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WO2024153619A1 - Protéines et procédés d'isolement de nucléosomes circulants - Google Patents

Protéines et procédés d'isolement de nucléosomes circulants Download PDF

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WO2024153619A1
WO2024153619A1 PCT/EP2024/050871 EP2024050871W WO2024153619A1 WO 2024153619 A1 WO2024153619 A1 WO 2024153619A1 EP 2024050871 W EP2024050871 W EP 2024050871W WO 2024153619 A1 WO2024153619 A1 WO 2024153619A1
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dna
nucleosomes
protein
histone
sample
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Priscilla VAN DEN ACKERVEKEN
Mark Edward Eccleston
Marielle HERZOG
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Belgian Volition SPRL
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
    • C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
    • C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57484—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites
    • G01N33/57488—Immunoassay; Biospecific binding assay; Materials therefor for cancer involving compounds serving as markers for tumor, cancer, neoplasia, e.g. cellular determinants, receptors, heat shock/stress proteins, A-protein, oligosaccharides, metabolites involving compounds identifable in body fluids
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6875—Nucleoproteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the invention relates to proteins and methods for detecting and isolating circulating, cell free nucleosomes, in particular nucleosomes of disease origin. Such methods allow for improved analysis of genetic and epigenetic markers associated with nucleosomes of disease origin.
  • Cellular DNA exists as a protein-nucleic acid complex called chromatin.
  • the nucleosome is the basic unit of chromatin structure and consists of DNA wound around a protein complex.
  • the DNA is wound around consecutive nucleosomes in a structure often said to resemble “beads on a string” and this forms the basic structure of open or euchromatin. In compacted or heterochromatin this string is coiled and super coiled in a closed and complex structure.
  • Each nucleosome in chromatin consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex are wrapped approximately 145 base pairs (bp) of DNA.
  • Another histone, H1 which may be located on the nucleosome outside of the core histones, binds a further 20bp of DNA to produce nucleosomes (or chromatosomes) containing approximately 165bp of DNA.
  • Histone H1 is said to act as a linker histone and the additional DNA is often referred to as “linker DNA”, i.e. the DNA connecting one nucleosome to another in chromosomes.
  • the linker DNA separating two nucleosomes in a chromosome is sometimes longer than 20bp and may be up to 80bp in length.
  • Circulating cell free nucleosomes are reported to comprise predominantly mononucleosomes together with associated DNA produced as chromatin fragments by digestion of chromatin on cell death.
  • DNA abnormalities are characteristic of all cancer diseases.
  • the DNA of cancer cells differs from that of healthy cells in many ways including, but not limited to, point mutations, translocations, gene copy number, micro-satellite abnormalities, DNA strand integrity and nucleotide modifications (for example methylation of cytosine at position 5).
  • These tumour- associated-alterations in DNA structure or sequence are investigated routinely in cancer cells or tissue removed at biopsy or surgery for clinical diagnostic, prognostic and treatment selection purposes. Tumour genetic and epigenetic characteristics vary between different tumour types and between different patients with the same tumour disease.
  • tumour DNA in cells removed at surgery or biopsy may help the clinician to assess minimal residual disease, predict patient prognosis, select appropriate treatments for the patient, monitor disease progression and detect any relapse or acquired treatment resistance at an early stage (possibly many months earlier than radiological detection) and allow potentially successful changes in treatment courses.
  • tissue DNA tests have limitations as invasive biopsy procedures cannot be performed repeatedly on patients. For some patients, biopsy may not be used at all. Biopsy is expensive to perform, uncomfortable for the patient, poses patient risk, and may lead to surgical complications.
  • a tumour in a patient may consist of multiple tumoural clones located within different areas of the same tumour or within different metastases (in metastatic cancer) not all of which may be sampled by biopsy.
  • a tissue biopsy DNA investigation therefore provides a snap-shot of the tumour, both in time and in space, amongst different tumour clones located within different areas of a tumour at one particular moment in time.
  • tumour DNA circulating tumour DNA
  • Tumour derived ctDNA circulates as small DNA fragments consistent with the size expected for mononucleosomes.
  • Investigation of matched blood and tissue samples from cancer patients shows that cancer associated mutations, present in a patient’s tumour (but not in his/her healthy cells) are also present in ctDNA in blood samples taken from the same patient (Newman et al, 2014).
  • DNA sequences that are differentially methylated (epigenetically altered by methylation of cytosine residues) in cancer cells can also be detected as methylated sequences in ctDNA in the circulation.
  • cfDNA cell-free circulating DNA
  • tumour burden so disease progression may be monitored both quantitatively by the proportion of ctDNA present and qualitatively by its genetic and/or epigenetic composition.
  • Analysis of ctDNA can produce highly useful and clinically accurate data pertaining to DNA originating from all or many different clones within the tumour and which integrates the tumour clones spatially. Moreover, repeated sampling over time is a much more practical and economic option. Analysis of ctDNA has the potential to revolutionize the detection and monitoring of tumours, as well as the detection of relapse and acquired drug resistance at an early stage for selection of treatments for tumours through the investigation of tumour DNA without invasive tissue biopsy procedures.
  • Such ctDNA tests may be used to investigate all types of cancer associated DNA abnormalities (e.g. point mutations, nucleotide modification status, translocations, gene copy number, micro-satellite abnormalities and DNA strand integrity) and would have applicability for routine cancer screening, regular and more frequent monitoring and regular checking of optimal treatment regimens (Zhou et al, 2012).
  • cancer associated DNA abnormalities e.g. point mutations, nucleotide modification status, translocations, gene copy number, micro-satellite abnormalities and DNA strand integrity
  • Blood, plasma or serum may be used as a substrate for ctDNA assays and any DNA analysis method may be employed including, without limitation, genetic DNA sequencing, epigenetic DNA sequencing analysis (e.g. for sequences containing 5-methylcytosine), PCR, BEAMing, NGS (targeted or whole genome), digital PCR, isothermal DNA amplification, cold PCR (coamplification at lower denaturation temperature-PCR), MAP (MIDI-Activated Pyrophosphorolysis), PARE (personalized analysis of rearranged ends) and Mass Spectrometry.
  • genetic DNA sequencing e.g. for sequences containing 5-methylcytosine
  • PCR e.g. for sequences containing 5-methylcytosine
  • BEAMing e.g. for sequences containing 5-methylcytosine
  • NGS targeted or whole genome
  • digital PCR isothermal DNA amplification
  • cold PCR coamplification at lower denaturation temperature-PCR
  • MAP MIDI-Activated Pyrophosphorolysis
  • ctDNA tests have potential applicability in all cancer diseases.
  • Cancers investigated include, without limitation, cancer of the bladder, breast, colorectal, melanoma, ovary, prostate, lung liver, endometrial, ovarian, lymphoma, oral, leukaemias, head and neck, and osteosarcoma (Crowley et al, 2013; Zhou et al, 2012; Jung et al, 2010).
  • the nature of ctDNA tests will now be illustrated by outlining some (non-limiting) example approaches.
  • the first example involves the detection of a cancer associated gene sequence mutation in ctDNA.
  • Blood tests involving the detection of a single gene mutation in ctDNA generally have low clinical sensitivity. There are two reasons for this. Firstly, although all cancers have mutations, the frequency of any particular mutation in a particular cancer disease is usually low. For example, although K-RAS and P53 mutations are regarded as two of the more frequent cancer mutations and have been studied in a wide range of cancers including bladder, breast, colon, lung, liver, pancreas, endometrial and ovarian cancers, they were detected in 23%-64% and 17%-54% of cancer tissue samples respectively.
  • K- RAS and P53 mutations could be detected in the ctDNA of 0%-75% of K-RAS and P53 tissue positive patients. The sum of these two effects meant that K-RAS or P53 mutations were detected in the blood of less than 40% of cancer patients (Jung et al, 2010).
  • a second example involves the detection of multiple cancer associated gene sequence mutations in ctDNA.
  • mutations of any particular gene such as K-RAS or P53 may be present in only a minority of cancers, all cancers contain mutations therefore a sufficiently large panel of mutations should in principle facilitate the detection of most or even all tumours.
  • One way to increase the clinical sensitivity of such tests is therefore to test for a wide range of mutations in many genes.
  • Newman et al. have taken this approach for non-small cell lung cancer (NSCLC) and investigated 521 exons and 13 intron sequences from 139 recurrently mutated genes.
  • the mutations studied encompassed multiple classes of cancer associated genetic alterations, including single nucleotide variation (SNV) and fusion genes. In this way the authors reported the detection of more than 95% of stage ll-IV tumours and 50% of stage I tumours with 96% specificity in ctDNA blood tests (Newman et al, 2014).
  • SNV single nucleotide variation
  • a third example involves the detection of cancer associated epigenetic alterations to particular gene sequences in ctDNA. This approach can be applied to any DNA or nucleotide modification.
  • a prime example of this approach is the detection of genes which are differentially methylated at cytosine residues in certain cancers. A large number of genes have been investigated for this purpose in a variety of cancers.
  • a few of these are SEPTIN-9, APC, DAPK, GSTP1, MGMT, P16, RASSF1A, T1G1, BRCA1, ERa, PRB, TMS1, MLH1, HLTF, CDKN2A, SOCS1, SOCS2, PAX5, PGR, PTGS2 and RAR[32 investigated in bladder, breast, colorectal, melanoma, ovarian and prostate cancers.
  • bisulfite conversion sequencing methods are used in which DNA is extracted from plasma and then treated with bisulfite which converts unmodified cytosine residues to uracil. Sequencing, PCR or other methods can then be applied to determine whether a particular methylated gene sequence is present.
  • a fourth example is the “fragmentomics” approach involving sequence analysis of circulating DNA fragments and comparison with the results of nuclease-accessible site analysis (also known as DNase hypersensitivity analysis) of tissues and cell lines.
  • nuclease-accessible site analysis also known as DNase hypersensitivity analysis
  • Protein bound DNA is protected from nuclease digestion and, following extraction, may be sequenced to identify the unique DNA protein occupancy pattern (or unoccupied open DNA pattern) of a cell type. Circulating cfDNA fragments are similarly protected by protein binding which may be histone in nature, as in nucleosomes, or may be by other proteins such as transcription factors. The boundaries of cfDNA fragments relate to their binding to nucleosomes, transcription factors or other proteins and the fragmentation patterns obtained by sequencing an individual’s cfDNA can be built into a map of nucleosome and other protein occupancy. Such cfDNA occupancy maps can be compared to the occupancy maps for known tissues or cancer cell lines derived as nuclease- accessible site maps.
  • a fifth example involves analysis of global genome DNA methylation patterns in cancer cells and healthy cells in subjects.
  • the DNA of a healthy cell displays a globally dispersed pattern of methylation across the entire genome which reflects the epigenetic state of the particular cell and tissue.
  • the transition of cells from a healthy state to a malignant cancer cell involves a global net loss of DNA methylation (5-methylcytosine) together with localised increases in the levels of 5-methylcytosine residues within regulatory (e.g. gene promoter) regions of the genome with abundant CpG sites clustered together within a short regions of DNA.
  • regulatory e.g. gene promoter
  • ctDNA fragments from cancer cells tend to be either very hypomethylated or very hypermethylated with relatively little in between (/.e. at both extremes of methylation level) whereas DNA from normal cells has a different methylation pattern between the two extremes and tends to be more modestly methylated.
  • These properties of cancer DNA have been used as the basis of a physicochemical test for cancer (Sina et al, 2018).
  • a major problem associated with all these ctDNA analysis methods is that the mutant allele fraction (MAF - the proportion of alleles at a specific genomic location which are mutant) of cfDNA in cancer patients is low.
  • the ctDNA analytic target of the methods is diluted in a larger quantity of circulating cfDNA of hematopoietic origin. This means that the proportion of cfDNA constituted by tumour derived ctDNA is low which places limitations on all ctDNA analysis methods. For this reason, increasing the MAF of patient samples has become a goal of workers in the ctDNA field.
  • the MAF of cfDNA fragments is size dependent and cfDNA fragments of sizes 90-150bp in length have a higher MAF, and hence higher ctDNA fractions, than larger cfDNA fragments.
  • Circulating cfDNA consists of DNA molecules of various sizes up to 20,000bp in length (Zhou et al, 2012). In agreement with the hypothesis that ctDNA circulates predominantly as mononucleosomes, measured levels of cell free nucleosomes in the circulation are, like DNA levels, higher in cancer patients than in healthy subjects (Holdenrieder et al, 2001).
  • nucleosomes are a non-specific product of cell death and raised levels are observed for many conditions involving elevated cell death including acute trauma (Holdenrieder and Stieber, 2009).
  • circulating nucleosome levels can also rise markedly on treatment with cytotoxic drugs or radiotherapy.
  • nucleosomes are also cleared from the circulation so levels may spike with treatment and then fall (Holdenrieder et al, 2001).
  • Phenol-chloroform and sodium iodide extraction methods provide the highest yield and extract small DNA fragments of less than 200bp in length.
  • Other methods tested are reported to have lower DNA extraction yields and may fail to extract small DNA fragments of less than 200bp in length (Fong et al, 2009).
  • Extraction of cfDNA from blood, serum or plasma for analysis of ctDNA is usually performed using commercially available DNA extraction products. Such extraction methods claim high recoveries of circulating DNA (>50%) and some products (for example, the QIAamp Circulating Nucleic Acid Kit produced by Qiagen) are claimed to extract DNA fragments of small size. Typical sample volumes used are in the range 1-5mL of plasma.
  • the level of cfDNA in cancer patients is often raised, the ctDNA fraction is low and the total ctDNA levels may be lower than the observed increase in cfDNA. This may indicate that at least some of the increase in cfDNA may be tumour associated, perhaps related to the tumour environment or stroma, rather than directly derived from malignant cancer cells containing cancer associated DNA mutations.
  • nucleosomes per se are an indicator of cell death and are released as part of the normal cell turnover process of the body as well as in conditions associated with elevated levels of cell death such as autoimmune diseases, stroke, sepsis, post trauma, burns, myocardial infarction, cerebral stroke, during graft rejection after organ transplantation and after severe exercise.
  • nucleosomes of tumour origin circulate together with other non-tumour nucleosomes of various cellular and tissue origins and may constitute a low fraction of all circulating nucleosomes.
  • Non-tumour nucleosomes will interfere in any method for the quantification or epigenetic analysis of tumour associated nucleosomes or of nucleosomes of tumour origin. A similar effect may occur in other body fluids.
  • Feces may contain nucleosomes and associated DNA of colorectal cancer cell origin together with nucleosomes originating in healthy colon or rectal cells.
  • Sputum may contain nucleosomes and associated DNA of lung cancer cell origin together with nucleosomes originating in healthy lung cells. Similar effects will occur in other body fluids.
  • tumour derived or tumour associated DNA and nucleosomes from body fluid samples.
  • analytical methods to distinguish tumour and non-tumour circulating cell free nucleosomes for improved detection of cancer disease states.
  • an isolated protein comprising: a histone H1 globular (H1g) domain; and optionally a histone H1 N-terminal domain; wherein the protein does not contain a histone H1 C-terminal domain.
  • the protein of the present invention comprises: a histone H1 globular (H1g) domain; and a histone H1 N-terminal domain; wherein the protein does not contain a histone H1 C-terminal domain.
  • H1g histone H1 globular domain
  • H1 N-terminal domain wherein the protein does not contain a histone H1 C-terminal domain.
  • the protein further comprises a tag or linker amino acid sequence and wherein the tag or linker amino acid sequence is not a histone H1 C-terminal domain.
  • the tag or linker amino acid sequence is a His tag and/or a SUMO tag.
  • the H1g domain is derived from H1.0, H1.1 , H1.2, H1.3, H1.4 or H1.5.
  • the H1g domain is derived from H1.0.
  • the H1g domain comprises or consists of the H1g domain as shown in Figure 1.
  • the H1 C-terminal domain comprises or consists of the H1 C-terminal domain shown in Figure 1.
  • the H1 N-terminal domain comprises or consists of the H1 N-terminal domain shown in Figure 1.
  • nucleic acid encoding the protein of the present invention.
  • an expression cassette comprising the nucleic acid according to the present invention.
  • a vector comprising the nucleic acid or the expression cassette according to the present invention.
  • a cell comprising the nucleic acid, the expression cassette or the vector according to the present invention.
  • a solid phase comprising the protein of the present invention, wherein the protein is covalently attached to the solid phase.
  • the solid phase is a magnetic bead
  • a seventh aspect of the present invention there is provided a method for making a protein according to the present invention by culturing a cell according to the present invention and purifying the protein from the supernatant.
  • a protein according to the present invention for use in medicine.
  • a protein according to the present invention for use in a diagnostic assay.
  • a method for separating circulating cell free nucleosomes comprising linker DNA from a biological fluid sample comprising the steps of:
  • step (ii) isolating nucleosomes from the sample which are bound to the protein in step (i).
  • a method for separating circulating cell free nucleosomes which do not comprise linker DNA from a biological fluid sample comprising the steps of:
  • step (ii) isolating nucleosomes from the sample which are not bound to the protein in step (i).
  • the method which additionally comprises:
  • step (iii) analysing the isolated cell free nucleosomes of step (ii) by immunoassay, protein analysis (e.g. western blot or mass spectrometry) and/or DNA analysis.
  • immunoassay protein analysis (e.g. western blot or mass spectrometry) and/or DNA analysis.
  • step (iii) comprises analysing the isolated cell free nucleosome for epigenetic nucleosome features that are selected from: histone type (for example, H2A, H2B, H3, H4 histone), post-translational modifications, histone isoforms, particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), proteins adducted to the nucleosome or combinations thereof.
  • histone type for example, H2A, H2B, H3, H4 histone
  • histone isoforms for example, particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), proteins adducted to the nucleosome or combinations thereof.
  • the DNA analysis comprises DNA sequencing including Next Generation Sequencing (targeted orwhole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature- PCR), isothermal amplification, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE).
  • DNA sequencing including Next Generation Sequencing (targeted orwhole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature- PCR), isothermal amplification, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE).
  • MAP MIDI-Activated Pyrophosphorolysis
  • PARE Personalized Analysis of Re-arranged Ends
  • the method additionally comprises contacting the sample bound in step (i) with a binding agent which binds to nucleosomes or a component thereof.
  • the biological fluid sample is selected from a blood, serum or plasma sample.
  • the sample is additional contacted with a further binding agent which binds to nucleosomes comprising linker DNA.
  • a method for isolating purified circulating tumour DNA (ctDNA) from a biological fluid sample comprising the steps of:
  • a method of diagnosing cancer comprising:
  • step (ii) comprises analysing the isolated circulating tumour nucleosomes for epigenetic nucleosome features that are selected from: histone type (for example, H2A, H2B, H3, H4 histone), histone post-translational modifications, histone isoforms, particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), proteins adducted to the nucleosome or combinations thereof.
  • histone type for example, H2A, H2B, H3, H4 histone
  • histone post-translational modifications for example, histone post-translational modifications
  • histone isoforms particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications)
  • the associated DNA is analysed using DNA sequencing, for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Rearranged Ends (PARE).
  • DNA sequencing for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Rearranged Ends (PARE).
  • MAP MIDI-Activated Pyrophosphorolysis
  • PARE Personalized Analysis of Rearranged Ends
  • FIG. 1 An alignment of somatic human H1 variants (Hergeth and Schneider (2015)) (SEQ ID NO. 1-6).
  • the globular domain is shown as a solid line.
  • the N- and C- terminal domains are shown as dotted and dashed lines, respectively.
  • FIG. 1 Results using H1 protein (full length or the truncated H1 protein) to bind nucleosomes.
  • the results show that both full length recombinant H1 (H1 FL-a and H1 FL-b) and truncated H1 protein (H1g) selectively bind to nucleosomes which contain linker DNA (167bp recombinant nucleosomes and HeLa cell derived mononucleosomes), but not to 147bp recombinant nucleosomes which do not contain linker DNA.
  • H1 protein full length or the truncated H1 protein
  • the truncated H 1 g protein is a more efficient or stronger binder of nucleosomes containing linker DNA than the full length protein H1 FL and therefore more efficiently removes nucleosomes containing linker DNA from a mixture of nucleosomes with and without linker DNA.
  • Figure 3 Results for the separation of a mixture of recombinant mononucleosomes with several DNA length (147bp, 167bp and 187bp) using a truncated Histone H1g protein.
  • the unbound supernatant fraction is highly enriched for 147bp DNA fragments achieved by highly efficient, and near total, removal of 167bp and 187bp DNA by the binding to truncated H1g protein.
  • Figure 4 Results for the separation of mononucleosomes with or without linker DNA in a HeLa cell mononucleosome preparation using a Histone H1g protein.
  • the DNA fragment size profile (using the Bioanalyzer) of a HeLa cell chromatin mononucleosome preparation (dotted line).
  • Each nucleosome in a chromosome in a living cell consists of a protein complex of eight highly conserved core histones (comprising of a pair of each of the histones H2A, H2B, H3, and H4). Around this complex are wrapped approximately 145-147 base pairs (bp) of DNA.
  • Another histone, H1 may be located on the nucleosome outside of the core histones and binds a further 20-80bp of DNA to produce nucleosomes (or chromatosomes) containing approximately 165bp of DNA.
  • This further DNA is referred to in the art as “linker DNA”, i.e. the DNA connecting one nucleosome to another.
  • the DNA separating two nucleosomes in a chromosome may be longer than 20bp, for example if adjacent nucleosomes both contain H 1 , and may be up to 80bp in length.
  • chromatin refers to the complex formed of DNA and histone proteins when the DNA is packaged within a cell. Therefore, histones are a component of chromatin.
  • cell free nucleosomes originating from a tumour less frequently contain histone H1 than nucleosomes originating in healthy cells (see WO 2017/068371). This difference may be exploited for immunoaffinity enrichment of disease associated or derived nucleosomes using antibody binders for H1 to separate nucleosomes that do, or do not, contain histone H1.
  • nucleosomes can be separated by other means (see WO 2021/038010). These means utilise separation of nucleosomes based on nucleosome features associated with the presence or absence of linker DNA. Underhill et al have studied the size range of ctDNA fragments produced by human cancer xenografts in rats compared to the size range of healthy rat cfDNA. Also as shown in WO 2021/038010) circulating DNA fragments occur in a range of sizes up to approximately 230bp in length.
  • circulating cfDNA In healthy subjects, circulating cfDNA is predominantly of haematopoietic origin and has a range of fragment sizes varying from approximately 150-230bp in length together with smaller amounts of larger DNA fragments corresponding to circulating oligonucleosomes.
  • the most common size of healthy haematopoietic cfDNA is approximately 167bp in length with only a very small proportion of fragments smaller than 150bp.
  • Haematopoietic cfDNA is also present in subjects with conditions such as cancer and pregnancy but additional shorter circulating DNA fragments of approximately 120-150bp are also present.
  • the ctDNA fragment size shows a 10bp periodicity which is related to the nucleosomal DNA helical periodicity and the most common sizes are 134bp and 144bp in length. Therefore, the majority of healthy cfDNA is more than 160bp in length and the majority of ctDNA is less than 150bp in length. Without being bound by theory, the inventors reasoned that most circulating cell free nucleosomes of non-tumour (and non- fetal) origin can be deemed to contain linker DNA whereas circulating cell free nucleosomes of tumour origin can be deemed to contain no linker DNA.
  • short approximately 150bp DNA (in particular about 145bp DNA) nucleosomes comprise a greater proportion of total circulating cell free nucleosomes in pregnant women than in non-pregnant healthy subjects and are thought to be of fetal or placental origin.
  • nucleosomes containing histone H1 could be isolated from blood samples using an anti-histone H1 antibody immobilised on a solid support as an immunosorbent (WO 2017/068371).
  • the protein histone H1 (itself) can be used to bind and remove nucleosomes containing linker DNA from plasma samples (W02021/038010).
  • a truncated histone H1 protein (or polypeptide) immobilised on a solid support as an immunosorbent can be used to bind and remove nucleosomes containing linker DNA from plasma samples.
  • binding proteins that bind to nucleosomes containing linker DNA can also be used in a similar manner to bind and remove nucleosomes containing linker DNA from serum or plasma or other body fluid samples. Therefore isolated proteins of the present invention find particular use in isolating nucleosomes with linker DNA from a sample (e.g. a biological fluid sample).
  • these proteins of the present invention can give rise to an improved effect compared to the full length histone H1 protein used in the prior art. Whilst not wishing to be bound by any theory we believe that the improved efficacy of the present invention derives from the increased stability of the truncated protein of the present invention over the native full length protein. We believe that this effect could not have been predicted by a skilled worker.
  • the present inventors have designed and generated proteins based on histone H1 , which can be used to provide an improved means of isolating and separating nucleosomes containing linker DNA from nucleosomes which do not contain linker DNA.
  • the present invention provides a protein derived from a histone H1 which consists essentially of the histone H1 globular (H1g) domain and optionally a tag or linker sequence which may optionally contain an enzymatic site sequence.
  • there present invention provides a protein derived from a histone H1 which consists essentially of the H1g domain and a histone 1 N-terminal domain and optionally a tag sequence.
  • the protein of the present invention does not contain the histone H1 C-terminal domain.
  • the protein of the present invention can be seen as a truncated form or a fragment of a histone H1 protein.
  • the protein of the present invention is suitable for use as a binding agent which binds to nucleosome associated linker DNA. Therefore, the protein of the present invention may also be referred to herein as a binding agent of the present invention or a binding agent which binds to nucleosomes comprising linker DNA.
  • the proteins of the invention may be referred to as isolated proteins. As used herein the term “isolated” refers to a protein purified to some degree from endogenous material.
  • an isolated protein therefore also refers to a protein that is free from the environment in which it may naturally occur.
  • an isolated protein could be part of composition of matter and still be “isolated” because that composition of matter is not the original, natural environment of the protein.
  • the protein of the present invention may be derived from any variant (also known as an isoform) of histone H 1 , such as H1.0, H1.1 , H1.2, H1.3, H1.4 or H1.5.
  • the protein of the present invention may be derived from variants which have been identified in specific tissue types such as Hit and H1T2 in testis.
  • the H1g is derived from H1.0.
  • an isolated protein comprising a histone H1.0 globular domain; and optionally a histone H1.0 N- terminal domain; wherein the polypeptide does not contain a histone H1.0 C-terminal domain.
  • histone H1 examples are known in the art.
  • the native sequences for these proteins may be found in publicly available databases, for example, The Universal Protein Resource “UniProt”, depicted under accession numbers P16403 (H1.2 human), P16402 (H1.3 human), P10412 (H1.4 human), P16401 (H1.5 human), P07305 (H1.0 human), Q02539 (H1.1 human).
  • H1 variants In human and mouse cells 11 H1 variants have been described and are encoded by single genes. Six of the variants are mainly expressed during the S phase and hence are replicationdependent. They are encoded by genes within histone cluster 1 located in human cells on chromosome 6. The five further variants are expressed over the whole cell cycle and their encoding genes are scattered in the genome.
  • Example of H1 variants which can be used in the invention are set out below in Table 1 with current, suggested nomenclature. Table 1 : Example H1 variants
  • histone H1 variants include H1.X, Hit and H1 FOO in isoform 1 and 2.
  • the present invention may also employ H5 from chicken erythrocytes which is a specific ortholog of H1.0.
  • FIG. 1 Examples of histone H1 amino acid sequences are shown in Figure 1 in which the globular domain is shown as a solid line. The N- and C-terminal domains are shown as dotted and dashed lines, respectively.
  • the present invention is useful with any other histone H1 variant existent at the time of filing or with any future variants that may emerge.
  • the present invention may also employ any histone H1 mutant which arises or is synthesised.
  • the nucleosome linker DNA binding protein of the present invention may be derived from any of these variants or mutants.
  • H1g or N-terminal domain or a fragment or truncated form of H1 comprising the amino acid sequence shown as Figure 1 are also intended to embrace functionally equivalent variants of the H1g or N-terminal domain or truncated forms of H1 of a fragment thereof comprising the amino acid sequence shown as Figure 1 and variants which have been modified in the amino acid sequence without adversely affecting, to any substantial degree, the capacity of H1g or N-terminal domain or H1 protein relative to those of the native molecule.
  • Said modifications include, the conservative (or non-conservative) substitution of one or more amino acids for other amino acids, the insertion and/or the deletion of one or more amino acids, provided that the capacity to interact with the nucleosome linker DNA is substantially maintained, i.e., the variant maintains the ability (capacity) to interact with the nucleosome linker DNA protein at physiological conditions.
  • variant refers to a protein differing from a specifically recited protein, i.e. reference or parent protein, by amino acid insertions, deletions, and/or substitutions, created using, for example, recombinant DNA techniques or by de novo synthesis. “Variant” and “mutant” are used indistinctly in the context of the present invention.
  • variants or mutants of the H1g, N-terminal domain or H1 protein or a fragment thereof comprising the amino acid sequence shown in Figure 1 may have at least 70%, or at least 80%, or at least 90%, or at least 95%, or at least 96%, or at least 97%, or at least 98%, or at least 99% sequence identity with the sequences shown as Figure 1 provided that the capacity to interact with the nucleosome linker DNA is substantially maintained.
  • variants may contain one or more conservative amino acid substitutions compared to the original amino acid or nucleic acid sequence.
  • Conservative substitutions are those substitutions that do not substantially affect or decrease the affinity of a H1 variant to bind nucleosome linker DNA.
  • a H1g or N-terminal domain variants that specifically binds nucleosome linker DNA may include up to 1 , up to 2, up to 5, up to 10, or up to 15 conservative substitutions compared to any of the sequences given as Figure 1 and retain specific binding to nucleosome linker DNA.
  • the percentage of sequence identity may be determined by comparing two optimally aligned sequences over a comparison window.
  • the aligned sequences may be polynucleotide sequences or polypeptide sequences.
  • the portion of the polynucleotide or amino acid sequence in the comparison window may comprise insertions or deletions (i.e., gaps) as compared to the reference sequence (that does not comprise insertions or deletions).
  • the percentage of sequence identity is calculated by determining the number of positions at which the identical nucleotide residues, or the identical amino acid residues, occurs in both compared sequences to yield the number of matched positions, then dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Sequence identity between two polypeptide sequences or two polynucleotide sequences can be determined, for example, by using the Gap program in the WISCONSIN PACKAGE version 10.0-UNIX from Genetics Computer Group, Inc.
  • the percentage of sequence identity between proteins and their corresponding functions may be determined, for example, using a variety of homology-based search algorithms that are available to compare a query sequence, to a protein database, including for example, BLAST, FASTA, and Smith-Waterman.
  • BLASTX and BLASTP algorithms may be used to provide protein function information. A number of values are examined in order to assess the confidence of the function assignment. Useful measurements include "E-value” (also shown as "hit_p"), "percent identity”, “percent query coverage”, and “percent hit coverage”.
  • the E-value, or the expectation value represents the number of different alignments with scores equivalent to or better than the raw alignment score, S, that are expected to occur in a database search by chance.
  • a "high" BLASTX match is considered as having an E- value for the top BLASTX hit of less than IE-30; a medium BLASTX is considered as having an E-value of IE- 30 to IE-8; and a low BLASTX is considered as having an E- value of greater than IE-8.
  • Percent identity refers to the percentage of identically matched amino acid residues that exist along the length of that portion of the sequences which is aligned by the BLAST algorithm. In setting criteria for confidence of polypeptide function prediction, a "high" BLAST match is considered as having percent identity for the top BLAST hit of at least 70%; a medium percent identity value is considered from 35% to 70%; and a low percent identity is considered of less than 35%.
  • Query coverage refers to the percent of the query sequence that is represented in the BLAST alignment, whereas hit coverage refers to the percent of the database entry that is represented in the BLAST alignment.
  • a protein of the invention is one that either (1) results in hit_p ⁇ le-30 or % identity >35% AND query _coverage>50% AND hit_coverage>50%, or (2) results in hit_p ⁇ le-8 AND query _coverage>70% AND hit_coverage>70%.
  • Variants of the H1g or N-terminal domain or truncated H1 or of a fragment thereof comprising the amino acid sequence shown as Figure 1 may maintain at least about 75%, for example at least about 80%, 90%, 95%, 96%, 97%, 98%, 99% or 100% of the capacity to interact with nucleosome linker DNA.
  • the protein of the present invention including variants thereof as described herein may have an increased capacity to interact with nucleosome linker DNA of about 105%, for example at least about 110%, 115%, 120%, 125%, 130%, 140%, 150% or more compared with that of the wild type H1 .
  • the interaction between protein of the present invention and nucleosome linker DNA can be determined by conventional methods.
  • in vitro binding of the protein to the nucleosome linker DNA may be determined by ELISA, Gel Shift Assay (EMSA), surface plasmon resonance (SPR), or by flow cytometry.
  • ESA Gel Shift Assay
  • SPR surface plasmon resonance
  • the protein of the present invention is coated on a solid support, such as sepharose, sephadex, plastic or magnetic beads.
  • a solid support such as sepharose, sephadex, plastic or magnetic beads.
  • said solid support comprises a porous material.
  • the histone H1 protein is derivatised to include a tag or linker which can be used to attach the histone H1 protein to a suitable support which has been derivatised to bind to the tag.
  • tags and supports are known in the art and may be used in the present invention e.g.
  • the histone H1 protein is derivatised with a His-SUMO tag.
  • the histone H1 protein coated support may be included within a device, for example a microfluidic device.
  • a solid phase comprising the protein of the present invention, wherein the protein is covalently attached to the solid phase.
  • the solid phase is a magnetic bead.
  • the proteins of the present invention may comprise a signal peptide at their N-terminus so that when the polypeptide is expressed inside a cell, the nascent protein is directed to the endoplasmic reticulum and subsequently secreted.
  • the core of the signal peptide or leader sequence may contain a long stretch of hydrophobic amino acids that has a tendency to form a single alpha-helix.
  • the signal peptide may begin with a short positively charged stretch of amino acids, which helps to enforce proper topology of the polypeptide during translocation.
  • At the end of the signal peptide there is typically a stretch of amino acids that is recognised and cleaved by signal peptidase.
  • Signal peptidase may cleave either during or after completion of translocation to generate a free signal peptide and a mature protein.
  • the free signal peptides are then digested by specific proteases.
  • the protein of the present invention may also be engineered or used in a composition which promotes protein stability and such proteins and composition form part of the invention.
  • Common approaches to stabilizing proteins and which may be used in the invention include adding stabilizing agents, modifying the environment such as changing the temperature, the pH, ionic strength, stabilizing mutations and forming protein complexes.
  • the present invention also envisages adding a cysteine residue(s) to the protein. Cysteine is an amino acid that contains a thiol group, which can form disulfide bonds with other cysteine residues. These disulfide bonds can help to stabilize a protein by crosslinking its subunits or by providing structural support. NUCLEIC ACID
  • the present invention also provides a nucleic acid encoding the protein of the invention, hereinafter "the nucleic acid of the invention”.
  • This nucleic acid may optionally encode a tag or linker for the protein of the present invention.
  • polynucleotide As used herein, the terms “polynucleotide”, “nucleotide”, and “nucleic acid” are intended to be synonymous with each other.
  • nucleic acid sequences and constructs of the invention may contain alternative codons in regions of sequence encoding the same or similar amino acid sequences, in order to avoid homologous recombination.
  • nucleic acid sequences and constructs of the invention may contain alternative codons in regions of sequence in order to improve the coating of the protein to the solid phase or the stability of the protein H1 or the protein function (e.g. DNA binding).
  • Nucleic acids according to the invention may comprise DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides which include within them synthetic or modified nucleotides. A number of different types of modification to oligonucleotides are known in the art. These include methylphosphonate and phosphorothioate backbones, addition of acridine or polylysine chains at the 3' and/or 5' ends of the molecule. For the purposes of the use as described herein, it is to be understood that the polynucleotides may be modified by any method available in the art. Such modifications may be carried out in order to enhance the in vivo activity or life span of polynucleotides of interest.
  • the terms "variant”, “homologue” or “derivative” in relation to a nucleotide sequence include any substitution of, variation of, modification of, replacement of, deletion of or addition of one (or more) nucleic acid from or to the sequence.
  • the nucleic acid of the invention can contain a regulatory sequence operatively linked for the expression of the nucleotide sequence encoding the protein of the invention, thereby forming a gene construct, hereinafter the "gene construct of the invention".
  • the term "operatively linked” means that the protein encoded by the nucleic acid sequence of the invention is expressed in the correct reading frame under control of the expression control or regulating sequences. Therefore, in another aspect, the invention provides an expression cassette, hereinafter “the expression cassette of the invention", comprising the gene construct of the invention operatively linked to an expression control sequence.
  • the gene construct of the invention can be obtained through the use of techniques that are widely known in the art.
  • the expression cassette may comprise one or more control sequences.
  • Control sequences are sequences that control and regulate transcription and, where appropriate, the translation of said protein, and include promoter sequences, transcriptional regulators encoding sequences, ribosome binding sequences (RBS) and/or transcription terminating sequences.
  • the expression cassette of the present invention may additionally include an enhancer, which may be adjacent to or distant from the promoter sequence and can function to increase transcription from the same.
  • the expression control sequence may function in prokaryotic cells or in eukaryotic cells and organisms, such as mammalian cells.
  • the expression cassette may comprise a promoter. Any promoter may be used in this methodology.
  • the present invention also provides a vector, or kit of vectors, which comprises a nucleic acid of the invention, or an expression cassette of the invention.
  • a vector may be used to introduce the nucleic acid or expression cassette into a host cell so that it expresses the protein of the invention.
  • vector is intended to refer to a molecule capable of transporting the nucleic acid or expression cassette.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments may be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian and yeast vectors). Other vectors (e.g.
  • non-episomal mammalian vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • the vector may, for example, be a plasmid or a viral vector, such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.
  • a viral vector such as a retroviral vector or a lentiviral vector, or a transposon-based vector or synthetic mRNA.
  • the choice of vector may be dependent upon the type of host cell to be used and the purpose of use.
  • Another aspect of the present invention relates to a cell, hereinafter "the cell of the invention", comprising the nucleic acid of the invention, or the vector of the invention.
  • the cell may comprise a nucleic acid, or an expression cassette, or a vector according to the present invention.
  • the cell may be prokaryotic or eukaryotic.
  • Cells suitable for performing the invention include, without limitation, mammalian, plant, insect, fungal and bacterial cells.
  • the present invention also relates to a method for making the protein of the invention by culturing a cell of the invention and purifying the protein from the supernatant.
  • the present inventors have found that separation can occur through use of the histone feature (i.e. at least the H1g) of the present invention to bind to linker DNA associated with a nucleosome - the protein of the present invention.
  • a method for separating circulating cell free nucleosomes comprising linker DNA from a biological fluid sample comprising the steps of:
  • the method of the invention can be used in a negative selection method for removing nucleosomes comprising linker DNA and isolating nucleosomes without linker DNA. Therefore, according to another aspect of the invention there is provided a method for separating circulating cell free nucleosomes which do not comprise linker DNA from a biological fluid sample, wherein said method comprises the steps of:
  • step (ii) isolating nucleosomes from the sample which are not bound to the protein in step (i).
  • a method for separating cell free nucleosomes with linker DNA from cell free nucleosomes without linker DNA from a biological fluid sample e.g. by affinity purification
  • said method comprises the steps of:
  • step (ii) isolating nucleosomes from the sample which are bound to the protein in step (i).
  • Methods of separation described herein may be used to isolate nucleosomes of disease (or fetal) origin due to the absence of linker DNA. Therefore, in one embodiment, there is provided a method for isolating circulating cell free nucleosomes of disease or fetal origin from a biological fluid sample (e.g. by affinity purification), wherein said method comprises the steps of:
  • step (ii) isolating nucleosomes from the sample which are not bound to the protein in step (i).
  • Methods of separation described herein may be used to isolate nucleosomes of disease (or fetal) origin due to the absence of linker DNA. Therefore, in one embodiment, there is provided a method for isolating circulating cell free nucleosomes of disease or fetal origin from a biological fluid sample (e.g. by affinity purification), wherein said method comprises the steps of:
  • step (i) contacting the sample with the protein of the present invention; and (ii) isolating nucleosomes from the sample which are not bound to the protein in step (i).
  • the binding between the linker DNA associated with the nucleosome and the protein of the invention may occur in the liquid phase (j.e. prior to binding the protein to the solid support). Binding of the protein to a solid support may then be achieved simultaneously or subsequently using a linker reaction.
  • nucleosomes with or without linker DNA may be defined by the length of DNA wrapped around the nucleosome. Therefore, according to a further aspect of the invention, there is provided a method for separating cell free nucleosomes with linker DNA from cell free nucleosomes without linker DNA from a biological fluid sample (e.g. by affinity purification), wherein said method comprises the steps of:
  • step (ii) isolating nucleosomes from the sample which are not bound to the protein in step (i).
  • a method for isolating circulating cell free nucleosomes of disease or fetal origin from a blood, serum or plasma sample by affinity purification comprising the steps of:
  • step (ii) isolating nucleosomes from the sample which are bound to the protein in step (i).
  • the subsets of nucleosomes produced by separation based on these various features may be different and can be useful for a variety of applications for research purposes and for clinical applications.
  • the structure of nucleosomes may vary in cancer cells compared to healthy cells.
  • Applications of the invention include separation of tumour derived nucleosomes, separation of maternal and fetal nucleosomes in samples taken from pregnant women, disease derived nucleosomes from normal circulating nucleosomes, nucleosomes containing methylated or unmethylated DNA, as well as a variety of subsets of nucleosomes useful for research or other purposes.
  • nucleosomes comprising approximately 150 or more base pairs of DNA in length refer to nucleosomes which have retained linker DNA.
  • the nucleosome core particle (/.e. the histone octamer comprising of a pair of each of the histones H2A, H2B, H3, and H4) consists of approximately 150bp of DNA, such as between 145 and 150 bp of DNA, in particular 147bp of DNA. Therefore, nucleosomes comprising more than 150bp of DNA (such as more than 155bp, 160bp or 165bp of DNA) contain linker DNA.
  • the binding agent i.e.
  • the protein of the invention binds to nucleosomes consisting of at least 165 base pairs of DNA.
  • the binding agent does not bind to nucleosomes comprising approximately 150bp or less of DNA, i.e. the binding agent selectively binds to nucleosomes containing linker DNA.
  • the binding agent binds to a region within linker DNA.
  • the binding agent binds (directly) to the region of DNA not in contact with the histone (core) octamer, i.e. the linker DNA.
  • the methods described herein may be used for detecting, isolating, enriching and/or purifying circulating cell free nucleosomes of disease origin, in particular from circulating cell free nucleosomes of healthy origin.
  • Disease origin refer to nucleosomes and chromatin fragments originating from diseased (e.g. abnormal or unhealthy) cells.
  • healthy origin refer to nucleosomes and chromatin fragments originating from healthy (e.g. normal or non-diseased) cells.
  • the binding agent is directed to bind features associated with linker DNA
  • this may be used to bind (and remove) nucleosomes of healthy origin from the sample, i.e. the nucleosomes of disease origin are obtained in the unbound portion of the sample.
  • This may therefore be referred to as a negative selection method.
  • a positive or negative selection method is used will determine which nucleosome fraction is isolated (i.e. the bound fraction for a positive selection method or the free/eluted fraction for a negative selection method).
  • the circulating cell free nucleosomes are of tumour origin. Therefore, the present method finds particular use as a method of purifying, isolating, separating or enriching a sample for circulating cell free nucleosomes of tumour origin. The sample may then be used for subsequent analysis and, for example, for use in diagnostic methods.
  • nucleosomes comprising linker DNA that may be used for separation of nucleosomes include the protein of the present invention which bind to linker DNA.
  • binders of linker DNA i.e. , the protein of the present invention, may be used directly to bind and isolate nucleosomes comprising linker DNA.
  • separation methods may include affinity chromatography or magnetic antibody beads. If using an affinity column chromatography set up, for example, it will be understood that the sample may be passed through an affinity column comprising the required binding agent(s). Either the nucleosomes bound to the solid phase or the flow through (i.e. the unbound nucleosomes) may be collected for further analysis, depending on whether a positive or negative selection method described herein is employed.
  • proteins of the invention may be used regardless of whether target nucleosomes already comprise a binder of linker DNA.
  • the isolation is based on substitution of that binder of linker DNA by binding to the binding agent used in the invention due to a high relative concentration and/or high relative binding affinity of the binder of the invention compared to the binder initially bound to the nucleosome, e.g. due to equilibrium and kinetic reasons.
  • tumour nucleosome and circulating tumour DNA (ctDNA) isolation is performed by an immunological affinity purification method employing a protein of the present invention.
  • protein of the present invention may be used in combination with another binding agent(s) capable of specific binding to the required target may be used for the methods of the invention.
  • binding agents may be used in the methods of the invention, i.e. to prepare a panel of binding agents to enrich for nucleosomes of different types. Therefore, in one embodiment, the sample is contacted with more than one type of binding agent which binds to nucleosomes comprising linker DNA.
  • additional binding agents may include without limitation, antibodies, affimers, aptamers or binding proteins (e.g. nucleosome binding proteins).
  • binding proteins e.g. nucleosome binding proteins.
  • histone H1 , macroH2A or chromatin binding proteins themselves are used as the additional binding agent in a method of the invention.
  • nucleosomes and/or associated DNA isolated by methods of the invention may be analysed by immunoassay, protein analysis using e.g. Western Blot or mass spectrometry, DNA sequencing (i.e. analysing the associated DNA for a particular genetic sequence or methylated genetic sequence), epigenetic signal structures or other characteristics.
  • the nucleosome fraction in particular the nucleosome fraction enriched for disease (e.g. tumour origin)
  • the nucleosome associated DNA may be analysed, e.g. for genetic or DNA sequence markers. Therefore, in one embodiment, the method comprises:
  • step (ii) analysing the DNA associated with the nucleosomes isolated in step (i).
  • a blood plasma sample is collected from a subject with suspected or diagnosed cancer.
  • Nucleosomes without linker DNA in the plasma sample are isolated or enriched by a method as described herein.
  • the DNA associated with the nucleosomes enriched for tumour origin is extracted to produce a size-selected DNA fragment library of approximately less than 150bp in length.
  • the DNA is analysed for cancer associated mutation abnormalities and/or mutant allele fraction and/or gene methylation abnormalities.
  • Any DNA analysis method may be employed including, without limitation, DNA sequencing including Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE).
  • DNA sequencing including Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis
  • BEAMing PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE).
  • MAP MIDI-Activated Pyrophosphorolysis
  • PARE Personalized Analysis of Re-arranged Ends
  • DNA analysis may include analysis for any genetic DNA markers including nucleotide substitutions, nucleotide insertions, nucleotide deletions, methylated DNA sequences or other DNA sequence mutations.
  • Typical cancer associated DNA abnormalities that may be investigated in such an analysis include, without limitation, point mutations, translocations, gene copy number mutations, microsatellite abnormalities, DNA strand integrity and gene methylation status.
  • DNA analysis may involve determining the mutant allele fraction (MAF), i.e. the proportion of alleles at a specific genomic location which are mutant.
  • MAF is generally expressed as a fraction or a percentage.
  • Such a panel might, without limitation, include one or more mutations in the ABL1, ACVR1, ACVR1B, ACVR2A, AJLIBA, AKT1, AKT2, AKT3, ALB, ALK, AMER1, APC, APEX1, APLNR, APOB, AR, ARAP, ARHGAP35, ARID1A, ARID2, ARID5B, ATF7IP, ATM, ATP11B, ATR, ATRX, ATXN3, AURKA, AXIN1, AXIN2, B2M, BAP1, BCL2, BCL2L1, BCL2L11, BCL9, BOOR, BIRC2, BIRC3, BRAF, BRCA 1, BRCA2, BRD7, BTG2, BTK, CARD11, CASP8, CBL, CCND1, CCND2, CCND3, CCNE1, CD44, CD70, CD79B, CDH1, CHD3, CHD8, CDK12, CDK2, CDK4, CDK6, CDKN2A, CDKN2B,
  • genes have been investigated as markers for differential cytosine methylation status in cancer.
  • a few of these are SEPTIN-9, APC, DAPK, GSTP1, MGMT, P16, RASSF1A, TIG1, BRCA1, ERa, PRB, TMS1, MLH1, HLTF, CDKN2A, S0CS1, S0CS2, PAX5, PGR, PTGS2 and RAR/32.
  • a method for isolating purified circulating tumour DNA (ctDNA) from a biological fluid sample comprising the steps of:
  • This aspect of the invention finds particular use in analysing nucleosomes without linker DNA because such nucleosomes are predicted to be of disease origin.
  • the DNA associated with the isolated nucleosomes is extracted prior to analysis. Many methods of analysis of extracted DNA are known in the art.
  • the DNA is directly analysed, i.e. without requiring extraction. If the cell free nucleosomes isolated are of tumour origin, the level of isolated tumour nucleosomes detected as a proportion of nucleosomes present in the original (untreated) sample may be used as an indicator of the proportion of DNA that comprises circulating tumour DNA (ctDNA) in a sample. Furthermore, the converse level is the proportion of DNA of healthy origin in the sample.
  • the methods described herein may be used to detect the level/proportion of ctDNA in a sample or changes to such levels over time.
  • a measure is similar to mutant allele fraction measures of cancer associated mutations in ctDNA and may be used as a measure of tumour burden and response to therapy.
  • Nucleosomes which have been enriched for disease (e.g. tumour) origin may also be analysed for epigenetic markers (“epigenetic epitopes”) or subjected to further enrichment methods. Therefore, in one embodiment, the method comprises:
  • step (ii) analysing the cell free nucleosomes isolated in step (i).
  • the analysing step comprises analysing the isolated cell free nucleosome for epigenetic nucleosome features that are selected from: histone type (for example, H1 , H2A, H2B, H3, H4 histones), histone post-translational modifications, histone isoforms, particular nucleotides, modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), or combinations thereof or for the presence of other proteins adducted to the nucleosome.
  • histone type for example, H1 , H2A, H2B, H3, H4 histones
  • histone post-translational modifications for example, histone post-translational modifications
  • histone isoforms particular nucleotides, modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), or combinations thereof or for the presence of other proteins adducted to the nucleosome.
  • the presence or level of enriched cell free nucleosomes of disease origin containing a modified nucleotide, histone post-translational modification, histone isoform or nucleosomeprotein adduct detected may be used as an indicator of disease status, disease prognosis, disease monitoring, treatment monitoring, minimal residual disease or disease susceptibility to particular treatments or for other clinical applications.
  • the analysis of the isolated nucleosomes of disease origin may involve any suitable method of analysis of which many are known in the art. These methods include without limitation analysis by immunoassay using an antibody or other binder to a common nucleosome epitope such as DNA or to an epigenetic structure of interest including a histone modification, histone variant, DNA modification or another molecule adducted to a nucleosome. These methods include all the methods described in WO 2005/019826, WO 2013/030577, WO 2013/030579 and WO 2013/084002, which are incorporated herein by reference. Without limitation, any of these methods may be employed in combination with the present invention. These methods also include multiplex methods for the analysis of multiple epitopes present in circulating nucleosomes of disease origin.
  • the analysis of nucleosomes of disease origin isolated by a method of the invention may also involve any proteomics method known in the art including, without limitation, electrophoresis methods, chromatographic methods and any method involving mass spectrometry including methods involving chromatography and mass spectrometry and/or stable isotope labelled mass spectrometry and/or methods involving protein digestion to produce peptides for identification and/or quantification by mass spectrometry or any combinatorial mass spectrometry method with any other method.
  • proteomics method known in the art including, without limitation, electrophoresis methods, chromatographic methods and any method involving mass spectrometry including methods involving chromatography and mass spectrometry and/or stable isotope labelled mass spectrometry and/or methods involving protein digestion to produce peptides for identification and/or quantification by mass spectrometry or any combinatorial mass spectrometry method with any other method.
  • the epitope is selected from a histone modification (e.g. a histone post- translational modification [PTM]), a modified nucleotide, a histone variant or isoform, or a nucleosome adduct or variant thereof.
  • a histone modification e.g. a histone post- translational modification [PTM]
  • PTM histone post- translational modification
  • the modified nucleotide comprises 5-methylcytosine.
  • Assays for further epigenetic epitopes may be performed in isolation or as part of an assay panel.
  • the isolated nucleosome sample obtained from the methods described herein are subject to further enrichment steps. Therefore, in one embodiment, a method of the invention additionally comprises contacting the isolated nucleosomes with a histone H3.1 and/or H3.2 and/or H3t binding agent. It has previously been shown that the histone 3 isoforms, H3.1 , H3.2 and H3t, may be used for ctDNA enrichment. IMMUNOASSAY METHODS
  • the proteins of the present invention which bind to nucleosomes can be used directly in isolation and detection methods for cell free nucleosomes. Therefore, according to a further aspect, there is provided the use of a binding protein for isolation of cell free nucleosomes from a body fluid sample.
  • an immunoassay method of detecting cell free nucleosomes in a biological fluid sample comprising the steps of:
  • step (ii) contacting the sample bound in step (i) with a second binding agent which binds to nucleosomes or a component thereof;
  • an immunoassay method of detecting cell free nucleosomes in a biological fluid sample comprising the steps of:
  • step (ii) contacting the sample bound in step (i) with a protein of the present invention
  • antibodies that are selective for binding to nucleosomes may be used in immunoassay methods of the invention as a direct measure of such nucleosomes.
  • Antibodies may be raised by a variety of methods known in the art including immunization and library methods such as phage display.
  • ECV extracellular vesicles
  • Exosomes are one example of an ECV that may comprise nucleosomes and/or DNA and/or other chromatin fragments.
  • Nucleosomes associated with exosomes or other ECVs, including nucleosomes containing linker DNA may be protected from, or less susceptible to, binding to chromatin proteins or other binders and may thus remain unbound when these moieties are used in methods of the invention. Any such ECV associated nucleosomes containing linker DNA that are not bound by proteins using methods as described herein may remain in the supernatant. This effect may be prevented by removal of exosomes and/or other ECVs from the sample.
  • a method for separating cell free nucleosomes with linker DNA from cell free nucleosomes without linker DNA from a biological fluid sample by affinity purification comprising the steps of:
  • step (iii) isolating nucleosomes from the sample which are not bound to the binding agent in step (ii).
  • steps (i) and (ii) above may be performed in any order or concurrently.
  • binders to ECVs are known in the art and may be used for the purposes of binding and removing ECVs from a sample.
  • Commercial products for the isolation of exosomes and other ECVs from samples are also available.
  • Lectins are proteins that are known to bind to most or all exosomes. Therefore, in one embodiment, exosomes or other ECVs are removed from the sample using lectin.
  • lectin proteins are attached to a solid phase support and used to bind to exosomes and other ECVs present in a sample. Following exposure to the lectin protein, the sample supernatant is depleted of exosomes/ECVs and hence of any exosome or ECV associated nucleosomes containing linker DNA.
  • a method for separating cell free nucleosomes with linker DNA from cell free nucleosomes without linker DNA from a biological fluid sample by affinity purification comprising the steps of:
  • step (iii) isolating nucleosomes from the sample which are not bound to the binding agent in step (ii).
  • the sample is a blood plasma sample.
  • an assay method for detecting or diagnosing the presence of a disease by measuring or detecting the presence and/or the level of nucleosomes containing little or no linker DNA in a body fluid, and using the detected level as a biomarker (either alone as a member of a panel of tests) of the disease status of a subject 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 subject susceptibility to treatment regimens.
  • body fluids used for diagnostic testing include without limitation blood, serum, plasma, urine, cerebrospinal fluid and other fluids.
  • the body fluid selected as the sample is blood, serum or plasma.
  • Isolated nucleosomes containing little or no linker DNA in a body fluid may be measured by a variety of methods including mass spectrometry and immunoassay.
  • the nucleosome associated DNA may be measured by physical or chemical methods including, without limitation, chromatography, electrophoresis, PCR and DNA sequencing methods.
  • the assay response, level, concentration or quantity of nucleosomes containing little or no linker DNA in a body fluid may be expressed in absolute terms or relative terms, for example without limitation as a proportion of the total nucleosome level present or as a ratio to the level of another nucleotide or histone variant or histone PTM(s) or to the level of total DNA.
  • the ratio of nucleosomes in a sample that do, or do not contain linker DNA is used as a marker of cancer.
  • a high ratio of [nucleosomes without linker DNA]/[nucleosomes with linker DNA] is indicative of cancer.
  • a ratio of the amount or concentration of [DNA fragments of length less than 150bp]/[DNA fragments of length greater than 150bp] may be determined or the mutant allele fraction may be determined.
  • the cut-off of 150bp for DNA fragment size is used here as an example. Other cut-offs may be used.
  • the methods described herein may be used in conjunction with methods of diagnosis. For example, a sample may be enriched to isolate DNA or cell free nucleosomes of tumour origin using the methods described herein and the enriched sample may then be used in a method of diagnosis by detecting further epigenetic markers which are associated with disease.
  • a method of diagnosing cancer comprising:
  • step (ii) comprises analysing the isolated circulating tumour nucleosomes for epigenetic nucleosome features that are selected from: histone type (for example, H2A, H2B, H3, H4 histone), histone post-translational modifications, histone isoforms, particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications), proteins adducted to the nucleosome or combinations thereof.
  • histone type for example, H2A, H2B, H3, H4 histone
  • histone post-translational modifications for example, histone post-translational modifications
  • histone isoforms particular nucleotides or modified nucleotides (for example methylated, hydroxyl-methylated or other nucleotide modifications)
  • the associated DNA is analysed using DNA sequencing, for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature- PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Rearranged Ends (PARE).
  • DNA sequencing for example a sequencing method selected from Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature- PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Rearranged Ends (PARE).
  • MAP MIDI-Activated Pyrophosphorolysis
  • PARE Personalized Analysis of Rearranged Ends
  • a method of diagnosing or detecting cancer in an animal or a human subject which comprises the steps of:
  • step (ii) contacting the isolated nucleosomes of tumour origin obtained in step (i) with a further binding agent which binds to an epigenetic epitope of tumour derived nucleosomes (the “biomarker”);
  • Detecting and/or quantifying may be performed directly on the purified or enriched nucleosome sample, or indirectly on an extract therefrom, or on a dilution thereof. Quantifying the amount of the biomarker present in a sample may include determining the concentration of the biomarker present in the sample.
  • Uses and methods of detecting, monitoring and of diagnosis according to the invention described herein are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease.
  • Uses and methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.
  • the methods of diagnosis described herein may further comprise comparing the level of the second binding agent present in the biological sample with one or more control(s).
  • the biological sample from the one or more control(s) is taken from healthy (or “normal”) patient(s) and/or patient(s) with an associated benign disease.
  • the biological sample from the one or more control(s) is taken from healthy patient(s).
  • a method of diagnosing cancer which comprises the steps of:
  • step (b) using the level of the nucleosomes measured in step (a) to determine if the patient has cancer.
  • a method of diagnosing cancer which comprises the steps of:
  • step (b) using the ratio measured in step (a) to determine if the patient has cancer.
  • the methods described herein may be used to monitor a patient for progression of relapse of cancer.
  • the methods described herein may be used to select a suitable therapy for the patient. For example, analysing the DNA associated with the isolated nucleosomes can determine the genotype of the cancer of the patient which may make them more or less responsive to a particular therapy.
  • the methods described herein may be used to monitor minimal residual disease (MRD), i.e. identify the presence of residual malignant cells in a patient who has been treated. The detection of MRD indicates that treatment is incomplete.
  • MRD minimal residual disease
  • a method of treating cancer which comprises the steps of:
  • step (c) using the level of the nucleosomes measured in step (b) to determine if the patient has cancer;
  • step (d) administering a treatment if the patient is determined to have cancer in step (c).
  • a method of treating cancer which comprises the steps of:
  • step (c) using the ratio of the nucleosomes measured in step (b) to determine if the patient has cancer;
  • step (d) administering a treatment if the patient is determined to have cancer in step (c).
  • the method of the invention is used to measure the level of nucleosomes containing short or long DNA fragments (i.e. of approximately less than 150bp and more than 150bp in length, respectively) in a sample is an immunoassay method of the invention as described herein.
  • a method of treating cancer which comprises the steps of:
  • step (c) using the level of DNA fragments isolated in step (b) to determine if the patient has cancer;
  • step (d) administering a treatment if the patient is determined to have cancer in step (c).
  • An important aspect of the method of the invention is the facilitation of improved detection and treatment of cancer diseases through improved methods for ctDNA detection and analysis.
  • step (c) comprises determining the mutant allele fraction of the DNA fragments.
  • step (c) comprises sequencing the DNA fragments. The nucleotide sequence may then be used to determine if a patient has cancer, e.g. analysing for any genetic DNA markers including nucleotide substitutions, nucleotide insertions, nucleotide deletions, methylated DNA sequences or other DNA sequence mutations, as described hereinbefore.
  • the treatment administered is selected from: surgery, radiotherapy, chemotherapy, immunotherapy, hormone therapy and biological therapy.
  • kits for detecting or isolating or measuring nucleosomes which comprises: (i) a protein of the present invention and (ii) a binding agent which specifically binds to nucleosomes or a component thereof, optionally together with instructions for use of the kit in accordance with the method as defined herein.
  • Kits of the invention may alternatively, or additionally, include reagents for the isolation and/or analysis of nucleosome associated DNA fragments.
  • a kit which comprises: (i) a protein of the present invention; and (ii) reagents for the isolation and/or analysis of nucleosome associated DNA fragments, optionally together with instructions for use of the kit in accordance with the method as defined herein.
  • kits as defined herein for the diagnosis of cancer.
  • the nucleosome is a cell free mononucleosome or oligonucleosome. It will be clear that the term “nucleosome” as used herein is intended to include mononucleosomes and oligonucleosomes and any such chromatin fragments that can be analysed in fluid media.
  • the cell-free nucleosome is a mononucleosome, oligonucleosome or other chromosome fragment.
  • the biological fluid (i.e. body fluid) sample is selected from a blood, serum or plasma sample.
  • a biological fluid sample is obtained from a subject and cell free nucleosomes in the sample are enriched for disease associated or disease derived nucleosomes as described herein.
  • the fluid sample may be any biological fluid (or body fluid) sample taken from a subject including, without limitation, cerebrospinal fluid (CSF), whole blood, blood serum, plasma, menstrual blood, endometrial fluid, urine, saliva, or other bodily fluid (stool, tear fluid, synovial fluid, sputum), breath, e.g. as condensed breath, or an extract or purification therefrom, or dilution thereof.
  • Biological samples also include specimens from a live subject or taken post-mortem. The samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.
  • the body fluid is selected from blood, serum and plasma.
  • the subject is a human or an animal, such as a human, a horse, a pig, a dog, a cat or a mouse.
  • the invention relates to veterinary uses including for livestock and companion animals such as cats, dogs, horses, sheep, goats, pigs, deer, llamas, cows and cattle. References to “subject” or “patient” are used interchangeably herein.
  • Immunoassay methods described herein refer to anti-nucleosome antibodies or binders or to antibodies or other binders that bind to an epitope present in a nucleosome. It will be clear to those skilled in the art that such binders may be directed to bind to any epitope present in a nucleosome or chromatin fragment.
  • epitopes include, without limitation, histone proteins (in particular core histone proteins), histone isoforms, modified histones (e.g. histone post- translational modifications), DNA associated with nucleosomes (e.g. nucleotides, modified nucleotides), conformational epitopes or histone adducts (i.e. proteins adducted to a nucleosome).
  • the binding agent is coated on a solid support, such as sepharose, sephadex, plastic or magnetic beads.
  • said solid support comprises a porous material.
  • the binding agent is derivatised to include a tag or linker which can be used to attach the binding agent to a suitable support which has been derivatised to bind to the tag.
  • tags and supports are known in the art (e.g. Sortag, Click Chemistry, biotin/streptavidin, his-tag/nickel or cobalt, GST-tag/GSH, SUMO, SUMO- His, antibody/epitope tags and many more).
  • the coated support may be included within a device, for example a microfluidic device.
  • the disease (from which the isolated, circulating, cell free nucleosomes originate) is selected from cancer, an autoimmune disease or inflammatory disease.
  • the disease is cancer.
  • the autoimmune disease is selected from: Systemic Lupus Erythematosus (SLE) and rheumatoid arthritis.
  • the inflammatory disease is selected from: Crohn’s disease, colitis, endometriosis and Chronic Obstructive Pulmonary Disorder (COPD).
  • COPD Chronic Obstructive Pulmonary Disorder
  • the isolated nucleosomes may be referred to as “tumour derived” or “tumour associated” nucleosomes.
  • the methods of the invention are suitable for use in all cancer diseases.
  • the tumour derived or tumour associated cell free nucleosomes originate from a cancer selected from: breast cancer, bladder cancer, colorectal cancer, skin cancer (such as melanoma), ovarian cancer, prostate cancer, gastric cancer, lung cancer, pancreatic cancer, bowel cancer, liver cancer, endometrial cancer, lymphoma, oral cancer, head and neck cancer, leukaemia and osteosarcoma.
  • a ligand or binder may comprise a peptide, an antibody or a fragment thereof, or a synthetic ligand such as a plastic antibody, or an aptamer, or an affimer or oligonucleotide, capable of specific binding to the nucleosome.
  • the antibody can be a monoclonal antibody or a fragment thereof capable of specific binding to the nucleosome.
  • a ligand or binder for example a chromatin binding agent as described herein, or a binding agent directed towards the nucleosome or a component thereof
  • a detectable marker such as a luminescent, fluorescent, enzyme or radioactive marker.
  • a ligand may be labelled with an affinity tag, e.g. a biotin, a SBP (streptavidin binding peptides), avidin, streptavidin, GST, Sumo or His (e.g. hexa-His) tag.
  • affinity tag e.g. a biotin, a SBP (streptavidin binding peptides), avidin, streptavidin, GST, Sumo or His (e.g. hexa-His) tag.
  • affinity tag e.g. a biotin, a SBP (streptavidin binding peptides), avidin, streptavidin, GST, Sumo or His (e.g. hexa-His)
  • biomarker means a distinctive biological or biologically derived indicator of a process, event, or condition. Biomarkers can be used in methods of diagnosis, e.g. clinical screening, and prognosis assessment and in monitoring the results of therapy, identifying subjects most likely to respond to a particular therapeutic treatment, drug screening and development. Such biomarkers include, for example, the level of nucleosomes isolated by a method of the invention(/.e. the level of nucleosomes comprising linker DNA) or the level of epigenetic features of the isolated cell free nucleosomes.
  • kits for performing methods of the invention.
  • Such kits will suitably comprise a ligand for detection and/or quantification of the target or biomarker, and/or a biosensor, and/or an array as described herein, optionally together with instructions for use of the kit.
  • a further aspect of the invention is a kit for detecting the presence of a disease state, comprising a biosensor capable of detecting and/or quantifying one or more of the biomarkers as defined herein.
  • detecting and “diagnosing” as used herein encompass identification, confirmation, and/or characterisation of a disease state.
  • Methods of detecting, monitoring and of diagnosis according to the invention are useful to confirm the existence of a disease, to monitor development of the disease by assessing onset and progression, or to assess amelioration or regression of the disease.
  • Methods of detecting, monitoring and of diagnosis are also useful in methods for assessment of clinical screening, prognosis, choice of therapy, evaluation of therapeutic benefit, i.e. for drug screening and drug development.
  • Efficient diagnosis and monitoring methods provide very powerful “patient solutions” with the potential for improved prognosis, by establishing the correct diagnosis, allowing rapid identification of the most appropriate treatment (thus lessening unnecessary exposure to harmful drug side effects), and reducing relapse rates.
  • Circulating cell free nucleosomes level is a non-specific indicator and occurs in a variety of conditions including inflammatory diseases, a large variety of benign and malignant conditions, autoimmune diseases, as well as following trauma or ischaemia (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 subjects. These include, without limitation, trauma (for example; severe injury or surgery), extreme exercise (for example running a marathon), stroke and heart attack, sepsis or other serious infection and endometriosis.
  • the immunoassays of the invention include immunometric assays employing enzyme detection methods (for example ELISA), fluorescence labelled immunometric assays, time- resolved fluorescence labelled immunometric assays, chemiluminescent immunometric assays, immunoturbidimetric assays, particulate labelled immunometric assays and immunoradiometric assays and competitive immunoassay methods including labelled antigen and labelled antibody competitive immunoassay methods with a variety of label types including radioactive, enzyme, fluorescent, time-resolved fluorescent and particulate labels. All of said immunoassay methods are well known in the art, see for example Salgame et al, 1997 and van Nieuwenhuijze et al, 2003.
  • Any DNA physical or chemical analysis method may be employed for methods of the invention including methods to measure DNA fragment length (e.g. chromatography or electrophoresis). Any physical or chemical method may also be employed for further DNA analysis such as that described by Sina et al, 2018. Similarly, any DNA sequencing method may be employed including Next Generation Sequencing (targeted or whole genome) and methylated DNA sequencing analysis, BEAMing, PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR), isothermal amplification, hybridization, MIDI-Activated Pyrophosphorolysis (MAP) or Personalized Analysis of Re-arranged Ends (PARE).
  • Next Generation Sequencing targeted or whole genome
  • BEAMing PCR including digital PCR and cold PCR (co-amplification at lower denaturation temperature-PCR)
  • isothermal amplification hybridization
  • MAP MIDI-Activated Pyrophosphorolysis
  • PARE Personalized Analysis of Re-arranged Ends
  • DNA analysis may include analysis for any genetic DNA markers including nucleotide substitutions, nucleotide insertions, nucleotide deletions, methylated DNA sequences or other DNA sequence mutations.
  • Typical cancer associated DNA abnormalities that may be investigated in such an analysis include, without limitation, point mutations, translocations, gene copy number mutations, microsatellite abnormalities, DNA strand integrity and gene methylation status.
  • the method of the invention is repeated on multiple occasions.
  • This embodiment provides the advantage of allowing the detection results to be monitored over a time period.
  • Such an arrangement will provide the benefit of monitoring or assessing the efficacy of treatment of a disease state.
  • Such monitoring methods of the invention can be used to monitor onset, progression, stabilisation, amelioration, relapse and/or remission.
  • the invention also provides a method of monitoring efficacy of a therapy for a disease state in a subject, suspected of having such a disease, comprising detecting and/or quantifying the biomarker present in a biological sample from said subject.
  • test samples may be taken on two or more occasions.
  • the method may further comprise comparing the level of the biomarker(s) present in the test sample with one or more control(s) and/or with one or more previous test sample(s) taken earlier from the same test subject, e.g. prior to commencement of therapy, and/or from the same test subject at an earlier stage of therapy.
  • the method may comprise detecting a change in the nature or amount of the biomarker(s) in test samples taken on different occasions.
  • a change in the level of the biomarker in the test sample relative to the level in a previous test sample taken earlier from the same test subject may be indicative of a beneficial effect, e.g. stabilisation or improvement, of said therapy on the disorder or suspected disorder.
  • the method of the invention may be periodically repeated in order to monitor for the recurrence of a disease.
  • Methods for monitoring efficacy of a therapy can be used to monitor the therapeutic effectiveness of existing therapies and new therapies in human subjects and in non-human animals (e.g. in animal models). These monitoring methods can be incorporated into screens for new drug substances and combinations of substances.
  • the monitoring of more rapid changes due to fast acting therapies may be conducted at shorter intervals of hours or days.
  • Identifying and/or quantifying can be performed by any method suitable to identify the presence and/or amount of a specific protein in a biological sample from a patient or a purification or extract of a biological sample or a dilution thereof.
  • Biological samples that may be tested in a method of the invention include those as defined hereinbefore.
  • the samples can be prepared, for example where appropriate diluted or concentrated, and stored in the usual manner.
  • the biomarker may be directly detected, e.g. by SELDI (-TOF), LS-MS/MS or MALDI (-TOF).
  • the biomarker may be detected directly or indirectly via interaction with a ligand or ligands such as an antibody or a biomarker-binding fragment thereof, or other peptide, or ligand, e.g. aptamer, affimer, or oligonucleotide, or molecularly imprinted polymer, capable of specifically binding the biomarker.
  • the ligand or binder may possess a detectable label, such as a luminescent, fluorescent or radioactive label, and/or an affinity tag.
  • detecting and/or quantifying can be performed by one or more method(s) selected from the group consisting of: SELDI (-TOF), MALDI (-TOF), a 1-D gel-based analysis, a 2-D gel-based analysis, Mass spectrometry (MS), reverse phase (RP) LC (liquid chromatography), size permeation (gel filtration), ion exchange, affinity, HPLC, LIPLC and other LC or LC-MS-based techniques.
  • SELDI SELDI
  • MALDI MALDI
  • HPLC high pressure liquid chromatography
  • LPLC low pressure liquid chromatography
  • nanoflow LC nanoflow LC
  • TMT tandem mass tags
  • TFT thin-layer chromatography
  • NMR nuclear magnetic resonance
  • Methods of diagnosing or monitoring according to the invention may comprise analysing a sample to detect the presence or level of the biomarker. These methods are also suitable for clinical screening, prognosis, monitoring the results of therapy, identifying patients most likely to respond to a particular therapeutic treatment, for drug screening and development, and identification of new targets for drug treatment.
  • Identifying and/or quantifying may be performed using an immunological method, involving an antibody, or a fragment thereof capable of specific binding to the biomarker.
  • Suitable immunological methods include sandwich immunoassays, such as sandwich ELISA, in which the detection of the analyte biomarkers is performed using two antibodies which recognize different epitopes on a analyte biomarker; radioimmunoassays (RIA), direct, indirect or competitive enzyme linked immunosorbent assays (ELISA), enzyme immunoassays (EIA), chemiluminescence immunoassays (ChLIA), Fluorescence immunoassays (FIA), western blotting, immunoprecipitation and any particle-based immunoassay (e.g. using gold, silver, or latex particles, magnetic particles, or Q-dots).
  • Immunological methods may be performed, for example, in microtitre plate, test-tube cuvettes or strip format.
  • biosensors for performing methods of the invention include “credit” cards with optical or acoustic readers. Biosensors can be configured to allow the data collected to be electronically transmitted to the physician for interpretation and thus can form the basis for e-medicine.
  • biosensor means anything capable of detecting the presence of the target.
  • kits for the diagnosis and monitoring of the presence of a disease state are described herein.
  • the kits additionally contain a biosensor capable of identifying and/or quantifying a biomarker.
  • a kit according to the invention may contain one or more components selected from the group: a ligand binder, or ligands, specific for the biomarker or a structural/shape mimic of the biomarker, one or more controls, one or more reagents and one or more consumables; optionally together with instructions for use of the kit in accordance with any of the methods defined herein.
  • biomarkers for a disease state permits integration of diagnostic procedures and therapeutic regimes. For example, detection of a biomarker can be used to screen subjects prior to their participation in clinical trials.
  • the biomarkers provide the means to indicate therapeutic response, failure to respond, unfavourable side-effect profile, degree of medication compliance and achievement of adequate serum drug levels.
  • the biomarkers may be used to provide warning of adverse drug response. Biomarkers are useful in development of personalized therapies, as assessment of response can be used to fine-tune dosage, minimise the number of prescribed medications, reduce the delay in attaining effective therapy and avoid adverse drug reactions.
  • biomarker can be used to titrate the optimal dose, predict a positive therapeutic response and identify those patients at high risk of severe side effects.
  • Biomarker-based tests provide a first line assessment of ‘new’ patients, and provide objective measures for accurate and rapid diagnosis, not achievable using the current measures.
  • biomarker tests are useful to identify family members or patients with mild or asymptomatic disease or who may be at high risk of developing symptomatic disease. This permits initiation of appropriate therapy, or preventive measures, e.g. managing risk factors. These approaches are recognised to improve outcome and may prevent overt onset of the disorder.
  • Biomarker monitoring methods, biosensors and kits are also vital as patient monitoring tools, to enable the physician to determine whether relapse is due to worsening of the disorder. If pharmacological treatment is assessed to be inadequate, then therapy can be reinstated or increased; a change in therapy can be given if appropriate. As the biomarkers are sensitive to the state of the disorder, they provide an indication of the impact of drug therapy.
  • This example used three H1 protein products. The first was a recombinant full-length H1 protein purchased from Sigma-Aldrich (Product No: H1917-100UG, referred to as H1 FL-a). The second was a tagged recombinant H1 protein full length (referred to as H1 FL-b). The third was a tagged recombinant truncated H1 protein (referred to as H 1g). This truncated form is composed of the first 96 amino acids of the H1 protein corresponding to the N-terminal and globular domain. All protein products were derived from H1.0.
  • H1 FL-b H1 FL-a/H1g per mg of magnetic beads in borate buffer 0.1 M, pH 9.5 with ammonium sulphate.
  • borate buffer 0.1 M borate buffer 0.1 M, pH 9.5 with ammonium sulphate.
  • the coated beads were then blocked for 1 h at 37°C with blocking buffer (PBS with 0.1% tween 20 and 1% of BSA, pH 7.4) and then washed 3 times with a wash buffer (PBS with 0.1% tween 20 with 0.1 % BSA, pH 7.4) before their use.
  • blocking buffer PBS with 0.1% tween 20 and 1% of BSA, pH 7.4
  • a wash buffer PBS with 0.1% tween 20 with 0.1 % BSA, pH 7.4
  • H1g-beads demonstrate that the H1 protein may be used to prepare immunosorbents that bind to nucleosomes containing linker DNA.
  • H1g protein may be used to capture nucleosomes containing linker DNA more efficiently than full length H1.
  • the results demonstrate that the protein of the present invention can be used as a binder in an immunoassay for nucleosomes generally and also to certain types of nucleosomes that are bound selectively by the protein.
  • H1g coated magnetic beads here referred after as H1g-beads
  • H1g-beads H1g coated magnetic beads
  • a mixture of 3 recombinant mononucleosomes with different DNA lengths i.e. , 147bp or 167bp or 187bp
  • the H1g-beads were then isolated using a magnet, washed and treated with Proteinase-K to release any bound nucleosomal DNA.
  • the supernatant remaining after the H1g-beads incubation was also treated with proteinase K to purify any unbound nucleosomal DNA.
  • nucleosomal DNA was extracted from the unprocessed fraction of the same HeLa nucleosome preparation.
  • the observed DNA size distribution of the unprocessed fraction (dotted line) consists of 2 bands corresponding to the size of mononucleosome (157bp) and the dinucleosome (331 bp).
  • the observed size distribution of the isolated DNA consists of 171 bp DNA (solid black line) whereas the unbound DNA is shorter with a peak at 150bp (grey line) ( Figure 4).
  • a histone H1g coated magnetic bead reagent is incubated with a plasma sample taken from a cancer patient suffering from a metastatic colorectal cancer.
  • the magnetic reagent is isolated using a magnet and the DNA present in the liquid phase and the magnetic beads is extracted. The extracted DNA is then sequenced.
  • the observed DNA size distribution of the unprocessed fraction (dotted line) consists of a mononucleosome (162bp) and a mixture of di-, tri- and poly-nucleosomes. After H1g-bead incubation, the observed size distribution of the isolated DNA consists of 173bp and the mixture of di-, tri- and poly-nucleosomes (solid black line) whereas the unbound DNA is shorter with a peak at mononucleosome size (168bp, grey line) ( Figure 5a).

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Abstract

L'invention concerne une protéine isolée comprenant un domaine globulaire d'histone H1 (H1g), ainsi que des procédés et des utilisations associés. L'invention peut être utilisée pour détecter et isoler des nucléosomes acellulaires circulants, en particulier des nucléosomes d'origine de maladie.
PCT/EP2024/050871 2023-01-16 2024-01-16 Protéines et procédés d'isolement de nucléosomes circulants Ceased WO2024153619A1 (fr)

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