use of a nucleosome-protein adduct and methods to detect the presence of a nucleosome-protein adduct

专利摘要:
USE OF A NUCLEOSOME-PROTEIN ADULT, METHODS TO DETECT THE PRESENCE OF A NUCLEOSOME-PROTEIN ADULT AND KIT. The invention relates to a method for detecting and measuring the presence of nucleosome-protein adducts and the use of such measures for the detection and diagnosis of disease. The invention also relates to a method of identifying nucleosome adduct biomarkers for the detection and diagnosis of the disease and for biomarkers identified by said method.
公开号:BR112014013082B1
申请号:R112014013082-5
申请日:2012-12-07
公开日:2021-02-02
发明作者:Jacob Vincent Micallef；Mark Edward Eccleston；Marielle Herzog
申请人:Singapore Volition Pte Limited；
IPC主号:

专利说明:

FIELDS OF THE INVENTION
[001] The invention relates to a method for detecting and measuring the presence of nucleosome-protein adducts and the use of such measures for the detection and diagnosis of disease. The invention also relates to a method of identifying the nucleosome adduct biomarkers for the detection and diagnosis of disease and to the biomarkers identified by said method. BACKGROUND OF THE INVENTION
[002] The human body comprises several hundred types of cells. All of these types contain the same genome, but widely different phenotypes and different functions in the body. This phenotypic diversity is due to the differential expression of the genome in different cell types. The control of differential gene expression is not fully understood, but the basic mechanisms include gene regulation by a number of interconnected epigenetic signals associated with the gene, including control of chromatin packaging such as euchromatin or heterochromatin, control of nucleosome positioning and accessible sites of nuclease, DNA methylation and variation in the structure of nucleosomes around which DNA is involved.
[003] The nucleosome is the basic unit of the chromatin structure and consists of a protein complex of eight highly conserved nuclear histones (comprising of a pair of each of the histones H2A, H2B, H3 and H4). Around 146 complex pairs of DNA are involved around this complex. Another histone, H1 or H5, acts as a linker and is involved in the chromatin compaction. DNA is involved in consecutive nucleosomes, in a structure often referred to as "beaded" and this forms the basic structure of euchromatin or open chromatin. In compacted chromatin or heterochromatin this chain is coiled and supercoiled in a closed and complex structure (Herranz and Esteller, 2007).
[004] Normal cell renewal in adult humans involves the creation by cell division of some 1011 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 these are removed and the level of circulating nucleosomes found in healthy patients is low. Elevated levels are found in patients with a variety of conditions including many cancers, autoimmune diseases, inflammatory conditions, stroke and myocardial infarction (Holdenrieder & Stieber, 2009).
[005] Mononucleosomes and oligonucleosomes can be detected by Enzyme-Linked Immunoadsorptive Assay (ELISA) and several methods have been reported (Salgame et al, 1997; Holdenrieder et al, 2001; van Nieuwenhuijze et al, 2003). These assays typically employ an antihistone antibody (for example, anti-H2B, anti-H3, or anti-H1, H2A, H2B, H3 and H4) as the capture antibody and an anti-DNA or anti-H2A- H2B-DNA as a detection antibody. However, we have found that the results of these tests do not agree with each other. In addition, although most of the 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 agree well. The correlation coefficient between ELISA results for circulating free nucleosome levels and circulating DNA levels as measured by real-time PCR (Polymerase Chain Reaction) has been reported to be r = 0.531 in serum and r = 0.350 in plasma (Holdenrieder et al, 2005).
[006] ELISA methods for nucleosome are used in cell culture, primarily as a method for detecting apoptosis (Salgame et al, 1997; Holdenderde et al, 2001; van Nieuwenhuijze et al, 2003), and are also used for the measurement of free circulating nucleosomes in serum and plasma (Holdenrieder et al, 2001). Levels of free nucleosome in serum and plasma, released into circulation by dying cells, have been measured by the ELISA method in studies of a variety of different cancers to assess their use as a potential biomacador (Holdenrieder et al, 2001). Average circulating nucleosome levels are reported to be high in most, but not all, cancers studied. The highest circulating nucleosome levels have been observed in patients with lung cancer. The lowest levels were seen in prostate cancer, which were within the normal range of healthy patients. However, patients with malignant tumors are reported to have serum nucleosome concentrations that varied considerably and some patients with advanced tumor disease were found to have low levels of circulating nucleosome, within the range measured for healthy patients (Holdenrieder et al, 2001 ). Because of this and the variety of non-cancer causes for increased levels of nucleosome, circulating nucleosome levels are not used clinically as a cancer biomarker (Holdenrieder and Stieber, 2009).
[007] The structure of nucleosomes can vary by Post-Transcriptional Modification (PTM) of histone proteins and by inclusion of histone variant proteins. Histone protein PTM typically occurs at the terminals of the eight nuclear histones and common modifications include acetylation, methylation or ubiquitination of lysine residues, as well as methylation of arginine residues and phosphorylation of serine residues. Histone modifications are known to be involved in the epigenetic regulation of gene expression (Herranz and Esteller, 2007). The structure of the nucleosome can also vary by including alternative isoforms or histone variants, which are different gene or splice products and have different amino acid sequences. Histone variants can be classified into a variety of families that are subdivided into individual types. The nucleotide sequences of a large number of histone variants are known and publicly available, for example, at 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 histones and histone fold-containing proteins. Database Vol. 2011. (Submitted) and http: //genome.nhgri.nih .gov / histones / complete.shtml), the GenBank (NIH genetic sequence) DataBase, the EMBL Nucleotide Sequence Database and the Japan Database (DDBJ).
[008] Histone variant and histone modification patterns present in healthy and diseased cells have been shown to differ in numerous studies (mostly immunohistochemicals) (Herranz and Esteller, 2007). A disadvantage of immunohistochemical methods for clinical use is that tissue sample collection is invasive involving surgery or biopsy.
[009] In addition to epigenetic signaling mediated by the structure and position of the nucleosome, control of gene expression in cells is also mediated by the state of DNA methylation (Herranz and Esteller, 2007). 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.
[010] The involvement of DNA methylation in cancer was reported as early as 1983 (Feinberg and Vogelstein, 1983). Patterns of DNA methylation observed in cancer cells differ from those in healthy cells. Repetitive elements, particularly around pericentromeric areas, are reported to be hypomethylated in cancer in relation to healthy cells, but specific gene promoters are reported to be hypermethylated in cancer. The balance of these two effects is reported to result in global hypomethylation of DNA in cancer cells (Rodriguez-Paredes & Esteller, 2011).
[011] Hypermethylation of certain specific genes can be used as a diagnostic biomarker for cancers. For example, a reported method for detecting the hypermethylation of the Septin 9 gene, by PCR amplification, of DNA extracted from plasma has been reported to detect 72% of colon cancers with a false positive rate of 10% (Grutzmann et al, 2008) . The DNA methylation status of specific genes or loci is usually detected by selective cytosine bisulfite deamination, but not 5-methylcytosine, for uracil, leading to the primary DNA sequence change, which can be detected by sequencing or other means (Allen et al, 2004).
[012] Global DNA hypomethylation is a characteristic of cancer cells (Esteller 2007 and Hervouet et al, 2010). Global DNA methylation can be studied in cells using immunohistochemistry techniques. Alternatively, DNA is extracted from the cells for analysis.
[013] It has been known for many years that, in addition to nucleic acid and histone proteins, chromatin comprises a large number of non-histone proteins linked to its constituent DNA and / or histones (Yoshida and Shimura 1972). These chromatin-associated proteins are of a wide variety of types and have a variety of functions including transcription factors, transcription enhancing factors, transcription repression factors, histone-modifying enzymes, DNA damage repair proteins and more. The study of chromatin-bound proteins has been largely carried out by methods of Chromatin Immune Precipitation (ChIP). These methods are well known in the art, but they are complex, laborious and expensive.
[014] In a typical ChIP method, cell chromatin is cross-linked so that all protein and nucleic acid components are covalently linked to each other. The chromatin is then cut to form a mononucleosome and oligonucleosome preparation. An antibody to the protein of interest is added to the cut chromatin to immunoprecipitate those chromatin fragments containing the protein. The antibody is normally bound to a solid phase (for example, plastic beads) to facilitate the isolation of the chromatin complex containing the protein of interest. The cross-linking is then reversed and the protein is removed by digestion with a proteinase. The DNA associated with the chromatin complex is isolated and analyzed to determine the DNA sequence, gene or locus associated with the binding of the particular protein, using any of a variety of techniques including PCR followed by gel electrophoresis, DNA sequencing (ChIP- Seq) or DNA microarray (ChIP-on-chip).
[015] These ChIP methods reveal the DNA sequences associated with histone proteins linked to chromatin. Derivatives of the ChIP method have been developed to facilitate studies of the association of non-histone proteins with histones and nucleosides - we include, for example, Histone Associated Assays (Ricke and Bielinsky, 2005). Many chromatin-binding proteins that are involved in the mechanisms of cancer and other diseases, but their abundance in the nucleosome adduct form, in circulation have not been previously investigated. Examples include the High Mobility Group 1 Protein (HMGB1), the homologous Zeste 2 enhancer polycomb protein (EZH2) and the nuclear receptor protein group.
[016] The High Mobility Protein Group is a component of chromatin present in about 3% of the weight of DNA or histones. They are structural proteins that bind to nucleosomes without any specificity for the underlying DNA sequence (Gerlitz et al; 2009). HMGB1 is a chromosomal protein of architecture and a pro-inflammatory mediator. She is involved in cell death, apoptosis and various diseases including various inflammatory and autoimmune conditions, sepsis, meningitis and neurodenegation. Overexpression of HMGB1 is associated with all central cancer markers (Tang et al; 2010). HMGB1 is strongly linked to the chromatin of apoptotic cells. Studies of nucleosome-HMGB1 complexes have shown that these adducts are found in the circulation of patients suffering from the autoimmune systemic lupus erythematosus disease (SLE) and that the adducts are involved in the development of anti-nuclear antibodies which is a key feature of SLE. Nucleosomes not bound to HMGB1 do not elicit an immune response. The binding of HMGB1 to the nucleosome in these adducts has been demonstrated by nucleosome immunoprecipitation with an antibody directed to DNA or histones followed by Western Blot using an anti-HMGB1 antibody to demonstrate the presence of HMGB1 in the immunoprecipitated nucleosomes (Urbonaviciute et al; 2008) .
[017] HMGB proteins interact with many other proteins known to affect chromatin function and chromatin complexes involving HMGB proteins plus additional proteins have been shown to occur (Gerlitz et al; 2009). Then, in addition to simple nucleosome-protein adducts, nucleosome-protein-complex adducts in which 2 or multiple proteins are associated with nucleosomes occur in chromatin.
[018] EZH2 is a member of the group-Polycomb (PcG) family that forms multimeric protein complexes involved in maintaining the transcriptional repressive state of genes. EZH2 is a histone-modifying enzyme (histone-lysine N-methyltransferase) that methylates the amino acid residue 27 of lysine in histone 3 of the nucleosomes. This histone modification is associated with chromatin condensation and gene silencing (Cao et al; 2002).
[019] Nuclear receptors are molecules that regulate gene expression under the control of hormones or ligands, for example, the estrogen receptor (ER) regulates the expression of estrogen-dependent genes. Many of these proteins are involved in pathological processes, for example, ER is involved in the progression of breast cancer and many breast cancer treatments target ER and / or prevent the interaction of ER with its estradiol ligand.
[020] In addition to the nucleosome-protein adducts that occur in the cell, there are other nucleosome-protein adducts that can be formed after the release of the cell's nucleosomes, after cell death. Such nucleosome adducts include the nucleosome-immunoglobulin adducts that are a key feature of SLE.
[021] We now report simple immunoassay methods for direct estimation of protein-nucleosome adducts in biological samples. We have developed simple methods for detecting nucleosomes linked to EZH2, HMGB1 and various nuclear receptors and have shown that such nucleosome adducts can be detected in serum samples and that they are used as biomarkers in the disease. SUMMARY OF THE INVENTION
[022] According to the first aspect of the invention, the use of a nucleosome-protein adduct is provided as a biomarker in the blood for the diagnosis of cancer, autoimmune disease or inflammatory disease.
[023] According to a second aspect of the invention, a method is provided to detect the presence of a nucleosome-protein adduct in a sample comprising the steps of: (i) contacting the sample with the first binding agent that is binds to nucleosomes or a component thereof; (ii) contacting the nucleosomes or sample with a second binding agent that binds to a protein added to a nucleosome; (iii) detecting or quantifying the binding of said second binding agent to the protein added in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the sample.
[024] In accordance with a third aspect of the invention, a method is provided to detect the presence of a nucleosome adduct in a sample comprising the steps of: (i) contacting a sample with a first binding agent that binds to a protein added to a nucleosome; (ii) contacting the nucleosomes or sample with a second binding agent that binds to the nucleosomes or a component thereof; (iii) detecting or quantifying the binding of said second nucleosome binding agent or a component thereof in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the sample.
[025] In accordance with a further aspect of the invention, a method is provided for detecting a nucleosome adduct in a cell comprising the steps of: (i) isolating chromatin from a cell; (ii) digest, sonicate or otherwise break chromatin to form mononucleosomes and / or oligo-nucleosomes; and (iii) detecting or measuring the presence of the nucleosome adduct according to an ELISA method of the invention described in the second and third aspects above.
[026] In accordance with a further aspect of the invention, a method is provided for detecting or diagnosing a disease state in an animal or a human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a fluid body of a patient; and (ii) use the detected nucleosome adduct level to identify the patient's disease state.
[027] In accordance with a further aspect of the invention, a method is provided for assessing an animal or human patient for suitability for a medical treatment comprising the steps of: (i) detecting or measuring a nucleosome adduct in a body fluid of the patient; and (ii) use the detected nucleosome adduct level as a parameter for selecting an appropriate treatment for the patient.
[028] In accordance with a further aspect of the invention, a method is provided for monitoring a treatment of an animal or human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a patient's body fluid; (ii) repeat the detection or measurement of a nucleosome adduct in a patient's body fluid on one or more occasions; (iii) use any changes in the nucleosome adduct level detected as a parameter for any changes in the patient's condition.
[029] In accordance with a further aspect of the invention, a method is provided to identify an adducting nucleosome biomarker to detect or diagnose a disease state in an animal or human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a patient's body fluid; (ii) detecting or measuring a nucleosome adduct in a body fluid of a healthy patient or a control patient; and (iii) use the difference between the levels detected in sick and control patients to identify whether a nucleosome adduct is useful as a biomarker for disease status.
[030] In accordance with a further aspect of the invention, a biomarker identified according to the methods defined herein is provided.
[031] According to a further aspect of the invention, a kit is provided for the detection of a nucleosome adduct comprising a specific ligand or linker for the nucleosome adduct or component part thereof, or a structure / shape mimic. of the base of DNA, nucleotide or nucleoside or component part thereof, together with instructions for using the kit. BRIEF DESCRIPTION OF THE FIGURES Figure 1: ELISA dose-response curve for the detection of EZH2 nucleosome adduct levels in digested chromatin extracted from Hela cells diluted in horse serum. Figure 2: ELISA results of the Nucleosome-EZH2 adduct for serum samples taken from 5 healthy patients and 11 patients with tumors. Figure 3: ELISA dose-response curve for the detection of nucleosome-HMGB1 adduct levels in digested chromatin extracted from Hela cells diluted in horse serum. Figure 4: Results of nucleosome-HMGB1 adduct ELISA for serum samples taken from 5 healthy patients and 11 patients with tumors. Figure 5: Nucleosome-HMGB1 adduct ELISA results for serum samples taken from 31 healthy patients and 74 patients with (A) colon cancer, (B) breast cancer or (C) lung cancer. Figure 6: ELISA dose-response curve for the detection of nucleosome-Progesterone Receptor adduct levels in free nucleosomes, prepared by the method of * Holdenrieder et al; 2001. Figure 7: ELISA results for the detection of levels of nucleosome-Androgen Receptor adducts in 2 cases of prostate cancer and a free nucleosome sample, prepared by the method of * Holdenrieder et al; 2001. Figure 8: ELISA dose-response curve for detecting levels of nucleosome-Estrogen Alpha Receptor (ERα) adducts in free nucleosomes, prepared by the method of * Holdenrieder et al; 2001. Figure 9: ELISA results for the detection of nucleosome-ERβ adduct levels in digested MCF7 chromatin. The test was carried out in two different formats. In the first format, the anti-nucleosome antibody was coated in the wells and the anti-ERβ antibody was biotinylated. In the second format, the anti-ERβ antibody was coated in the wells and the anti-nucleosome antibody was biotinylated. Figure 10: Results of H2AZ-ERβ nucleosome adduct ELISA. Figure 11: Results of nucleosome-ERβ adduct ELISA for serum samples taken from 12 healthy patients and 16 patients with tumors. DETAILED DESCRIPTION OF THE INVENTION
[032] According to a first aspect of the invention, the use of a nucleosome-protein adduct is provided as a biomarker in the blood for the diagnosis of cancer, autoimmune disease or inflammatory disease. In one embodiment, the biomarker is used for the diagnosis of cancer. We have shown that two such adducts containing HMGB1 and EZH2 are present in the circulation of cancer patients, but are not detected in the circulation of healthy patients.
[033] It is well known in the art that cancers can be hormone-dependent and require the presence of growth hormone. Nuclear hormones are also known to function by nuclear localization of the hormone complex linked to the receptor and binding to specific hormone response elements in the genome. The expression of the genes associated with the elements is regulated by the binding of the hormone complex linked to the receptor to the genomic response element. In one embodiment, the invention provides hormone-nucleosome receptor adduct and hormone-nucleosome receptor adduct biomarkers to characterize a patient's tumor state. These adducts can be circulating adducts present in the blood or other body fluid, or they can be produced by digesting chromatin from a sample of tumor tissue.
[034] It is well known in the art that nuclear hormone receptors regulate gene expression under hormone or ligand control. For example, the estrogen receptor works by binding its substrate (the steroid hormone estrogen) to the cell surface membrane. Binding is followed by the internalization of the hormone-receptor complex and intranuclear localization, where the receptor binds to specific elements of hormonal response in the genome. The specific gene sequence to which the estrogen receptor binds is known as the Estrogenic Response Element (ERE). Expression of genes associated with ERE can be regulated by the receptor and, therefore, by the presence or level of estrogen in a patient's circulation. It is also well known in the art that breast cancer growth is often under estrogen control and such cancer is often referred to as estrogen-dependent. As these tumors overexpress the Estrogen Receptor (ER) they are often called ER + tumors. The growth of estrogen-dependent tumors can be slowed or prevented by therapeutic interventions aimed at preventing estrogen binding to the estrogen receptor and this is a common method of treating breast cancer. Examples of such treatments include the drug Tamoxifen, which acts as an estrogen antagonist in estrogen-dependent breast cancer, and aromatase inhibitors that decrease or prevent estrogen production. However, over time, cancers develop into estrogen-independent tumors, which will grow even in the absence of estrogen stimulation and require different treatments. The diagnosis of estrogen-dependent and estrogen-independent tumors is currently routinely made by immunostaining tumor biopsy tissue to determine whether or not the estrogen receptor is abundant in tumor cells. Doctors may need to retest a tumor's estrogen dependence again and again during the course of tumor treatment to determine whether additional estrogen-dependent treatment is appropriate or not, or whether the patient's treatment regimen should be changed to reflect the change in tumor nature as the disease progresses. Unfortunately, current tests are suboptimal and require repeated painful biopsies each time the test is done. In an embodiment of the invention, the detection of estrogen-receptor nucleosome adducts in the circulation of patients with breast cancer is used as an indicator of estrogen receptor binding to ERE in the nucleus of tumor cells, as an indicator for estrogen dependence of a tumor to assist in the selection of appropriate treatment and for predictive prognosis information. This method has the advantages of being indicative of the estrogen ERE-receptor binding in the tumor, rather than a simple indicator of the presence or abundance of the estrogen receptor, and of being able to be repeated as often as desired by a simple blood test without the need for biopsy. We have developed simple ELISA methods for the detection and quantification of ER nucleosome adducts containing both ERα and ERβ forms of the receptor. Surprisingly, these adducts are present in the circulation of cancer patients
[035] It will be clear to those skilled in the art that the same principles can be applied for the detection of estrogen-nucleosome receptor adducts in digested cell chromatin produced from the tumor cell itself. This method for assessing a tumor's estrogen dependence is superior to current methods because it is indicative of the estrogen ERE-receptor binding in the tumor, rather than a simple indicator of the presence or abundance of the estrogen receptor.
[036] In another embodiment of the invention, the detection of the presence of steroidal estrogen alone in an estrogen-estrogen receptor-nucleosome complex adduct or in the circulation, or in another body fluid, or in nucleosomes produced as a digestion of chromatin from tumor tissue is used as an indicator of a tumor's estrogen-dependent state.
[037] Circulating nucleosomes are reported to be elevated in endometriosis (Holdenrieder et al; 2001) and, as endometriosis tissue is estrogen-responsive, binding of the estrogen receptor in endometriosis cell chromatin can lead to estrogen-nucleosome receptor adducts or estrogen-estrogen-nucleosome receptor adducts in the circulation. In an additional embodiment of the invention, estrogen receptor-nucleosome adducts or estrogen receptor-estrogen-nucleosome complexes are detected in a body fluid as a biomarker for the presence of an estrogen-dependent gynecological condition including, for example, endometriosis.
[038] In a form similar to estrogen-dependent breast cancer, the growth of androgen-dependent prostate cancer requires, or is accelerated by androgen. Androgen-dependent prostate tumors are similarly treated by methods that prevent androgen binding to the androgen receptor (AR). Androgen-dependent prostate tumors can also develop to become androgen-independent and then resistant to treatments including physical or chemical drug castration to prevent the androgen from binding to its receptor. The androgen-dependent state of a tumor can be determined by the level of androgen receptor binding to androgen response elements (ARE) in the genome and this can be determined by analyzing the levels of the androgen-nucleosome receptor adduct present in the circulation of a patient or in chromatin digested from the prostate tissue. Modalities of the invention for this purpose include the detection of androgen-nucleosome receptor adducts or androgen-nucleosome receptor adducts in the circulation or in a patient's body fluid or in nucleosomes produced by chromatin digestion from tumor tissue of a patient. We have now developed simple ELISA methods for the detection and quantification of nucleosome-AR adducts and have demonstrated their usefulness. We have also developed simple ELISA methods for detecting and quantifying nucleosome-Progesterone Receptor adducts. Other hormone-dependent diseases can be addressed with similar modalities to the method of the invention. Such modalities include the detection of other nucleosome-receptor adducts including, for example, glucocorticoid nucleosome-receptor adducts, thyroid hormone receptor and retinic acid receptor for the detection of tumors including, for example, various types of leukemia involving retinoic acid receptor.
[039] In accordance with a further aspect of the invention, the methods described here above can be used to detect adducts to the hormone-hormone-nucleosome receptor complex. In one embodiment, the hormone-hormone-nucleosome receptor complex adducts comprise a thyroxine-nucleosome hormone receptor-adduct complex, a triodothyronine-thyroid hormone-nucleosome complex adduct, a retinoic acid-retinoic acid-nucleus adduct complex adduct. - somo, an androgen-androgen-nucleosome receptor adduct or an estrogen-estrogen-nucleosome receptor adduct. This aspect of the invention has the advantage of distinguishing hormone-activated adducts as well as adducts containing wild or normal hormone receptor from hormone receptor that does not bind to its ligand, for example, due to mutation in the course of disease progression ( for example, in estrogen-independent breast cancer). This aspect of the invention can be accomplished in multiple ways. In one embodiment, an antibody or other linker targeted to bind to the hormone alone is used in place of the antibody targeted to bind to the hormone receptor. In an alternative modality, hormone is extracted from a hormone-nucleosome receptor complex adduct captured by an antibody and quantified by established methods, for example, immunoassay methods, spectrographic methods or chromatographic methods, including high performance liquid chromatography. performance (HPLC), liquid chromatography followed by mass spectroscopy (LC / MS) or gas chromatography followed by mass spectroscopy (GC / MS). For example, the androgen-androgen-nucleosome receptor adduct is captured by immobilized antibodies directed to bind to an epitope present in the adduct (for example, the androgen receptor or a nucleosome). The hormone is then extracted from the solid phase bound to the adduct in an organic solvent (for example, diethyl ether). The solvent is transferred, dried and the androgen is redissolved in assay buffer and its concentration is measured (for example, by competitive immunoassay). It will be clear to those skilled in the art that this modality will have particular application for small molecule hormones such as steroid and thyroid hormones.
[040] The present invention aims at the detection of proteins that are linked to nucleosomes. This can be done through a double antibody ELISA test in which one antibody is directed to bind to nucleosomes and the other is directed to bind to the protein bound to the nucleosome. However, the antibody selected to bind to the nucleosome does not have to target the entire nucleosome complex, but may target a protein or nucleic acid component part of the nucleosome. In this embodiment of the invention, the antibody employed to bind to the nucleosome can be directed to bind to any component part of a nucleosome, including, for example, a particular histone, histone modification, histone variant or isoform, or to a particular nucleotide or modified nucleotide. We have shown that this assay design works well using the example of using an antibody targeted to bind histone variant H2AZ as a nucleosome linker. It will be very clear to those skilled in the art that this method has an additional advantage of selectively binding only to those nucleosomes that contain both, the protein of interest in the adduct and H2AZ. This design provides a method for an assay to test any combination of the adduced protein with any particular histone, histone modification, histone variant, nucleotide, modified nucleotide or other nucleosome structure.
[041] According to a second aspect of the invention, a method is provided to detect the presence of a nucleosome-protein adduct in a sample comprising the steps of: (i) contacting the sample with a first binding agent that binds to nucleosomes or a component thereof; (ii) contacting the nucleosomes or sample with a second binding agent that binds to a protein added to a nucleosome; (iii) detecting or quantifying the binding of said second adducting protein binding agent in the sample; and (iv) use the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the sample.
[042] It will be clear to those skilled in the art that the binding agent to be detected can be selected to be either the antibody targeting the adduced protein or the nucleosome or a component part of the nucleosome.
[043] In accordance with a third aspect of the invention, a method is provided for detecting the presence of a nucleosome adduct in a sample comprising the steps of: (i) contacting a sample with a first binding agent that binds to a protein added to a nucleosome; (ii) contacting the nucleosomes or samples with a second binding agent that binds to nucleosomes or a component thereof; (iii) detecting or quantifying the binding of said second nucleosome binding agent or a component thereof in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the sample.
[044] In one embodiment, the nucleosome adduct includes a proinflammatory protein, a High Mobility Group Protein, a polycomb protein, a chromatin-modifying enzyme, a nuclear receptor or a hormone. In an alternative embodiment, the nucleosome adduct includes a High Mobility Group Protein, a polycomb protein, a chromatin-modifying enzyme, a hormone receptor or a hormone. In an additional embodiment, the nucleosome adduct includes a chromatin-modifying enzyme, a nuclear receptor or a hormone. In an additional modality, the Protein of the High Mobility Group is HMGB1. In one embodiment, when the biomarker is used for the diagnosis of cancer, the nucleosome-protein adduct includes a Protein from the High Mobility Group.
[045] In one embodiment, the chromatin-modifying enzyme is an enzyme for acetylation, deacetylation, methylation, demethylation, phosphorylation, dephosphorylation, ubiquitination, deubiquitination, sumoylation, histone dehydration or DNA methyltransferase. In an alternative embodiment, the chromatin-modifying enzyme is EZH2.
[046] In one embodiment, when the nucleosome-protein adduct includes a nuclear receptor, said nuclear receptor is the estrogen receptor, androgen receptor, progesterone receptor, thyroid hormone receptor, glucocorticoid receptor or retinoic acid receptor. . In an alternative embodiment, when the nucleosome-protein adduct includes a nuclear receptor, said nuclear receptor is the estrogen receptor, androgen receptor or retinoic acid receptor.
[047] In one embodiment, when the nucleosome-protein adduct includes a hormone, that hormone is a thyroid hormone, a glucocorticoid hormone or a steroid hormone including an estrogen, an androgen, a progestogen, a corticosteroid or retinoic acid . In an alternative modality, when the nucleosome-protein adduct includes a hormone, that hormone is a steroid hormone including an estrogen, an androgen, a corticosteroid or retinoic acid.
[048] In one embodiment, when the nucleosome-protein adduct includes a hormone receptor, said hormone receptor is the estrogen receptor, androgen receptor, progesterone receptor, thyroid hormone receptor or retinoic acid receptor.
[049] We have shown that the method can be done using an antibody targeting the nucleosome itself in combination with an antibody targeting to bind the adduced protein to the nucleosome or using an antibody targeting a component of a nucleosome, again in combination with an antibody directed to bind the adduced protein to the nucleosome. In one embodiment, the antibody or nucleosome linker or component of the nucleosome is targeted to bind to a particular epigenetic nucleosome epitope; for example, any histone variant (for example, H2AZ), any histone modification (for example, trimethyl H3K9) or any modified nucleotide or nucleotide (for example, 5-methylcytosine). In an alternative embodiment, the nucleosome linker or nucleosome component is targeted to bind to a particular epigenetic signal structure such that only a particular subgroup of nucleosome adducts containing said epigenetic signal structure is detected.
[050] In one embodiment, the binding agent used is an antibody, an antibody fragment or an aptamer. In an additional embodiment, the binding agent used is an antibody.
[051] In one embodiment, the sample is a biological fluid. In an additional embodiment, the sample is blood or serum or plasma. It will be clear to those skilled in the art that the detection of nucleosome adducts in body fluid has the advantage of being a minimally invasive method that does not require biopsy.
[052] In some cases, however, it may be preferable to assess the state of a cell's nucleosome adduct directly by producing nucleosomes from that cell and to analyze the nucleosomes for the presence of particular nucleosome adducts.
[053] In accordance with a further aspect of the invention, a method is provided for detecting a nucleosome adduct in a cell comprising the steps of: (i) isolating a cell's chromatin; (ii) digestion, sonification or otherwise breaking the chromatin to form mono-nucleosomes and / or oligo-nucleosomes; and (iii) detecting or measuring the presence of the nucleosome adduct according to an ELISA method of the invention described in any of the second to sixth aspects above.
[054] In accordance with a further aspect of the invention, a method is provided for detecting or diagnosing a disease state in an animal or human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a body fluid of a patient; and (ii) use the level of the detected nucleosome adduct to identify the patient's disease status.
[055] In one embodiment of the invention the presence of a nucleosome adduct in a sample is used to determine the optimal treatment regimen for a patient in need of such treatment. An example of such a modality is the detection of a nuclear hormone-nucleosome receptor adduct or adduct of a hormone-nucleosome receptor-hormone complex to assess the hormone dependence of a tumor.
[056] In accordance with a further aspect of the invention, a method is provided for evaluating an animal or human patient for suitability of a medical treatment comprising the steps of: (i) detecting or measuring a nucleosome adduct in a body fluid of the patient; and (ii) use the detected nucleosome adduct level as a parameter for selecting an appropriate treatment for the patient.
[057] In accordance with a further aspect of the invention, a method is provided for monitoring a treatment of an animal or human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a patient's body fluid; (ii) repetition of the detection or measurement of a nucleosome adduct in a patient's body fluid on one or more occasions; (iii) use any changes in the detected nucleosome adduct level as a parameter for any changes in the patient's condition.
[058] In one embodiment, the nucleosome adduct is detected or measured as one of a measurement panel.
[059] In accordance with a further aspect of the invention, a method is provided for detecting or measuring a nucleosome adduct, either alone or as part of a measurement panel, for the purposes of detecting or diagnosing a disease state, or for evaluation of an animal or human patient for suitability for a medical treatment, or for monitoring a treatment for an animal or human patient, for use in patients with current or suspected cancer, benign tumors, inflammatory disease, autoimmune disease, endometriosis, disease infection, sepsis, stroke or myocardial infarction.
[060] In accordance with a further aspect of the invention, a method is provided to identify an adducting nucleosome biomarker to detect or diagnose a disease state in an animal or human patient comprising the steps of: (i) detecting or measuring a nucleosome adduct in a patient's body fluid; (ii) detecting or measuring a nucleosome adduct in a body fluid of a healthy patient or a control patient; (iii) use the difference between the levels detected in sick and control patients to identify whether a nucleosome adduct is useful as a biomarker for disease status.
[061] In accordance with a further aspect of the invention, a kit is provided for the detection of a nucleosome adduct comprising a specific ligand or linker for the nucleosome adduct or component part thereof, or a structure / shape mimetic. of the base DNA, nucleotide or nucleoside or component part thereof, along with instructions for using the kit.
[062] In addition to the components histone and nucleic acid, chromatin is known to contain a wide variety of proteins that perform a wide variety of functions. We selected HMGB1, EZH2 and several nuclear receptors as examples of these proteins and we have developed simple ELISA methods for the detection of mononucleosome and oligonucleosome adducts to these proteins. We did these ELISA methods directly on serum samples taken from healthy and sick patients and the methods do not require any sample extraction or other sample pretreatment. Surprisingly, we have shown that these nucleosome adducts can be detected in the serum of cancer patients and that nucleosome adduct ELISA assays are useful in detecting and diagnosing disease states.
[063] HMGB1 is a damage-associated molecular standard protein (DAMP) associated with cell death, apoptosis and several diseases including various inflammatory and autoimmune conditions, sepsis, meningitis, neurodegeneration, SLE and cancer (Tang et al; 2010). Elevated expression of HMGB1 occurs in many cancers and is said to be associated with invasion and metastasis (Sims et al, 2010). Elevated levels of HMGB1 also occur in the blood of cancer patients, as well as in a variety of other conditions (Stoetzer et al, 2012). Circulating HMGB1 can be measured by ELISA, but such measures are not used in routine clinical practice, because circulating HMGB1 occurs in bound and free forms and the Western immunoblot methods currently available to distinguish these are not suitable for routine use. Thus, there is a need for a reliable method to distinguish between free HMGB1 and HMGB1 complexes (Urbonaviciute and Voll, 2011). An important class of circulating HMGB1 complexes is HMGB1-nucleosome adduct and one embodiment of the present invention is directed to the detection of HMGB1-nucleosome adducts and other HMG-nucleosome adducts. We have shown that HMG-nucleosome adducts can be measured in the blood of cancer patients using a fast and simple ELISA method.
[064] HMGB1 is tightly bound to apoptotic cell chromatin. Studies of nucleosome-HMGB1 complexes have shown that these adducts are found in the circulation of patients suffering from autoimmune SLE disease and that the adducts are involved in the development of anti-nuclear antibodies which is a major characteristic of SLE. The presence of these adducts in the circulation has not been used for the purpose of clinical diagnosis because the Western blot methods used for these detections are expensive, slow and laborious, and are not suitable for routine clinical use. The present invention overcomes these shortcomings.
[065] EZH2 is a chromatin-modifying enzyme (histone-lysine N-methyltransferase) that methylates the amino acid lysine 27 of histone 3 of the nucleosomes leading to chromatin condensation and gene silencing (Cao et al; 2002) . This protein is known to bind chromatin to the nucleus of living cells. Surprisingly, we have shown that EZH2 remains linked to nucleosomes after cell death and mononucleosome-EZH2 and oligonucleosome-EZH2 adducts can be detected in the serum of cancer patients using the novel ELISA methods of the present invention.
[066] Chromatin-modifying enzymes are known to be involved in cancer (Fullgrabe et al, 2011) and inhibiting the activity of these enzymes through the use of target drugs is a major form of cancer therapy. These drugs include, for example, and without limitation Histone Deacetylation Complex Inhibitors (HDACi), Histone Methyl Transferase Inhibitors (HMTi) and DNA Methyl Transferase Inhibitors (DNMTi). Although the presence of HMGB1 adducts in the circulation is known to be pathological and associated with anti-nuclear antibodies, the discovery that chromatin-nucleosome-modifying enzyme adducts are present in the circulation has not been previously reported. Assays for chromatin-nucleosome-modifying enzyme adducts have multiple uses in cancer, including, for example, in assessing cancer disease states and in determining the effectiveness of chromatin-modifying enzyme-inhibiting drugs, for example, to determine whether the level of the chromatin-nucleosome-modifying enzyme adduct is altered by treatment with particular drugs. The method of the invention can be used to determine circulating chromatin-nucleosome-modifying enzyme adduct levels for a wide variety of disease diagnostic purposes including detection, monitoring, prognosis, differential diagnosis and choice of treatment regimen for disease. We have shown that nucleosome adducts containing the HMT EZH2 enzyme can be detected in the circulation of cancer patients. It will be clear to those skilled in the art that the method of the invention can be applied to other chromatin-modifying enzymes including the aforementioned HDAC and DNMT enzymes, as well as many other enzymes including, for example, enzymes for acetylation, demethylation, phosphorylation, dephosphorylation, ubiquitination, deubiquitination, sumoylation and dehumylation of histone.
[067] Nuclear receptors exert their regulatory gene effects on the nucleus under the control of the hormonal ligand. Examples include steroid hormone receptors, thyroid receptor, glucocorticoid receptor and vitamin D receptor and retinoic acid. These receptors are involved in a variety of mechanisms of cancers and other diseases. Some examples include the involvement of the Retinoic Acid Receptor (RAR) in leukemia, the Estrogen Receptor (ER) in breast cancer and endometriosis, the Androgen Receptor (RA) in prostate cancer and the Thyroid Hormone Receptor in the disease and thyroid cancer.
[068] Surprisingly, we have shown that nuclear-nucleosome receptor adducts can be detected in the circulation of cancer patients.
[069] So, all the proteins associated with intracellular chromatin that we chose to study can be found in the serum of cancer patients in the form of nucleosome adducts. These findings indicate that such adducts may not be unusual and that many of the intracellular protein-nucleosome adducts, involving many different chromatin-associated proteins, may retain their integrity after cell death and be amenable to detection in the serum of patients with cancer, disease autoimmune and inflammatory disease by the method of the present invention.
[070] We have used an antihistone antibody as the capture antibody for these assays in combination with an appropriate specific anti-chromatin protein antibody (anti-HMGB1, anti-EZH2 or anti-nuclear receptor). We have used assays to show that nucleosome adducts containing specific proteins can be measured in blood samples taken from cancer patients and are broken down for use as non-invasive or minimally invasive biomarkers. The levels of nucleosome-adducts detected in serum samples taken from these sick patients differ from those detected in serum samples from healthy patients.
[071] We measured the levels of nucleosome-HMGB1 and nucleosome-free EZH2 adducts in blood samples taken from 3 colon cancer patients, 6 lung cancer patients and 2 pancreatic cancer patients and compared these with levels present in the samples blood samples from 5 healthy patients as well as an artificially produced serum nucleosome preparation from healthy patients prepared as described in the literature (* Holdenrieder et al, 2001) and with commercially available preparation of nucleosomes prepared by digesting chromatin extracted from cells Hela.
[072] Normal ranges were calculated from results for the 5 healthy patients (mean result ± 2 standard deviation from mean) for the nucleosome-HMBG1 and nucleosome-EZH2 adducts and the results for cancer patients were examined to see whether they fall within or outside the respective normal range. The data show that 2 out of 3 colon cancer samples, 4 out of 6 lung cancer samples and 1 out of 2 pancreatic cancer samples had elevated nucleosime-HMBG1 adduct levels and similarly that 2 out of 3 colon cancer samples, 4 of 6 lung cancer samples and 1 of 2 pancreatic cancer samples had high nucleosome-EZH2 adduct levels (Optical Density results higher than the maximum of the normal range).
[073] We have similarly measured the levels of nuclear receptor nucleosome adducts in healthy and sick patients and have shown that these are present in the serum of cancer patients.
[074] Chromatin-binding proteins include, without limitation, nuclear receptors, High Mobility Group proteins (such as HMGB1), polycomb proteins, chromatin-modifying enzymes (such as EZH2), DNA-modifying enzymes, nuclear receptors, transcription factors, architectural or structural proteins, transcription enhancing factors, transcription repression factors, replication proteins, DNA damage repair proteins and any other proteins involved in the control of gene expression, chromatin packaging or replication.
[075] Nucleosome adducts can also occur due to the binding of nucleosomes present in a biological fluid after cell death. An example of such an adduct may be a nucleosome-antibody adduct formed in an autoimmune disease such as SLE.
[076] Then, in one embodiment of the invention, a method is provided to detect or measure the presence of a nucleosome-protein complex or adduct. The nucleosome adducts to be measured can be of any origin including, without limitation, naturally occurring nucleosome adducts present in biological fluids as a consequence of a healthy or diseased condition or nucleosome adducts can be produced by the digestion of chromatin extracted from cells, or they can be produced by cell-induced apoptosis or necrosis (for example, by the method of * Holdenrieder et al; 2001). Surprisingly, we have shown that nucleosome adducts occur in all of these situations and can be detected by the method of the invention.
[077] In another embodiment of the invention, a method is provided to detect or measure the presence of a nucleosome-protein adduct in a biological fluid.
[078] In a further embodiment of the invention, a method is provided to detect or diagnose the presence, type, recurrence or severity of a disease or to evaluate the optimal drug or other treatment options by testing the patient's sample, for the presence or level of one or more nucleosome-protein complexes or adducts.
[079] In a further embodiment of the invention, a method is provided to detect or diagnose the presence, type, recurrence or severity of a disease or to evaluate the optimal drug or other treatment options by testing a sample taken from a patient, for presence or level of a nucleosome-protein complex or adduct as part of a test panel. An ELISA method for detecting free nucleosomes containing different histone modifications has been reported (Bawden et al; 2005).
[080] So, such a test panel can consist, for example, of two or more measurements of nucleosomes containing different nucleosome epitopes; including without limitation different adducts and / or histone modifications and / or histone variants and / or modified nucleotides and / or nucleosome measurements per se, or any combination or proportion of any of these and any other nucleosome epitopes, such as a indicator of a patient's healthy or sick state.
[081] We conclude that the method of the present invention is a successful method for the detection and measurement of nucleosome adducts containing particular proteins, and that this method is a superior method for the detection of nucleosome adducts to the methods of the current technique. The method is fast, inexpensive and suitable for use in complex biological media and fluids including blood and blood products. We have demonstrated that the method of the current invention can be used to detect nucleosome adducts in blood, and that this can be used as a biomarker for cancer. It will be clear to a person skilled in the art that a biomarker present in the blood has value for a wide variety of diagnostic and disease selection purposes for cancer and other diseases that are associated with high circulating nucleosomes (Holdenrieder et al, 2001).
[082] According to one aspect of the invention, a double antibody, immunometric or sandwich immunoassay method is provided for detecting and measuring free nucleosome adducts in a sample. One embodiment of this aspect is an immunoassay that comprises the steps of: (i) contacting a sample that may contain nucleosome adducts with a first antibody or another linker that binds to nucleosomes or a component thereof; (ii) contacting the nucleosomes or sample with a second antibody or other linker that binds to a protein that may be present as a nucleosome-protein adduct; (iii) detecting and / or quantifying the binding of said second antibody or other linker to a nucleosome-protein adduct in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of a nucleosome-protein adduct in a sample.
[083] In accordance with another aspect of the invention, a method is provided for detecting and measuring a free nucleosome adduct in a sample by an immunometric immunoassay comprising the steps of: (i) contacting a sample that may contain nucleosome adducts containing a particular protein with a first antibody or other linker that binds to the protein of interest; (ii) contacting the nucleosomes or sample with a second antibody or other linker that binds to nucleosomes or a component thereof; (iii) detecting and / or quantifying the binding of the second antibody or other linker to nucleosome samples with a second antibody or other linker that binds to the nucleosomes or a component thereof in the sample; and (iv) using the presence or degree of such a bond as a measure of the presence of a nucleosome adduct in the sample.
[084] It will be clear to those skilled in the art that the antibody or other linker used binds to nucleosomes or a component thereof in stage (i) of the first aspect above and stage (ii) of the second aspect above may be an antibody (or other ligator) directed against intact nucleosomes or any component part of a nucleosome including without limitation against a histone, a histone variant, a histone modification, a nucleotide, a modified nucleotide or other part of a nucleosome's DNA component. Then in a further aspect of the invention, a method is provided to detect (only) those nucleosome-protein adducts that additionally contain another characteristic to which this linker is directed including without limitation a modification of histone, histone variant or particular nucleotide. An advantage of this design is that the nucleosome component epitope and protein adduct epitope of the assay can be selected to be epitopes whose levels both differ greatly in healthy or ill patients, or in other states under investigation. So it is likely to reduce the proportion of nucleosomes detected by the assay, but to increase the clinical selectivity or specificity of the assay.
[085] We have done this assay design using an antibody targeting the H2AZ nucleosome component as the anti-nucleosome antibody in conjunction with anti-EZH2 antibodies and shown that nucleosome-EZH2 adducts specifically associated with H2AZ can be detected by such assays and that these assays can be used to discriminate samples taken from healthy and sick patients.
[086] In a further aspect of the invention the nucleosome adduct to be detected may contain more than one protein. Additional proteins in an adduct can be directly or indirectly linked to the nucleosome. For example, a nucleosome can be linked to an HMGB protein and in addition to an additional protein or proteins. The additional protein (s) can be linked directly to the nucleosome or can be linked to the HMGB protein, and then indirectly to the nucleosome. Nucleosome adducts can contain large protein complexes consisting of multi-protein components where the attachment of a particular protein in the complex adduct to the nucleosome can be through the connection of multiple intermediate bonds. It will be clear to those skilled in the art that a protein attached to a nucleosome in a nucleosome adduct, either directly or indirectly, can be detected by a method of the present invention.
[087] It will be clear to those skilled in the art that the methods of the invention described include a variety of modalities including biosensor type assays and free labeling assays of the type marketed, for example, by ForteBio Incorporated North American.
[088] In accordance with a further aspect of the invention, a method is provided for detecting the proportion of nucleosomes comprising a particular nucleosome adduct in a sample comprising the steps of: (i) detecting or measuring the level of nucleosomes in a sample; (ii) detecting or measuring the level of a nucleosome adduct according to a method of the current invention; and (iii) use both measures to determine the proportion of nucleosomes that comprise the nucleotide adduct.
[089] We have shown that the detection and measurement of nucleosome adducts in the blood of patients can be used as a diagnostic method to identify cancer patients and to differentiate them from healthy patients. According to a further aspect of the invention, a method is provided to detect or diagnose the presence of a disease by measuring or detecting the presence and / or the level or concentration of free nucleosome adducts in a body fluid, and using the level detected as a biomarker of a patient's disease state including, without limitation, a clinical diagnosis of a disease, a differential diagnosis of a disease type or subtype, or a disease prognosis, or a disease relapse, or a diagnosis of susceptibility to patient for 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 a sample is blood, serum or plasma. The response of the level, concentration or quantity of a nucleosome adduct in a body fluid can be expressed in absolute terms or relative terms, for example, without limitation as a proportion of the total nucleosome level present or as a proportion to the level of nucleosomes containing another nucleosome structure such as histone modification or at the level of total DNA.
[090] In an embodiment of the invention the measurement of the nucleosome adduct is used as a member of a diagnostic test panel or measures for the detection or diagnosis of a patient'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 patient's susceptibility to treatment regimens.
[091] In accordance with another aspect of the invention, a method is provided to detect or measure the presence and / or the level of chromatin binding of a protein in a cell comprising the steps of: (i) isolating chromatin from a cell : (ii) break down chromatin to form mono-nucleosomes and / or oligo-nucleosomes; and (iii) detecting or measuring the presence of a nucleosome adduct in the mono-nucleosomes and / or oligo-nucleosomes by means of an immunoassay method of the invention.
[092] Methods for producing mono-nucleosomes and / or oligo-nucleosomes from chromatin are well known in the art and include enzymatic digestion and sonification (Dai et al, 2011). We have demonstrated this aspect for nucleosomes produced from Hela and MCF7 cells.
[093] It will be clear to those skilled in the art that the terms antibody, linker or linker in relation to any aspect of the invention is not limiting, but intended to include antibody fragments, aptamers or any linker capable of binding to particular molecules or entities and that any suitable linker 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 such chromatin fragments that can be analyzed in the fluid medium.
[094] In accordance with another aspect of the invention, a kit is provided for detecting or measuring adducts comprising a specific ligand or linker for the nucleosome adduct or a component part thereof, or a structure / shape mimic of the adduct of nucleosome or component part thereof, along with instructions for using the kit in accordance with any of the methods defined herein.
[095] In accordance with another aspect of the invention, a method is provided to identify an adduct nucleosome biomarker for detecting or diagnosing disease status in animals or humans comprising the steps of: (i) detecting or measuring the level of an adduct of free nucleosome in a body fluid of sick patients; (ii) detecting or measuring the level of a free nucleosome adduct in the body fluid of control patients; and (iii) use the difference between the levels detected in the disease and control patients to identify whether a nucleosome adduct is useful as a biomarker for this disease.
[096] It will be clear to those skilled in the art that control patients can be selected on a variety of bases that may include, for example, patients known to be disease-free or may be patients with a different disease (for example, for research differential diagnosis).
[097] In accordance with a further aspect of the invention, a method is provided to identify an adduct nucleosome biomarker to assess the prognosis of a sick animal or human patient comprising the steps of: (i) detecting or measuring the level of an adduct free nucleosome in a body fluid of sick patients; and (ii) correlate the level of free nucleosome adduct detected in a body fluid of sick patients with the outcome of the patients' disease.
[098] In accordance with a further aspect of the invention, a method is provided to identify an adducting nucleosome biomarker to be used for the selection of a treatment regimen for a sick animal or human patient in need of treatment comprising the steps of: (i) detecting or measuring the level of a free nucleosome adduct in a body fluid of sick patients; and (ii) correlating the level of the free nucleosome adduct detected in a body fluid in sick patients with the observed effectiveness of a treatment regimen in these patients.
[099] In accordance with a further aspect of the invention, a method is provided to identify an adducting nucleosome biomarker to be used to monitor the treatment of a sick animal or human patient comprising the steps of: (i) detecting or measuring the level of free nucleosome adduct in a body fluid of a sick patient; (ii) repeat said detection or measurement on one or more occasions during the progression of the patient's disease; (iii) correlate the level of free nucleosome adduct detected in a patient's body fluid with the disease progression in the patient.
[0100] In accordance with a further aspect of the invention, a biomarker identified by the method as defined herein is provided.
[0101] A further aspect of the invention provides ligands or linkers, such as naturally occurring or chemically synthesized compounds, capable of binding specifically to the biomarker. A linker or linker according to the invention can 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 specifically binding to the biomarker. The antibody can be a monoclonal antibody or a fragment thereof capable of specifically binding to the biomarker. A linker according to the invention can be labeled with a detectable label, such as luminescent, fluorescent, enzyme or radioactive labels; alternatively or additionally a linker according to the invention can be labeled with an affinity label, for example, a biotin, avidin, streptavidin or His (e.g. hexa-His) label. Alternatively, ligand binding can be determined using a free tagging technology, for example, that of ForteBio Inc.
[0102] A biosensor according to the invention can comprise the biomarker or a structure / shape mimic capable of specifically binding to an antibody against the biomarker. Also provided is an arrangement comprising a binder or mimic as described herein.
[0103] Also provided by the invention is the use of one or more ligands as described herein, which can be naturally occurring or chemically synthesized, and is suitably a peptide, antibody or fragment thereof, aptamer or oligo-nucleotide, or the use of a biosensor of the invention, or an arrangement 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 done on a biological sample as defined herein.
[0104] Diagnostic or monitoring kits are provided to carry out the methods of the invention. Such kits will suitably comprise a binder according to the invention, for detection and / or quantification of the biomarker, and / or a biosensor, and / or an arrangement as described herein, optionally together with instructions for using the kit.
[0105] A further aspect of the invention is a kit for detecting the presence of a disease state, comprising a bionicensor capable of detecting and / or quantifying one or more biomarkers as defined herein.
[0106] 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 the response to the drug, the biomarker is useful for the identification of new therapeutic compounds in in vitro and / or in vivo assays. Biomarkers of the invention can be employed in methods for selecting compounds that modulate the activity of the biomarker.
[0107] Then, in a further aspect of the invention, the use of a linker or linker, as described, is provided, which can 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 arrangement 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.
[0108] Also provided is a method of identifying a substance capable of promoting or suppressing the generation of the biomarker in a patient, comprising administering the test substance to an animal patient and detecting and / or quantifying the level of the biomarker present in a test sample of the patient.
[0109] The term "biomarker" means a biological or biologically derived indicator that is distinctive of a process, event or condition. Biomarkers can be used in diagnostic methods, for example, clinical selection, and assessment of prognosis and in monitoring therapy results, identifying patients most likely to respond to a particular therapeutic treatment, drug selection and development. Biomarkers and their uses are valuable for identifying new treatment drugs and for discovering new targets for treatment drugs.
[0110] The terms "detecting" and "diagnosing" as used herein include identification, confirmation, and / or characterization of a disease state. Detection, monitoring and diagnosis methods according to 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. Detection, monitoring and diagnostic methods are also useful in methods for assessing therapeutic benefit, that is, for drug selection and drug development.
[0111] Efficient diagnostic and monitoring methods provide powerful "individual 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 harmful drug side effects), and reducing relapse rates.
[0112] In one embodiment, said biomarker is released from tumor cells. Then, in accordance with a further aspect of the invention, a method for detecting tumor growth is provided which comprises the steps of (i) measuring a biomarker in a biological sample that is associated with or released from cells of a tumor and (ii) demonstrate that the level of the referred biomarker is associated with the size, stage, aggressiveness or spread of the tumor.
[0113] Increased cell turnover, cell death and apoptosis are known to lead to increased circulatory levels of free nucleosomes (Holdenrieder et al, 2001). Level of free circulating nucleosomes is a non-specific indicator and occurs in a variety of conditions including inflammatory diseases, a wide variety of benign and malignant conditions, autoimmune diseases, as well as after trauma or ischemia (Holdenrieder et al, 2001). It will be clear to those skilled in the art that the invention will have application in a variety of disease areas where circulating nucleosomes have been found in patients. These include, without limitation, trauma (eg, severe injury or surgery), extreme exercise (eg, running and marathon), stroke and heart attack, sepsis or other serious infections and endometriosis.
[0114] Immunoassays of the invention include immunometric assays employing enzymatic detection methods (eg ELISA), fluorescence labeling immunometric assays, time-resolved fluorescence labeled immunometric assays, chemiluminescent immunometric assays, immuno-turbidimetric assays , labeled particulate immunometric assays and immunoradiometric assays and competitive immunoassay methods including competitive immunoassay methods with labeled antigen and labeled antibody with a variety of labeling types including radioactive, enzymatic, fluorescent, time-resolved and particulate fluorescence labels. All such immunoassay methods are well known in the art, see, for example, Salgame et al, 1997 and van Nieuwehnhuijze et al, 2003.
[0115] 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 body fluid (stool, tear, synovial fluid , sputum), breathing, for example, as condensed breathing, or an extract or purification of them, or dilution of them. Biological samples also include specimens from the patient alive, or post-mortem withdrawals. Samples can be prepared, for example, where appropriate diluted or concentrated, and stored in the usual manner.
[0116] In one embodiment, the method of the invention is repeated on multiple occasions. This modality provides the advantage of allowing the detection of results to be monitored over a period. Such an arrangement provides the benefit of monitoring or evaluating the effectiveness of treating a disease state. Such monitoring methods of the invention can be used to monitor onset, progression, stabilization, improvement, relapse and / or remission.
[0117] Then, the invention also provides a method of monitoring the effectiveness of a therapy for a disease state in a patient suspected of having such a disease, comprising detecting and / or quantifying the biomarker present in a biological sample of said patient. In monitoring methods, test samples can be taken on two or more occasions. The method may also include comparing the level of the biomarker (s) present in the test sample (s) with one or more controls and / or with one more test sample (s) ) earlier take (s) of the same test patient, for example, before the start of therapy, and / or of the same test patient at an earlier stage of therapy. The method may comprise detecting a change in the nature or quantity of the biomarker (s) in the test sample (s) taken on different occasions.
[0118] Then, according to a further aspect of the invention, a method is provided for monitoring the effectiveness of therapy for a disease state in a human or animal patient, comprising: (a) quantifying the amount of the biomarker as defined herein; and (b) comparing the amount of the referred biomarker in a test sample with the amount present in one or more controls and / or one or more test samples at an early time from the same test patient.
[0119] A change in the level of the biomarker in the test sample in relation to the level in a previous test sample taken previously from the same test patient may be indicative of a beneficial effect, for example, stabilization or improvement of said therapy of the suspected disorder or disorder. In addition, once the treatment has been completed, the method of the invention can be periodically repeated in order to monitor for disease recurrence.
[0120] Methods for monitoring the effectiveness of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human patients and in non-human animals (for example, in animal models). These monitoring methods can be incorporated into selections for new substances and combinations of drug substances.
[0121] In an additional modality, the monitoring of faster changes due to fast-acting therapies can be conducted at shorter intervals of hours or days.
[0122] In accordance with a further 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 done directly on the sample, or indirectly on an extract of the sample, or on a dilution of the sample.
[0123] In alternative aspects of the invention, the presence of the biomarker is assessed by detecting and / or quantifying antibodies or fragments thereof capable of specifically binding to the biomarker that are generated by the patient's body in response to the biomarker and are then present in a biological sample from a patient having a disease state.
[0124] Identification and / or quantification can be done by any suitable method to identify the presence and / or quantity of a specific protein in a biological sample from a patient or a purification or extract from a biological sample or a dilution thereof. In methods of the invention, quantification can be done 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 herein above. Samples can be prepared, for example, where appropriate diluted or concentrated, and stored in a usual manner.
[0125] Identification and / or quantification of biomarkers can be done by detecting the biomarker or a fragment thereof, for example, a fragment with a truncated C-terminus, or with a truncated N-terminus. 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. It is noted in particular that peptides of the same sequence or sequence related to those histone terminals are particularly useful fragments of histone proteins.
[0126] 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 biomarker-binding fragment thereof, or another peptide, or ligand, for example aptamer, or oligonucleotide, capable of specifically binding to the biomarker. The linker or linker may have a detectable marker, such as a luminescent, fluorescent or radioactive marker, and / or an affinity marker.
[0127] For example, detection and / or quantification can be done by one or more methods selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), 1-D gel based analysis, 1-D analysis based on 2-D gel, spec. mass (MS), reverse phase LC (RP), size permeation (gel filtration), ion exchange, affinity, HPLC, UPLC and other techniques based on LC or LC MS. Appropriate LC MS techniques include ICAT® (Applied Biosystems, CA, USA), or iTRAQ® (Applied Biosystems, CA, USA). Liquid chromatography (for example, high performance liquid chromatography (HPLC) or low pressure liquid chromatography (LPLC)), thin layer chromatography, NMR spectroscopy (nuclear magnetic resonance) can be used.
[0128] Diagnostic or monitoring methods according to the invention may comprise analysis of a sample by SELDI TOF or MALDI TOF to detect the presence or level of the biomarker. These methods are also suitable for clinical selection, prognosis, monitoring of therapy results, identification of patients most likely to respond to a particular therapeutic treatment, for drug selection and development, and identification of new drugs for drug treatment.
[0129] Identification and / or quantification of analyte biomarkers can be done using an immunological method, involving an antibody, or a fragment of it capable of specifically binding to the biomarker. Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the analyte biomarkers is done using two antibodies that recognize different epitopes in an analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle based immunoassay (for example, using particle particles of gold, silver, or latex particles, magnetic particles, Q-dots). Immunological methods can be done, for example, in a microtiter plate or in the form of strips.
[0130] In one embodiment, one or more of the biomarkers can be replaced by a molecule or a measurable fragment of the molecule, found upstream or downstream of the biomarker in a biological pathway.
[0131] The identification of disease-specific key biomarkers is central to integrating diagnostic procedures and therapeutic regimens. Use of diagnostic tools of appropriate predictive biomarkers such as biosensors can be developed; consequently, in methods and uses of the invention, identification and quantification can be done using a bionic sensor, microanalytic system, microengineering system, micro separation system, immunochromatography system or other suitable analytical devices. The biosensor can incorporate an immunological method for detecting the biomarker (s), electrical, technical, magnetic, optical (for example, hologram) or acoustic technologies. Using such biosensors, it is possible to detect the target biomarker (s) at the anticipated concentrations found in biological samples.
[0132] As used herein, the term "biosensor" means anything capable of detecting the presence of the biomarker. Examples of biosensors are described here.
[0133] Biosensors according to the invention can comprise a linker linker or linkers, as described herein, capable of specifically binding to the biomarker. Such biosensors are useful in the detection and / or quantification of a biomarker of the invention.
[0134] The biomarker (s) of the invention can be detected using a biosensor incorporating technologies based on "intelligent" holograms, or high frequency acoustic systems, such systems are particularly amenable to "code" configurations bar "or arrangement.
[0135] In smart hologram sensors (Smart Holograms Ltd, Cambridge, UK), a holographic image is stored on a thin polymeric film that is sensitized to react specifically with the biomarker. On display, the biomarker reacts with the polymer leading to a change in the image revealed by the hologram. Reading the test result can be a change in the brightness, image, color and / or position of the image. For qualitative and semi-quantitative applications, a sensory hologram can be read visually, thus removing the need for detection equipment. A simple color sensor can be used to read the signal when quantitative measurements are required. Opacity or color of the sample does not interfere with sensor operation. The shape of the sensor allows multiplexing for simultaneous detection of various substances. Reversible and irreversible sensors can be designed to meet different requirements, and continuous monitoring of a particular biomarker of interest is feasible.
[0136] Suitably, biosensors for detecting one or more biomarkers of the invention combine biomolecular recognition with appropriate means to convert detection of the presence, or quantification of the biomarker in the sample into a signal. Biosensors can be adapted for diagnostic testing of "alternative site", for example, in the unit, departments without patients, surgery, home, field and work.
[0137] Biosensors to detect one or more biomarkers of the invention include acoustic sensors, plasmon resonance, halography, Bio-Layer Interferometry (BLI) and microengineering. Printed recognition elements, thin film transistor technology, magnetic acoustic resonator devices and other new acoustic-electric systems can be used in biosensors to detect one or more biomarkers of the invention.
[0138] Methods involving identification and / or quantification of one or more bi-markers of the invention can be done on bench instruments, or they can be incorporated in disposable, diagnostic or monitoring platforms that can be used in a non-laboratory environment, for example for example, in doctors 'offices or at patients' bedside. Suitable biosensors for developing methods of the invention include "credit" cards with optical or acoustic readers. Biosensors can be configured to allow the collected data to be electronically transmitted to the physician for interpretation and then can form the basis for e-medicine.
[0139] 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 linker, or linkers, specific to the biomarker or a structure / shape mimic of the biomarker, one or more controls les, one or more reagents and one or more consumables; optionally together with instructions for using the kit in accordance with any methods defined here.
[0140] The identification of biomarkers for a disease state allows integration of diagnostic procedures and therapeutic regimes. Detection of a biomarker of the invention can be used to select patients before participating in clinical trials. Biomarkers provide the means to indicate therapeutic response, failure of response, unfavorable side effect profile, degree of drug compliance and achievement of adequate serum drug levels. Biomarkers can be used to provide warnings of adverse drug response. Biomarkers are useful in the development of personalized therapies, just as response assessment can be used to adjust the dose, minimize the number of prescribed drugs, reduce the delay in reaching effective therapy and avoid adverse drug reactions. Then, by monitoring a biomarker of the invention, the patient's care can be precisely adapted to suit the needs determined by the disorder and the pharmacogenomic profile of the patient, the biomarker can then be used to titrate the optimal dose, predict a response positive therapy and identify those patients at high risk for severe side effects.
[0141] Tests based on biomarkers provide a first line of assessment for "new" patients, and provide objective measures for accurate and rapid diagnosis, not achievable using current measures.
[0142] In addition, diagnostic biomarker tests are useful for identifying family members or patients with mild or asymptomatic disease or who may be at high risk for developing symptomatic disease. This allows initiation of appropriate therapy, or preventive measures, for example, management of risk factors. These approaches are recognized for improving the outcome and for preventing the explicit onset of the disorder.
[0143] Methods of monitoring biomarkers, biosensors and kits are also vital as tools for monitoring patients, to enable the physician to determine whether the recurrence is due to the worsening of the disorder. If pharmacological treatment is judged to be inadequate, then therapy can be reinstalled or increased; a change in therapy can be given if appropriate. Because biomarkers are sensitive to the condition of the disorder, they provide an indication of the impact of drug therapy.
[0144] The invention will now be illustrated with reference to the following non-limiting examples. EXAMPLE 1
[0145] Serum samples were taken from 5 healthy patients, 3 patients with colon cancer, 6 patients with lung cancer and 2 patients with pancreatic cancer. A commercially available preparation produced by digesting chromatin extracted from Hela cells, in which the DNA and proteins in the nucleosome are cross-linked for stability, has been serially diluted in horse serum. A nucleosome preparation in human blood was prepared according to the Holdenrieder method (* Holdenrieder et al; 2001). These samples and preparations were evaluated in duplicate for nucleosome-EZH2 adduct by the method of the invention. Commercially available purified horse serum produced for use in tissue culture was also evaluated as a negative control sample containing no nucleosomes or nucleosome adducts.
[0146] The ELISA method used a solid-phase anti-histone capture antibody that binds to intact nucleosomes and a biotinylated monoclonal anti-EZH2 detection antibody as follows: a solution of the anti-histone antibody in 0.1 M 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 antihistone antibody was decanted. A solution of bovine serum albumin (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, 0.05 M TRIS / HCl pH 7.5 buffer containing 1% Tween 20). Serum sample (10 μL / well) and assay buffer (50 pL / well, 0.05 M TRIS / HCl pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and 1% substitute Nonidet P40 ) was added to the wells incubated overnight at 4 ° C. The serum and assay buffer mixture was decanted and the wells were washed three times with wash buffer (200 μL / well). A biotinylated anti-EZH2 detection antibody solution was added (50 μL / well) and incubated for 90 minutes at room temperature with light 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 light 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-sulphonic acid] -diammonium salt) was added and incubated for 20 minutes at room temperature with light agitation. The optical density (OD) of the wells was measured at a wavelength of 405 nm using a standard microtiter plate reader. A color dose-response curve increasing with increased concentration of nucleosome-EZH2 adduct was observed, with a low background signal in the absence of the nucleosome adduct (horse serum). The positive ELISA signal indicates that the EZH2 detected by the ELISA is incorporated into a nucleosome-EZH2 adduct comprising both histone protein and EZH2 since (i) the capture antibody binds to the histones in the sample and (ii) the detection binds to the adduct's EZH2 component. The results are shown in Figures 1 and 2. EXAMPLE 2
[0147] Serum samples were taken from 5 healthy patients, 3 colon cancer patients, 6 lung cancer patients and 2 pancreatic cancer patients. A commercially available nucleosome preparation produced by digesting the chromatin extracted from Hela cells was serially diluted in horse serum. A nucleosome preparation in human blood was prepared according to the Holdenrieder method (* Holdenrieder et al; 2001). These samples and preparations were analyzed in duplicate for nucleosome-HMGB1 adduct by the method of the invention. Purified horse serum was also evaluated as a negative control sample containing no nucleosome or nucleosome adducts.
[0148] The ELISA method used a solid-phase anti-histone capture antibody that binds to intact nucleosomes and a biotinylated monoclonal anti-HMGB1 detection antibody as follows: An anti-histone antibody solution in 0.1 M buffer phosphate 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 antihistone antibody was decanted. A solution of bovine serum albumin (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 washing buffer (200 μL / well, 0.05 M TRIS / HCl pH 7.5 buffer containing 1% Tween 20). Serum sample (10 μL / well) and assay buffer (50 pL / well, 0.05 M TRIS / HCl pH 7.5 containing 0.9% NaCl, 0.05% sodium deoxycholate and 1% substitute Nonidet P40 ) were added to the wells incubated overnight at 4 ° C. The serum and assay buffer mixture was decanted and the wells were washed three times with wash buffer (200 μL / well). A biotinylated anti-HMGB1 detection antibody solution was added (50 μL / well) and incubated for 90 minutes at room temperature with light 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 light 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-ethylbenzo-triazoline-6-sulfonic acid] -diammonium salt) was added and incubated for 20 minutes at room temperature with light 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 increasing HMGB1 nucleosome-adduct concentration was observed, with a low background signal observed in absence of the nucleosome adduct (horse serum) The positive ELISA signal indicates that the HMGB1 detected by ELISA is incorporated into a nucleosome-HMGB1 adduct comprising both histone protein and HMGB1 since (i) the capture antibody is binds to histones in the sample and (ii) the detection antibody binds to the adduct's HMGB1 component, the results are shown in Figures 3 and 4.
[0149] In a larger experiment, serum samples were taken from 25 colon cancer patients, 25 breast cancer patients and 24 lung cancer patients, as well as samples from 31 healthy patients. The samples were tested for nucleosome-HGB1 level and, using the mean of the healthy result plus 2 standard deviations from the mean as a limit, the following results were obtained for colon, breast and lung cancer: • Colon: 76% of cancers were detected (19 out of 25 patients) and 90% specificity (3 false positives from 31 healthy samples); • Breast: 96% of cancers were detected (24 out of 25 patients) and 90% specificity (3 false positives from 31 healthy samples); and • Lung: 100% of cancers were detected (24 out of 24 patients) and 86% specificity (4 false positives from 28 healthy samples); where a nucleosome-HMGB1 adduct level measured above the cutoff level is considered a positive result and a lower level is considered a negative result. The results are shown in Figure 5.
[0150] The assay for nucleosome-HMGB1 adduct levels was also performed in the reverse format where the anti-HMGB1 antibody was coated into wells as a capture antibody and the anti-nucleosome antibody was biotinylated and used as a detection antibody. This assay format also successfully detected nucleosome-HMGB1 adducts in the positive controls used (DO405nm = 1.15) but not in horse serum or buffer (both DO406 nm = 0.13). EXAMPLE 3
[0151] An ELISA assay for nucleosome-PR was performed using the method of Example 1 above, except that the biotinylated antibody used was targeted to bind to the progesterone receptor (PR). The results are shown in Figure 6. EXAMPLE 4
[0152] Serum samples taken from two prostate cancer patients, as well as positive and negative controls, were evaluated using an ELISA assay for nucleosome-AR adduct performed using the method in EXAMPLE 1 above, except that the biotinylated antibody used was targeted to bind to the androgen receptor (AR). The results are shown in Figure 7. EXAMPLE 5
[0153] An ELISA assay for nucleosome-ERα adduct was performed using a nucleosome sample prepared by the method of * Holdenrieder et al; 2001 by the method of Example 1 above, except that the biotinylated antibody used was targeted to bind to the alpha form of the estrogen receptor (ERα). The results are shown in Figure 8. EXAMPLE 6
[0154] A nucleosome sample was prepared by digesting the chromatin extracted from MCF7 cells and evaluated for nucleosome-ERβ adduct by ELISA. The assay was performed by a method similar to that of Example 1 above, except that the assay was done using a different anti-nucleosome antibody and an antibody targeted to bind to the beta form of the estrogen receptor (ERβ). The test was carried out in two different formats. In the first format, the anti-nucleosome antibody was coated in the wells and the anti-ERβ antibody was biotinylated. In the second format, the anti-ERβ antibody was coated in the wells and the anti-nucleosome antibodies were biotinylated. The trial was a success in both formats. Interestingly, the assay appeared to perform poorly when MCF7 chromatin was cross-linked, as is often done in ChIP methods. The results are shown in Figure 9. EXAMPLE 7
[0155] An ELISA assay for the nucleosome-H2AZ-ERβ adduct was performed using a nucleosome sample prepared by the method of * Holdenrieder et al; 2001 by the method of Example 6 above where the anti-ERβ antibody was coated in the wells, except that the biotinylated antibody used was directed to bind the histone variant H2AZ such that only the subgroup of nucleosome-ER β adducts containing H2AZ were detected. Using this method, where the nucleosome linker or nucleosome component is directed to link to an epigenetic signal structure, it is possible to detect a particular subset of nucleosome adducts containing only that epigenetic signal. The results are shown in Figure 10. EXAMPLE 8
[0156] Serum samples were taken from 12 healthy patients, 3 colon cancer patients, 6 breast cancer patients, 3 lung cancer patients and 4 pancreatic cancer patients. A nucleosome preparation in human blood was prepared according to the Holdenrieder method (* Holdenrieder et al; 2001) and was serially diluted in horse serum. These samples and preparations were evaluated in duplicate for nucleosome-ERβ adduct by the method of the invention. Purified horse serum commercially available for use in tissue culture was also evaluated as a negative control sample containing no nucleosomes or nucleosome adducts. The assay was performed using the method of Example 1 above, except that the detection antibody used was directed against the estrogen receptor (ERβ). Using a calculated limit as the mean of the healthy result plus 2 standard deviations from the mean; 2 out of 3 colon cancer samples, 3 out of 6 breast cancer samples, 2 out of 3 lung cancer samples and 4 out of 4 pancreatic cancer samples were found positive for ER-nucleosome adducts. The results are shown in figure 11. EXAMPLE 9
[0157] An ELISA assay for a steroid estrogen-estrogen receptor nucleosome is performed using the method of Example 6 above, except that the detection antibody used was targeted against estrogen steroid. This assay thus detected only nucleosome-estrogen receptor adducts that additionally contained steroid hormone. EXAMPLE 10
[0158] An ELISA assay with an estrogen-estrogen receptor-nucleosome adduct is performed using a method similar to that of the Example above, except that the assay is done on a microtiter plate or tube that is resistant to organic solvents and, after anti-nucleosome capture antibody to capture the nucleosome-estrogen receptor adducts on the surface of the wall, the liquid content of the well is decanted and diethyl ether is added to dissolve any steroid present in the captured adduct. The ether is transferred to another well or tube and dried. The dry extract is redissolved in assay buffer and the estrogen concentration is determined using a classic competitive immunoassay method for estrogen analysis. This assay thus detects only nucleosome-estrogen receptor adducts that additionally contain estrogen. EXAMPLE 11
[0159] An ELISA assay for retinoic acid-retinoic acid receptor nucleosome is performed using the method of Example 10 above, except that the competitive steroid immunoassay used was directed against retinoic acid. This assay thus detects only nucleosome-retinoic acid adducts that additionally contain steroidal retinoic acid. EXAMPLE 12
[0160] Nucleosome adduct assays similar to those described in Examples 1-10 are performed, except that the solid phase coated antibody used was directed against 5-methylcytidine. These assays thus detect only nucleosome-hormone receptor adducts and nucleosome-hormone-receptor complex adducts that are additionally associated with methylated DNA. REFERENCES Allen et al, A simple method for estimating global DNA methylation using bisulfite PCR of repetitive DNA elements. Nucleic Acids Research: 32 (3) e38DOI: 10.1093 / nar / gnh032, 2004 Bawden et al, Detection of histone modification in cell-free nucleosomes. WO 2005/019826, 2005 Cao et al, Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing SCIENCE 298, 1039-1043, 2002 Dai et al, Detection of Post-translational Modifications on Native Intact Nucleosomes by ELISA. http://www.jove.com/details.php id=2593 doi: 10.3791 / 2593. J Vis Exp. 50 (2011). Esteller, Cancer epigenomics: DNA methylomes and histone-modification maps Nature Reviews Genetics: 8, 286-298, 2007. Feinberg and Vogelstein, Hypomethylation distinguishes genes of some human cancers from their normal counterparts. Nature: 301, 89-92, 1983. Fullgrabe et al, Histone onco-modifications. Oncogene: 30, 3391-3403, 2011. Gerlitz et al, The dynamics of HMG protein-chromatin interactions in living cells. Biochem Cell Biol, 87, 127-137, 2009. Grutzmann et al, Sensitive Detection of Colorectal Cancer in Peripheral Blood by Septin 9 DNA Methylation Assay. PLoS ONE 3 (11): e3759. doi: 10.1371 / newspaper.pone.0003759, 2008 Herranz and Esteller, DNA methylation and histone modifications in subjects with cancer: potential prognostic and therapeutic targets. Methods Mol Biol.361: 25-62, 2007. Hervouet et al, Disruption of Dnmt1 / PCNA / UHRF1 Interactions Promotes Tumorigenesis from Human and Mice Glial Cells PLoS ONE 5 (6): e11333. doi: 10.1371 / journal.pone.0011333, 2010. Holdenrieder et al, Nucleosomes in serum of subjects with benign and malignant diseases. Int. J. Cancer (Pred. Oncol.): 95, 114-120, 2001. * Holdenrieder et al, Nucleosomes in Serum as a Marker for Cell Death. Clin Chem Lab Med; 39 (7), 596-605, 2001. Holdenrieder et al, Cell-Free DNA in Serum and Plasma: Comparison of ELISA and Quantitative PCR. Clinical Chemistry: 51 (8), 1544-1546, 2005. Holdenrieder and Stieber, Clinical use of circulating nucleosomes. Critical Reviews in Clinical Laboratory Sciences; 46 (1): 1-24, 2009. Ricke and Bielinsky, Easy detection of chromatin binding proteins by the histone association assay. Biol Proced Online; 7 (1), 60-69, 2005. Rodriguez-Paredes and Esteller, Cancer epigenetics reaches mainstream oncology. Nature Medicine: 17 (3), 330-339, 2011. Salgame et al, An ELISA for detection of apoptosis. Nucleic Acids Research, 25 (3), 680-681, 1997. Sims et al, HMGB1 and RAGE in inflammation and cancer. Annu. Rev. Immunol. 28, 367-388, 2010. Stoetzer et al, Circulating nucleosomes and biomarkers of immunogenic cell death as predictive and prognostic markers in cancer patients undergoing cytotoxic therapy. Expert Opin Biol Ther. 12 (Suppl. 1): S217-S224, 2012. Tang et al, High-mobility Group Box 1 [HMGB1] and Cancer. Biochim Biophys Acta. 1799 (1-2) 131, 2010. Urbonaviciute et al, Induction of inflammatory and immune responses by HMGB1 - nucleosome complexes: implications for the pathogenesis of SLE. J Exp Med, 205 (13), 3007-3018, 2008. Urbonaviciute and Voll, High-mobility group box 1 represents a potential marker of disease and novel therapeutic target in systemic lupus erythematosus. J Internal Medicine, 270, 309-318, 2011. van Nieuwenhuijze et al, Time between onset of apoptosis and release of nucleosomes from apoptotic cells: putative implications for sysytemic lupus erythematosus. Ann Rheum Dis; 62: 10-14, 2003. Yoshida and Shimura, Isolation of nonhistone chromosomal protein from calf thymus. Biochimica et Biophysica Acta (BBA) - Protein Structure; 263 (3), 690-695, 1972.

权利要求:
Claims (13)
[0001]
1. Use of a nucleosome-protein adduct CHARACTERIZED by the fact that it is used as a biomarker in the blood for the diagnosis of cancer, autoimmune disease or inflammatory disease, in which the nucleosome-protein adduct includes a chromatin-modifying enzyme or a nuclear receptor of hormone.
[0002]
2. Method for detecting the presence of a nucleosome-protein adduct in a body fluid sample, in which the nucleosome-protein adduct includes a chromatin-modifying enzyme or a nuclear hormone receptor, CHARACTERIZED by the fact that it comprises the steps of: (i) contacting the body fluid sample with a first binding agent that binds to the nucleosomes or a component thereof; (ii) contacting the nucleosomes linked in step (i) with a second binding agent that binds to a protein added to the nucleosome; (iii) detecting or quantifying the binding of said second protein binding agent added to the body fluid sample; and (iv) using the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the body fluid sample.
[0003]
3. Method for detecting the presence of a nucleosome-protein adduct in a body fluid sample, in which the nucleosome-protein adduct includes a chromatin-modifying enzyme or a nuclear hormone receptor, CHARACTERIZED by the fact that it comprises the steps of: (i) contacting a sample of body fluid with a first binding agent that binds to a protein added to a nucleosome; (ii) contacting the nucleosomes linked in step (i) with a second binding agent that binds to the nucleosomes or a component thereof; (iii) detecting or quantifying the binding of said second binding agent to the nucleosomes or a component thereof in the body fluid sample; and (iv) using the presence or degree of such a bond as a measure of the presence of the nucleosome adducts in the body fluid sample.
[0004]
4. Use or method according to any one of claims 1 to 3, CHARACTERIZED by the fact that the chromatin modifying enzyme is an acetylation, deacetylation, methylation, demethylation, phosphorylation, dephosphorylation, ubiquitination, deubiquitination, sumoilation, dehumilation histone or DNA methyl transferase.
[0005]
5. Use or method according to any one of claims 1 to 4, CHARACTERIZED by the fact that the chromatin-modifying enzyme is EZH2.
[0006]
6. Use or method according to any one of claims 1 to 5, CHARACTERIZED by the fact that the nuclear hormone receptor is an estrogen receptor, androgen receptor, progesterone receptor, thyroid hormone receptor or acid receptor retinoic.
[0007]
7. Use or method according to any one of claims 1 to 6, CHARACTERIZED by the fact that the nuclear hormone receptor is additionally linked to the hormone that is selected from a thyroid hormone, a estrogen, an androgen or retinoic acid.
[0008]
8. Method according to any one of claims 2 to 7, CHARACTERIZED by the fact that the nucleosome linker or nucleosome component is directed to bind to a particular epigenetic signal structure such that only a particular subgroup of adducts of nucleosome containing said epigenetic signal structure is detected.
[0009]
Method according to any one of claims 2 to 8, CHARACTERIZED by the fact that the binding agent is an antibody, an antibody fragment or an aptamer.
[0010]
10. Method according to any one of claims 2 to 9, CHARACTERIZED by the fact that the body fluid sample is selected from blood, serum or plasma.
[0011]
11. Method according to any one of claims 2 to 10, CHARACTERIZED by the fact that it is for use in the detection of adducts of the hormone-hormone-nucleosome receptor complex.
[0012]
12. Method according to claim 11, CHARACTERIZED by the fact that the adducts of the hormone-hormone-receptor-nucleosome complex comprise an adduct of the thyroxine-receptor-thyroid-nucleosome complex, an adduct of the triiodothyronine-receptor complex of thyroid-nucleosome hormone, an adduct of the retinoic acid-retinoic acid-nucleosome receptor complex, an adduct of the androgen-androgen receptor-nucleosome complex or an adduct of the estrogen-estrogen receptor-nucleosome complex.
[0013]
13. Method according to claim 11 or 12, CHARACTERIZED by the fact that it comprises the step of extracting the hormone from an adduct of the hormone-nucleosome receptor complex captured by an antibody followed by a quantification step.

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法律状态:
2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-08-13| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

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2020-07-28| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2020-11-17| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-02-02| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 07/12/2012, 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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US201161568090P| true| 2011-12-07|2011-12-07|

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GB1121040.8|2011-12-07|

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US61/568,090|2011-12-07|

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GB201121040A|GB201121040D0|2011-12-07|2011-12-07|Method for detecting nucleosome adducts|

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GBGB1121230.5A|GB201121230D0|2011-12-12|2011-12-12|Methods of detecting nucleosome adducts|

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GB1121230.5|2011-12-12|

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PCT/GB2012/053057|WO2013084002A2|2011-12-07|2012-12-07|Method for detecting nucleosome adducts|

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