 concentration was observed with a low background signal seen in the absence of histone macroH2A2 variant (fetal calf serum). The positive ELISA signal indicates that the histone macroH2A2 variant detected by the ELISA is incorporated within a nucleosome as (i) none of the free histones are present in the sample and (ii) the capture antibody used binds to histones within intact nucleosomes and does not bind to histone H2. The results are shown in Figure 2. EXAMPLE 3
[00166] A commercially available nucleosome preparation produced by digestion of chromatin extracted from MCF7 cells, in which DNA and proteins were cross-linked for stability (ensuring that all histones present are incorporated into intact nucleosomes) was tested using an ELISA method for nucleosome-associated histone H2AZ variant using a solid phase anti-histone capture antibody that binds to intact nucleosomes and does not bind to histone H2 and a biotinylated polyclonal affinity-purified anti-hitone variant detection antibody. Details of the test procedure are similar to those described in Example 2 above. A color reproducible dose-response curve increased with increased nucleosome-associated histone variant H2AZ concentration was observed with a low background signal seen in the absence of nucleosome-associated histone H2AZ variant (fetal calf serum). The positive ELISA signal indicates that the detected macroH2A2 histone variant is incorporated within a nucleosome as (i) none of the free histones are present in the sample and (ii) the capture antibody used binds to histones within intact nucleosomes and does not binds to histone H2. The results are shown in Figure 3. EXAMPLE 4
[00167]We used two nucleosome ELISA methods of the current technique to measure the circulating cell-free nucleosome content of blood serum and plasma samples taken from 20 healthy individuals. The first current ELISA method (ELISA 1) was the Roche Cell Death ELISA and the other (ELISA 2) an ELISA that uses an anti-histone capture antibody and anti-histone-DNA detection antibody complex. The nucleosome levels detected by both current nucleosome ELISA methods were both lower in normal plasma than in normal serum. The normal range (expressed in optical density units) for the serum level of nucleosomes was calculated (mean ± 2 standard deviations of the mean of the results from 20 healthy serum subjects) to be 0 to 4.3 OD units for ELISA 1 and 0 to 1.4 for ELISA 2. The respective ranges for plasma were 0 to 0.95 and 0 to 0.96. The results are shown in Figure 4.
[00168] We also measured the levels of nucleosomes containing a histone MTP and two nucleotides, as well as the three histone variants mH2A1.1, mH2A2 and H2AZ associated with the nucleosome, in the same samples. The results show that serum samples taken from healthy individuals have uniformly low levels of nucleosomes containing histone or PTM variants or nucleotides. The normal ranges (expressed as optical density) for the serum level of nucleosomes containing histone, PTM, or nucleotide variants were; (a) 0 to 0.36 for mH2A1.1, (b) 0.05 to 0.78 for mH2A2, (c) 0.11 to 0.58 for H2AZ, (d) 0.06 to 0.61 for P-H2AX (Ser139), (e) 0.06 to 0.36 for 5-methylcytosine and (f) 0.03 to 0.36 for 5-hydroxymethylcytosine. Plasma measured with EDTA results were highest for all 20 healthy subjects. The results are shown in Figures 5, 6, 7, 8, 9 and 10. EXAMPLE 5
[00169] We measured plasma cell-free nucleosomes with EDTA taken from 13 healthy subjects and 55 subjects with cancer of the stomach, large intestine, rectum, lung (small cell carcinoma and various non-small cell carcinomas), breast, ovary , prostate, kidney and various oral cancers (oral cavity, palate, pharynx and larynx). All samples from healthy individuals and cancer patients were positive for cell-free nucleosomes. However, the levels detected in samples taken from individuals with cancer were higher than those found in samples from healthy individuals and the results showed that healthy individuals and those with cancer can be discriminated against. For example, the normal range calculated in terms of OD as the mean of ± 2 standard deviations from the mean for the nucleosome H2AZ assay was 0 to 0.95. Using this cutoff level of 0.95; all 13 healthy subjects were negative for elevated nucleosome H2AZ levels. In contrast, a positive result for elevated nucleosome H2AZ levels was found for 46 of 55 cancer samples (an overall clinical sensitivity of 84 %) including cancer samples being 100% (8 of 8) from stomach, 100% (5 of 5) large intestine, 67% (2 of 3) rectum, 83% (5 of 6) small cell lung, 79% non-small cell lung, 50% (3 of 6) of breast, 100% (1 of 1) ovary, 100% (1 of 1) pancreas, 80% (4 of 5) prostate, 100% (1 of 1) kidney and 100% (5 of 5) mouth. The results are shown in Figure 19.
[00170]Similarly, for the nucleosome-associated mH2A1.1 assay, the normal range was 0 to 0.91. Using this cut-off value, all 13 healthy specimens were negative and 64% (35 of 55) of cancer specimens were positive. For the nucleosome-associated P-H2AX(Ser139) assay, the normal range was 0 to 1.08. Using this cut-off value, all 13 healthy specimens were negative and 60% (33 of 55) of cancer specimens were positive. Nucleosome-associated 5-methylcytosine was also measured and in the assay the normal range was 0 to 1.41. Using this cut-off value, all 13 healthy specimens were negative and 55% (30 of 55) of cancer specimens were positive.
[00171] We also use the methods of the invention to measure a variety of other nucleosome-associated structures in the same samples. The results of these immunoassays were compiled to provide a profile of nucleosome structures in samples taken from cancer patients normalized to detected levels of nucleosomes containing 5-methylcytosine. We compared the resulting profiles to the nucleosome structure of samples taken from healthy individuals. The nucleosome structure profile of cell-free nucleosomes was found to be different from that of healthy individuals. The results are shown in Figure 23. We similarly compiled nucleosome structure profiles for samples taken from a variety of non-cancerous diseases and compared this to the nucleosome profile in samples taken from cancer patients and healthy individuals. The results are shown in Figure 24.
[00172] We then performed another similar experiment including samples from 10 healthy individuals and another 62 patients with cancer of various types. The results were similar to the first experiment. For example, using the results for the nucleosome-associated histone variant H2AZ and a mean cutoff of + 2 standard deviations from the mean of the results for healthy individuals, negative results were obtained for all 10 healthy individuals and positive results were obtained for 95% (59 of 62) of cancer patients including 9 of 9 patients with prostate cancer, 5 of 5 patients with skin cancer, 6 of 8 patients with esophageal cancer, 12 of 13 patients with bladder cancer, 2 of 2 patients with cervical cancer and 1 of 1 patients with colon cancer, 4 of 4 patients with breast cancer, 7 of 7 patients with ovarian cancer, 7 of 7 patients with laryngeal cancer, 3 of 3 patients with lung cancer and 3 of 3 patients with kidney cancer. This result indicates that nucleosome-associated histone variants are clinically sensitive biomarkers for cancer. The results are shown in Figure 20. EXAMPLE 6
[00173] We used two nucleosome ELISA methods of the current technique to measure the circulating cell-free nucleosome content of EDTA plasma samples taken from 3 individuals with colon cancer, 13 individuals with lung cancer, 2 individuals with cancer pancreatic, 1 individual with oral cancer and a nucleosome sample produced from healthy individuals according to the Holdenrieder method (*Holdenrieder et al, 2001). The first current ELISA method (ELISA 1) was the Roche Cell Death ELISA and the other (ELISA 2) an ELISA using an anti-histone capture antibody and anti-histone-DNA detection antibody complex.
[00174] We also measured the levels of nucleosomes containing three histone variants, one histone MTP and two nucleotides. The results show that, although low nucleosome results for current technique ELISA methods were detected for most individuals, particularly for patients with pancreatic and oral cancer, most of these samples have higher detectable levels of nucleosomes than contain one or more nucleosome-associated histone variants. The results for samples taken from 3 individuals with colon cancer, 13 individuals with lung cancer, 2 individuals with pancreatic cancer, and 1 individual with oral cancer are shown in Figures 11, 12, 13, and 14, respectively. of significant nucleosome-associated histone were detected in 16 of 19 cancer specimens (all but 3 lung cancer specimens). In addition, nucleosome-associated 5-hydroxymethylcytosine was detected in 12 of 19 cancer samples and nucleosome-associated 5-methylcytosine was detected in all 19 cancer samples.
[00175] Furthermore, the pattern of nucleosome levels containing different histone variant levels is not uniform for all individuals, but exhibits different patterns for the different cancers tested. To facilitate comparison between results for individuals with the same or different cancers; the results for the nucleosome tests (for nucleosomes containing macroH2A1.1, macroH2A2, H2AZ, P-H2AX(Ser139), 5-methylcytosine and 5-hydroxymethylcytosine) were normalized as a ratio of the observed OD signal to nu - cleosomes containing 5-methylcytosine. The normalized results (with error bars showing the standard deviation in results where samples from more than one individual were tested) are shown for each cancer in Figure 15, as well as the same results for the nucleosome sample produced from individuals (mH2A2 and 5-hydroxymethylcytosine were not measured for this sample). Figure 15 shows that the pattern distribution of nucleosomes containing the different normalized histone variants, nucleotides or MTP in all four cancers investigated differs quite markedly from the distribution of nucleosomes in the sample from healthy individuals. Thus, the present invention can be used as a method for detecting cancer in a simple blood-based screening test. It will be clear to those skilled in the art that the invention includes testing nucleosomes containing other histone variants, nucleotide and/or histone modifications to further or better discriminate between free nucleosomes from circulating cells of tumor or other disease origin.
[00176] Furthermore, the pattern of nucleosome types observed differs from different cancer types. For example; a sample taken from an individual with oral cancer has lower normalized levels of either nucleosomes containing mH2A2 or P-H2AX(Ser139) than any of the other three types of cancer. Similarly, samples from individuals with pancreatic cancer can be distinguished from samples from individuals with colon cancer on the basis of a different normalized level of nucleosomes containing macroH2A1.1 variant. Thus, the present invention can be used as a method to diagnose cancer generally and to distinguish a particular type of cancer. It will be clear to those skilled in the art that the invention includes testing nucleosomes containing other histone variants and/or histone and/or nucleotide modifications to further, or better, discriminate between free nucleosomes of circulating cells of different or specific tumor origin another source of the disease. EXAMPLE 7
We measured nucleosome-associated histone variant levels in serum samples taken from 4 healthy subjects and 20 serum samples taken from subjects with pancreatic cancer using the method of the invention as described above. Using a cut-off level of 0.27 (mean + 2 standard deviations from levels found in healthy patients), nucleosome-associated H2AZ levels were elevated in 80% (16 of 20) of samples taken from cancer patients pancreatic and none of the healthy individuals. The results are shown in Figure 21. EXAMPLE 8
We measured the nucleosome-associated histone H2AZ levels of some human samples taken from cancer patients using a biotinylated anti-H2AZ detection antibody as described in Example 3. The method was performed twice using two anti-histone capture antibodies different monoclonal clonals to determine if the H2AZ results were repeated for different capture antibodies. The results in Figure 16 show that the nucleosome-associated histone H2AZ levels of the two assays are linearly related to a best-fit line that intercepts at approximately zero. Drives are single optical density reads. EXAMPLE 9
[00179] We measured the nucleosome-associated histone H2AZ levels of human EDTA plasma samples taken from lung cancer patients as described in Example 3. The detected levels were correlated with the patients' disease progression. The results shown in Figure 22, indicate that nucleosome-associated histone H2AZ levels increase with disease severity in terms of size, stage, nodal spread and distant metastatic spread, and nucleosome-associated histone H2AZ levels can be used, alone or as part of a diagnostic panel, as indicators of nodal disease, size, stage, or metastatic progression. REFERENCES
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权利要求:
Claims (17)
[0001]
1. Use of a histone variant or isoform associated with a cell-free nucleosome CHARACTERIZED by the fact that it is a biomarker in a body fluid sample for the diagnosis of cancer, cardiomyopathy, systemic lupus erythematosus, colitis, disorder chronic obstructive pulmonary disease, Crohn's disease and rheumatoid arthritis.
[0002]
2. Use, according to claim 1, CHARACTERIZED by the fact that the nucleosome is a mononucleosome or an oligonucleosome.
[0003]
3. Use, according to claim 1 or 2, CHARACTERIZED by the fact that the cell-free nucleosome comprising a histone variant or a histone isoform is measured in a blood sample.
[0004]
4. Use, according to any one of claims 1 to 3, CHARACTERIZED by the fact that the cancer is a cancer of the bladder, breast, colon, cervix, esophagus, kidney, large intestine, lung, oral cavity, ovary, pancreas , prostate, rectum, skin or stomach.
[0005]
5. Use, according to claim 4, CHARACTERIZED by the fact that the cancer is a cancer of the colon, lung, oral cavity or pancreas.
[0006]
6. Method for detecting the presence of a histone variant or a histone isoform associated with a cell-free nucleosome in a body fluid sample CHARACTERIZED by the fact that it comprises the steps of: (i) contacting the body fluid sample with a binding agent that binds to the histone variant or histone isoform; (ii) detecting or quantifying the binding of said binding agent to the histone variant or histone isoform in the body fluid sample; and (iii) use the presence or degree of such binding as a measure of the presence of the histone variant or histone isoform or nucleosomes containing the histone variant or histone isoform in the body fluid sample.
[0007]
7. Method for detecting the presence of a histone variant or a histone isoform associated with a cell-free nucleosome in a body fluid sample CHARACTERIZED by the fact that it comprises the steps of: (i) contacting the body fluid sample with a first binding agent that binds to nucleosomes; (ii) contacting the nucleosomes bound in step (i) with a second binding agent that binds to the histone variant or histone isoform; (iii) detecting or quantifying the binding of said second binding agent to the histone variant or histone isoform in the body fluid sample; and (iv) using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the body fluid sample.
[0008]
8. Method for detecting the presence of a histone variant or a histone isoform associated with a cell-free nucleosome in a sample CHARACTERIZED by the fact that it comprises the steps of: (i) contacting the body fluid sample with a first agent a binding agent that binds to the histone variant or histone isoform; (ii) contacting the sample, bound in step (i), with a second binding agent that binds to the nucleosomes; (iii) detecting or quantifying the binding of said second binding agent to nucleosomes in the body fluid sample; and (iv) using the presence or degree of such binding as a measure of the presence of nucleosomes containing the histone variant or histone isoform in the body fluid sample.
[0009]
9. Method according to any one of claims 6 to 8, CHARACTERIZED by the fact that the histone isoform is a common histone isoform occurring in all or most nucleosomes to test the nucleosomes per se.
[0010]
10. Method according to any one of claims 6 to 9, CHARACTERIZED by the fact that the binding agent used to bind a histone is directed to bind a region of said histone that is common to all or most variants or histone isoforms of said histone moieties and occurs in all or most nucleosomes to test the nucleosomes per se.
[0011]
11. Method according to any one of claims 6 to 10, CHARACTERIZED by the fact that the binding agent is an antibody.
[0012]
12. Method, according to any one of claims 6 to 11, CHARACTERIZED by the fact that the sample is blood, serum or plasma.
[0013]
13. In vitro method for detecting or diagnosing the state of a disease in an animal or human individual CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring a histone variant or a histone isoform associated with a cell-free nucleosome in a body fluid of a subject using the method as defined in any one of claims 6 to 8; and (ii) using the histone variant or histone isoform associated with a level detected nucleosome to identify the individual's disease state.
[0014]
14. In vitro method for evaluating an animal or human subject for the adequacy of a medical treatment CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring a histone variant or a histone isoform associated with a nucleosome free of cells in a body fluid of a subject using the method as defined in any one of claims 6 to 8; and (ii) using the histone variant or histone isoform associated with a detected level nucleosome as a parameter for selecting an appropriate treatment for the individual.
[0015]
15. In vitro method for monitoring a treatment of an animal or human subject CHARACTERIZED by the fact that it comprises the steps of: (i) detecting or measuring a histone variant or a histone isoform associated with a cell-free nucleosome in a bodily fluid from a subject using the method as defined in any one of claims 6 to 8; (ii) repeat detection or measurement of the histone variant or histone isoform associated with cell-free nucleosomes in an individual's bodily fluid on one or more occasions; and (iii) using any of the modifications in the histone variant or histone isoform associated with a detected level nucleosome as a parameter for any of the modifications in the individual's condition.
[0016]
16. In vitro method according to any one of claims 13 to 15, CHARACTERIZED by the fact that the histone variant or histone isoform associated with a nucleosome is detected or measured as one of a panel of measurements using the method, as defined in any one of claims 6 to 8.
[0017]
17. Kit for the detection of a histone variant or a histone isoform associated with a cell-free nucleosome CHARACTERIZED by the fact that it comprises a specific ligand or ligand for the histone variant or a histone isoform or a component part thereof, or a structural/shape mimicry of the histone variant or a histone isoform or a component part thereof, together with instructions for use of the kit, according to any of the methods as defined in any one of claims 6 to 16 .

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同族专利:

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RU2018101257A|2019-02-21|

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EP2751569B1|2017-12-27|

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PL2751569T3|2018-06-29|

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JP2017215338A|2017-12-07|

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KR20140078638A|2014-06-25|

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HK1199758A1|2015-07-17|

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RU2014112348A|2015-10-10|

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CN108107215A|2018-06-01|

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JP2014529405A|2014-11-13|

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EP2751569A1|2014-07-09|

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SG11201400202SA|2014-03-28|

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CA2845994C|2020-10-13|

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DK2751569T3|2018-03-12|

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WO2013030579A1|2013-03-07|

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BR112014004584A2|2017-05-16|

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EP3301449A1|2018-04-04|

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US20190120853A1|2019-04-25|

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PT2751569T|2018-03-28|

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JP6494703B2|2019-04-03|

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MX2014002444A|2014-10-23|

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AU2012300643B2|2017-06-15|

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MX349561B|2017-08-02|

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ZA201401296B|2014-12-23|

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HK1253354A1|2019-06-14|

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ES2662380T3|2018-04-06|

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AU2012300643A1|2014-03-06|

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SG10201601168QA|2016-03-30|

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GB201115098D0|2011-10-19|

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CA2845994A1|2013-03-07|

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US20160299152A1|2016-10-13|

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US20200341006A1|2020-10-29|

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CN104053993A|2014-09-17|

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RU2716494C2|2020-03-12|

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HRP20180362T1|2018-06-01|

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US10184945B2|2019-01-22|

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US9400276B2|2016-07-26|

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US20140206014A1|2014-07-24|

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KR102130855B1|2020-07-09|

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HUE038517T2|2018-10-29|

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RU2018101257A3|2019-02-21|

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法律状态:
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2019-07-09| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NAO 10196/2001, QUE MODIFICOU A LEI NAO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUAANCIA PRA VIA DA ANVISA. CONSIDERANDO A APROVAA AO DOS TERMOS DO PARECER NAO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NAO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDAANCIAS CABA-VEIS. |

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2021-05-11| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-07-06| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/08/2012, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US201161530304P| true| 2011-09-01|2011-09-01|

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GBGB1115098.4A|GB201115098D0|2011-09-01|2011-09-01|Method for detecting nucleosomes containing histone variants|

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