WO2019212364A1 - Test for heart failure - Google Patents

Test for heart failure Download PDF

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Publication number
WO2019212364A1
WO2019212364A1 PCT/NZ2019/050046 NZ2019050046W WO2019212364A1 WO 2019212364 A1 WO2019212364 A1 WO 2019212364A1 NZ 2019050046 W NZ2019050046 W NZ 2019050046W WO 2019212364 A1 WO2019212364 A1 WO 2019212364A1
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WIPO (PCT)
Prior art keywords
erfe
peptide
heart failure
patient
level
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PCT/NZ2019/050046
Other languages
French (fr)
Inventor
Christopher Joseph Pemberton
Arthur Mark Richards
Sarah APPLEBY
Richard TROUGHTON
Original Assignee
Christopher Joseph Pemberton
Arthur Mark Richards
Appleby Sarah
Troughton Richard
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Application filed by Christopher Joseph Pemberton, Arthur Mark Richards, Appleby Sarah, Troughton Richard filed Critical Christopher Joseph Pemberton
Publication of WO2019212364A1 publication Critical patent/WO2019212364A1/en

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    • 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
    • G01N33/74—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving hormones or other non-cytokine intercellular protein regulatory factors such as growth factors, including receptors to hormones and growth factors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
    • G01N2333/575—Hormones
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/325—Heart failure or cardiac arrest, e.g. cardiomyopathy, congestive heart failure
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/32—Cardiovascular disorders
    • G01N2800/326—Arrhythmias, e.g. ventricular fibrillation, tachycardia, atrioventricular block, torsade de pointes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/50—Determining the risk of developing a disease
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/52—Predicting or monitoring the response to treatment, e.g. for selection of therapy based on assay results in personalised medicine; Prognosis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/54—Determining the risk of relapse

Definitions

  • the present invention is concerned with the diagnosis or prediction of acute decompensated heart failure in a patient.
  • the present invention provides test kits, assays and methods useful for diagnosing acute decompensated heart failure in a patient, or for predicting a patient's risk of acquiring acute decompensated heart failure within one year of diagnosis with any medical condition, including a cardiac disease or disorder.
  • ADHF Acute decompensated heart failure
  • dyspnea difficulty breathing
  • leg or feet swelling a common and potentially serious cause of acute respiratory distress.
  • the condition is caused by severe congestion of multiple organs by fluid that is inadequately circulated by the failing heart.
  • An attack of decompensation can be caused by underlying medical illness, such as myocardial infarction, an abnormal heart rhythm, infection, or thyroid disease.
  • Treatment consists of reducing the fluid level with diuretics and improving heart function with nitrates, or levosimendan; other treatments such as aquapheresis ultra- filtration may also be required.
  • ADHF patients have typically been achieved using a jugular venous distension. However, this process can be laborious and requires specialist knowledge to interpret the results. As such there is an ongoing need to develop more useful clinical tools in the diagnosis or prognosis of acute decompensated heart failure in patients presenting to a clinic or emergency department.
  • the present invention seeks to address these requirements.
  • the present invention is predicated on the surprising and unexpected discovery of the correlation between levels of erythroferrone (ERFE) as a biomarker for the prediction or diagnosis of acute decompensated heart failure in a patient. Accordingly, in one aspect of the present invention there is provided a method for diagnosing acute decompensated heart failure in a patient, the method comprising the steps of:
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
  • a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of a diagnosis with any medical condition comprising the steps of:
  • the biological sample has been obtained from the patient within one year of diagnosis with any medical condition, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of the diagnosis with any medical condition.
  • a method for monitoring or assessing a patient diagnosed with acute heart failure comprising:
  • an assay for measuring the level of an erythroferrone (ERFE) peptide from a patient with, or at risk of acquiring, heart failure comprising a binding agent that selectively binds to an ERFE peptide, which binding agent can be quantitatively measured upon binding to the ERFE peptide in a biological sample from the subject.
  • ERFE erythroferrone
  • test kit or article of manufacture for diagnosing or predicting acute decompensated heart failure in a patient
  • the test kit or article of manufacture comprising an erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, together with instructions for how to diagnose heart failure in a patient, or instructions for how to predict a patient's risk of acquiring heart failure within one year of a diagnosis with any medical condition.
  • ERFE erythroferrone
  • a method of diagnosing atrial fibrillation in a patient comprising the steps of:
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of atrial fibrillation in the patient.
  • Figure 2 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and acute decompensated heart failure, (ii) NT-proBNP and acute decompensated heart failure, and (iii) ERFE+NT-proBNP and acute decompensated heart failure in patients, where in all patients haemaglobin levels [Hb] > 129 g/L.
  • ERFE was elevated in the acute decompensated heart failure patients as compared to ERFE levels from patients in a control population.
  • Figure 3 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and acute decompensated heart failure, (ii) NT-proBNP and acute decompensated heart failure, (iii) high sensitivity Troponin T (hsTnT) and acute decompensated heart failure, (iv) proadrenomedullin (proADM) and acute decompensated heart failure, and (v) ERFE+NT-proBNP and acute decompensated heart failure in patients, where in all patients haemaglobin levels [Hb] > 140 g/L.
  • ERFE 0.810 ⁇ 0.053
  • NT-proBNP 0.841 ⁇ 0.051
  • hsTnT 0.616 ⁇ 0.059
  • proADM 0.728 ⁇ 0.053
  • ERFE+NT-proBNP 0.835 ⁇ 0.046.
  • Figure 4 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and atrial fibrillation.
  • ERFE was elevated in atrial fibrillation patients as compared to ERFE levels from patients in a control population.
  • Figure 5 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between ERFE and acute decompensated heart failure in those patients with atrial fibrillation.
  • ERFE was elevated in atrial fibrillation patients as compared to ERFE levels from patients in a control population.
  • composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
  • STMI ST-elevation myocardial infarction
  • N-STEMI non ST-elevation myocardial infarction
  • UAP or "UA” as used herein means unstable angina (pectoris).
  • peptide and “polypeptide” or “protein” may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds.
  • Polypeptides useful in the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof.
  • Polypeptides herein may have chain lengths of at least 4 amino acids, at least 5 amino acids, or at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all 23 amino acids of the full-length EPOsp and/or CNPsp.
  • Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.
  • fragment or “functional derivative” in relation to a polypeptide is a subsequence of a polypeptide that may be detected using a binding agent.
  • the term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.
  • isolated as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment.
  • An isolated polypeptide may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques.
  • the polypeptide sequences may be prepared by at least one purification step.
  • purified does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample. The term should be similarly understood in relation to other molecules and constructs described herein.
  • variants refers to polypeptide sequences different from the specifically identified sequences, wherein 1 to 6 or more or amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of one, two, three, four, five or six amino acids are contemplated. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides.
  • variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about
  • identity is found over a comparison window of at least 5 to 7 amino acid positions.
  • Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance.
  • function may be as either a polypeptide, or antigenic polypeptide, or both.
  • Polypeptide sequence identity and similarity can be determined in the following manner.
  • the subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • BLASTP from the BLAST suite of programs, version 2.2.18 [April 2008]
  • bl2seq which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/).
  • NCBI ftp://ftp.ncbi.nih.gov/blast/
  • the similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp.
  • the parameter -F F turns off filtering of low complexity sections.
  • the parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match.
  • Variant polypeptide sequences commonly exhibit an E value of less than 1 x 10-5, less than 1 x 10-6, less than 1 x 10-9, less than 1 x 10-12, less than 1 x 10-15, less than 1 x 10-18 or less than 1 x 10-21 when compared with any one of the specifically identified sequences.
  • Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS- needle (available at http:/www. ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention.
  • biological sample includes biological fluids selected from blood including venous blood and arterial blood, plasma, serum, intertistial fluid, or any other body fluid.
  • biological sample also includes heart tissue sample.
  • biological sample and body fluid sample refers to a biological sample or a sample of bodily fluid obtained for the purpose of, for example, diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient.
  • such a sample may be obtained for diagnosing acute decompensated heart failure, for performing risk stratification of acute decompensated heart failure, for making a prognosis of a disease course in a patient with acute decompensated heart failure, for identifying a patient with elevated risk of acute decompensated heart failure, or combinations thereof.
  • body fluid samples would be more readily analysed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
  • level as used herein is intended to refer to the amount per weight or weight per weight of erythoferrone (ERFE). It is also intended to encompass “concentration” expressed as amount per volume or weight per volume.
  • concentration expressed as amount per volume or weight per volume.
  • circulating level is intended to refer to the amount per weight or weight per weight or concentration of an ERFE peptide present in the circulating fluid, such as whole blood, serum or plasma.
  • An “increase” or “decrease” in the level of an ERFE peptide (or any other biomarker for that matter) compared with a control, or a “change” or “deviation” from a control (level) in one example is statistically significant.
  • An increased level, decreased level, deviation from, or change from a control level or mean or historical control level can be considered to exist if the level differs from the control level by about 5% or more, by about 10% or more, by about 20% or more, or by about 50% or more compared to the control level.
  • Statistically significant may alternatively be calculated as P ⁇ 0.05.
  • Increased levels, decreased levels, deviation, and changes can also be determined by recourse to assay reference limits or reference intervals. These can be calculated from intuitive assessment or non-parametric methods.
  • these methods may calculate the 0.025, and 0.975 fractiles as 0.025* (n+ 1) and 0.975 (n+ 1). Such methods are well known in the art. Presence of a marker absent in a control may be seen as a higher level, deviation or change. Absence of a marker present in a control may be seen as a lower level, deviation or change.
  • reference interval as used herein is intended to refer to a figure within a statistical band of a representative concentration or alternatively a figure with an upper or lower concentration.
  • the reference interval will typically be obtained from subjects that do not have any pre-existing conditions that could result in artificially elevating the level of circulating ERFE.
  • delta % as used herein is understood to refer to a percentage change in a given variable (i.e. the level or concentration of ERFE peptide).
  • the delta % is determined by taking the final concentration of ERFE peptide in a biological sample, subtracting the initial concentration of ERFE peptide and dividing it by the initial concentration of ERFE peptide where the result is presented as a percentage.
  • an increase of 300 pmol/mL from an initial concentration of 800 pmol/mL represents a 37.5% change.
  • an increase of 31-39 delta % includes for example, 31, 32, 33, 34, 35, 36, 37, 38 or 39 delta % increase, as well as fractions there between.
  • subject or “patient” may used interchangeably herein to refer to a human or non-human primate.
  • the subject is a human.
  • suitable control population refers to the mean circulating ERFE peptide level from sex- and age-matched subjects for which their cardiac disease or disorder status is known.
  • the control population is used to provide a suitable reference interval by which a measured ERFE peptide level is compared.
  • binding agent as used herein is intended to refer to any molecule that binds (e.g.) an ERFE peptide, including small molecules, antibodies from any species whether polyclonal or monoclonal, antigen-binding fragments such as Fab and Fab2, humanized antibodies, chimeric antibodies, or antibodies modified in other ways including substitution of amino acids, and/or fusion with other peptides or proteins (e.g. PEG). It further includes aptamers (i.e. 40-80 polynucleotide molecules) which have been selectively evolved to have binding affinity for a target antigen, for example, ERFE peptide or fragmen thereof. It also includes receptors or binding proteins from any species or modified forms of them. In one example, the binding agent specifically binds to ERFE.
  • aptamers i.e. 40-80 polynucleotide molecules
  • the binding agent specifically binds to ERFE.
  • binding agent specifically binds to ERFE binds that protein/peptide or an epitope or immunogenic fragment thereof with greater affinity, avidity, more readily, and/or with greater duration than it binds to unrelated protein/peptide and/or epitopes or immunogenic fragments thereof. It is also understood by reading this definition that, for example, a binding agent that specifically binds to a first target (e.g.
  • ERFE may or may not specifically bind to a second target.
  • specific binding does not necessarily require exclusive binding or non-detectable binding of another molecule.
  • reference to binding means specific binding.
  • antibody refers to an immunoglobulin molecule capable of selectively binding to a target, such as ERFE, by virtue of an antigen binding site contained within at least one variable region.
  • This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, half antibodies, bispecific antibodies) and single domain antibodies such as domain antibodies and heavy chain only antibodies (e.g., camelid antibodies or cartilaginous fish immunoglobulin new antigen receptors (IgNARs)).
  • An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc).
  • Preferred forms of antibodies comprise a four-chain structure as their basic unit.
  • Full-length antibodies comprise two heavy chains ( ⁇ 50-70 kDa) covalently linked and two light chains ( ⁇ 23 kDa each).
  • a light chain generally comprises a variable region and a constant domain and in mammals is either a k light chain or a l light chain.
  • a heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s).
  • Heavy chains of mammals are of one of the following types a, d, e, g, or m.
  • Each light chain is also covalently linked to one of the heavy chains.
  • the two heavy chains and the heavy and light chains are held together by inter- chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies.
  • Each chain has an N-terminal variable region (VH or VL wherein each are ⁇ 110 amino acids in length) and one or more constant domains at the C- terminus.
  • the constant domain of the light chain (CL which is ⁇ 110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH which is -330-440 amino acids in length).
  • the light chain variable region is aligned with the variable region of the heavy chain.
  • the antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region can be identified between the CHI and Cm constant domains.
  • Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • the antibody is a murine (mouse or rat) antibody or a primate (preferably human) antibody.
  • the term "antibody” encompasses not only intact polyclonal or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion with an antigen binding site, humanised antibodies, human antibodies, chimeric antibodies, primatised antibodies, de-immunised antibodies or veneered antibodies.
  • antigen-binding fragment or "antigen-binding antibody fragment” shall be taken to mean any fragment of an antibody that retains the ability to bind to ERFE and preferably one which specifically binds to ERFE. This term includes a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain antibody (SCA or SCAB) amongst others.
  • An "Fab fragment” consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain .
  • An "Fab' fragment” of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner.
  • An "F(ab')2 fragment” of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction.
  • An "Fv fragment” is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains.
  • a “single chain antibody” (SCA) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.
  • 'chimeric antibody refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl Acad. Sci USA 81 :6851-6855).
  • a particular species e.g., murine, such as mouse
  • primate such as human
  • humanized antibody shall be understood to refer to a chimeric molecule, generally prepared using recombinant techniques, having an epitope binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin.
  • the antigen-binding site preferably comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate framework regions in the variable domains of human antibodies and the remaining regions from a human antibody.
  • Epitope binding sites may be wild type or modified by one or more amino acid substitutions.
  • variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs.
  • CDRs complementarity-determining regions
  • FRs framework regions
  • the variable regions can be "reshaped” or “humanized” by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
  • epitope includes any antigenic (e.g., a protein) determinant capable of specific binding to an antibody and/or a T cell receptor. That is, a site on an antigen to which B and/or T cells respond.
  • Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.
  • An epitope typically includes, for example, at least 3, 5 or 8-10 amino acids. The amino acids may be contiguous, or non-contiguous amino acids juxtaposed by tertiary folding. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
  • antigenic variant refers to polypeptide sequences different from the specifically identified sequences, wherein one or more amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of 1, 2, 3 or 4 amino acids are specifically contemplated. Variants may be naturally-occurring allelic antigenic variants, or non-naturally occurring antigenic variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, antigenic variants of the polypeptides useful in the invention have biological activities including hormone function or antigenic-binding properties that are the same or similar to those of the parent polypeptides.
  • antigenic variant with reference to (poly)peptides encompasses all forms of polypeptides as defined herein. The term “antigenic variant” encompasses naturally occurring, recombinantly and synthetically produced polypeptides.
  • polypeptide antigenic variants may be identified by physical methods known in the art, for example, by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) by recombinant DNA techniques also described by Sambrook et al. or by identifying polypeptides from natural sources with the aid of such antibodies.
  • the term "marker” or “biomarker” in the context of an analyte means any antigen, molecule or other chemical or biological entity that is specifically found in circulation or associated with a particular tissue (e.g. heart muscle) that it is desired to be identified in or on a particular tissue affected by a disease or disorder, for example heart failure.
  • the marker is a circulating peptide (e.g.) ERFE.
  • the marker is a cell surface antigen or a nuclear antigen that is differentially or preferentially expressed by specific cell types.
  • the marker is an intracellular antigen that is differentially or preferentially expressed by specific cell types.
  • ROC Receiver Operating Curve and a ROC plot depicts the overlap between two distributions by plotting the sensitivity versus 1 -specificity for a complete range of decision thresholds.
  • AUC means Area Under the Curve which yields information about the strength of a correlation determined by the Receiver Operating Curve analysis. Typical ROC values where the AUC > 0.70 yields a statistically significant correlation.
  • the term "effective amount” refers to the amount of a therapy that is sufficient to result in the prevention of the development, recurrence, or onset of a disease or condition and one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity, the duration of disease, ameliorate one or more symptoms of the disease or condition, prevent the advancement of the disease or condition, cause regression of the disease or condition, and/or enhance or improve the therapeutic effect(s) of another therapy.
  • the terms “manage”, “managing”, and “management” in the context of the administration of a therapy to a subject refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) or a combination of therapies, while not resulting in a cure of the disease or condition.
  • a subject is administered one or more therapies (e.g., one or more prophylactic or therapeutic agents) to "manage” the disease or condition so as to prevent the progression or worsening of the disease or condition.
  • therapeutic agent refers to any molecule, compound, and/or substance that is used for the purpose of treating and/or managing a disease or disorder, such as unstable angina.
  • therapeutic agents include, but are not limited to, proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs, Ig fragments, polyclonal antibodies and their fragments, monoclonal antibodies and their fragments), peptides (e.g., peptide receptors, selectins), binding proteins, biologies, proliferation-based therapy agents, hormonal agents, radioimmunotherapies, targeted agents, epigenetic therapies, differentiation therapies, biological agents, and small molecule drugs.
  • proteins include, but are not limited to, proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs, Ig fragments, polyclonal antibodies and their fragments, monoclonal antibodies and their fragments), peptides (e.g., peptide receptors, selectins), binding proteins, biologi
  • therapies and “therapy” can refer to any method(s), composition(s), and/or agent(s) that can be used in the prevention, treatment and/or management of a disease or condition or one or more symptoms thereof.
  • the terms “treat”, “treatment” and “treating” in the context of the administration of a therapy to a subject refer to the reduction, inhibition, elimination or amelioration of the progression and/or duration of (e.g.) acute decompensated heart failure, the reduction, inhibition, elimination or amelioration of the severity of (e.g.) acute decompensated heart failure, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies.
  • presentation and “index presentation” as used herein refers to those patients who have presented to an emergency department, to a hospital, to a medical clinic, to a health or well-being clinic, to a doctor, to a general practitioner, to a surgery etc. Description of the Present Invention
  • Erythroferrone (ERFE) protein is produced in bone marrow erythroblasts which promote increased intestinal absorption of iron and the release of iron from stores. ERFE performs this function by suppressing the hepatic synthesis of the master iron regulator known as hepcidin (Ganz et al. Blood 2017; Pautz et al. Nat Genetics 2014). ERFE is also known as myonectin and is produced by the gene known as FAM132B. Iron deficiency is a known co-morbidity in patients suffering heart failure and as such, Applicants hypothesized that the response or potential biomarker utility for ERFE in these patients warranted further investigation.
  • ERFE erythroferrone
  • Applicants have therefore identified a clinical utility for ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient.
  • a method for diagnosing acute decompensated heart failure in a patient comprising the steps of:
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
  • measurement of the ERFE levels is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
  • ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient significantly improved where the haemaglobin levels approximated or exceeded what is considered to be 'normal range'. For example, where [Hb] >129 g/L for a male patient and [Hb]>115 g/L for a female patient. Applicants rationalised this observation based on the role of ERFE in iron metabolism, and role of haemaglobin in iron trafficking.
  • the threshold haemaglobin level was [Hb] > 140 g/L
  • the performance of ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient was further enhanced.
  • ERFE is a superior biomarker for diagnosis or prediction of ADHF in patients that hsTnT and proADM.
  • the method further comprises measuring the haemaglobin levels of the patient to determine whether the patient should be included or excluded from a diagnostic analysis for acute decompensated heart failure involving ERFE.
  • Example 9 when read in conjunction with Figure 5, demonstrates a utility for ERFE in diagnosing acute decompensated heart failure in patients who have or present with atrial fibrillation.
  • a method for diagnosing acute decompensated heart failure in a patient who has atrial fibrillation comprising the steps of:
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
  • ERFE may be used to predict an episode of ADHF in a patient within one year of diagnosis with any medical condition, including cardiac related disease and disorders (as well as recurrent ADHF).
  • a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of diagnosis with any medical condition comprising the steps of:
  • the biological sample has been obtained from the patient within one year of diagnosis with any medical condition, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of the diagnosis with any medical condition.
  • a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of index presentation comprising the steps of:
  • the biological sample has been obtained from the patient within one year of index presentation, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of index presentation.
  • measurement of the ERFE peptide is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
  • the medical condition is selected from a cardiac related disease or disorder, diabetes, smoking related disease, chronic pulmonary artery disease, pulmonary artery disease, coronary artery disease, acute and chronic kidney disease.
  • the cardiac related disease or disorder is selected from myocardial infarction, unstable angina, atrial fibrillation, cardiac hypertrophy, mitral regurgitation, valve disorders, heart failure, pericarditis, disorders of nerve conduction, vasovagal syncope or any combination thereof.
  • ERFE could be used to predict hospital readmission for any reason within 30 or 90 days by determining levels or ERFE, where elevated levels meant readmission (following an episode of ADHF or any medical condition) was more likely. For example, refer to Tables IX and X in Example 6.
  • ERFE as a biomarker to monitor disease progression, with or without response to treatment
  • the methods, test kits and assays as described herein could be used to (i) establish the ADHF disease state of an individual where elevated levels relative to a reference interval obtained from a control population is diagnostic (or prognostic) of ADHF in the patient, (ii) monitor or assess disease progression as a function of time by establishing ERFE levels in multiple samples taken from the same patient at different time points, and (iii) monitor or assess whether a treatment regime has been successful (i.e.) a measured decrease in the levels of ERFE following an intervention/treatment would indicate that the patient has responded well to the intervention/treatment; the converse being that no reduction in the ERFE levels (or even an increase) would mean that the intervention/treatment has had little or no beneficial effect on the patient.
  • a method for monitoring or assessing a patient diagnosed with acute heart failure comprising:
  • the present invention further contemplates test kits and assays for detecting the level of ERFE in a sample, such as a biological sample obtained from a patient of interest.
  • an assay for measuring the level of an erythroferrone (ERFE) protein from a patient with, or at risk of acquiring, heart failure comprising a binding agent that selectively binds to an ERFE peptide, which binding agent can be quantitatively measured upon binding to the ERFE peptide in a biological sample from the subject.
  • ERFE erythroferrone
  • the assay may be selected from an immunoassay, an enzyme assay, a flourescence assay and a chemiluminescence assay.
  • Example 3/Table VI demonstrates that ERFE is a useful biomarker in a multivariate analysis for the diagnosis of acute decompensated heart failure when using the clinical variables of age, gender, body mass index, history of myocardial infarction, history of diabetes, NT-proBNP among others.
  • NT-proBNP is the current preferred marker used by clinicians for the diagnosis of ADHF, and these data demonstrate the utility of ERFE is identifying a select patient cohort who may otherwise avoid detection if only NT-proBNP was used.
  • NT-proBNP N-Terminal B-Type Natriuretic Peptide
  • ERFE erythroferrone
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic that the patient has acute decompensated heart failure.
  • Examples 7 and 8 when read in conjunction with Tables XI-XIV demonstrate a further utility for ERFE as a biomarker in uni- and multi-variate equations to predict mortality within 1 year of index presentation, or to predict a subsequent episode of heart failure within one year of index presentation.
  • a method for predicting mortality in a patient within one year of index presentation comprising the steps of:
  • an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is predictive of the mortality within one year of index presentation.
  • index presentation includes, but is not limited to, presentation to an emergency department, presentation to a hospital, presentation to a medical clinic, presentation to a health or well-being clinic, presentation to a doctor, presentation to a general practitioner and presentation to a surgery.
  • test kit or article of manufacture for diagnosing or predicting acute decompensated heart failure in a patient
  • the test kit or article of manufacture comprising an erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, together with instructions for how to diagnose heart failure in a patient, or instructions for how to predict a patient's risk of acquiring heart failure within one year of index presentation or diagnosis with any medical condition, or instructions for how to predict mortality within one year of index presentation .
  • ERFE erythroferrone
  • the ERFE peptide is a human ERFE protein as defined by SEQ ID NO: 1, as follows:
  • control population is sex and age-matched subjects who do not have acute decompensated heart failure, or who are not predisposed to acute decompensated heart failure, as measured by one or more risk factors.
  • the reference interval is the mean ERFE peptide level from the control population.
  • the sample is a biological sample, and may be selected from plasma, serum, whole blood, arterial blood, venous blood, saliva, bone marrow tissue, heart tissue, vascular tissue and interstitial fluid.
  • the biological sample is preferably plasma, serum, whole blood, arterial blood and venous blood.
  • the methods, assays and test kits described herein may be performed in conjunction with the analysis of one or more other risk factors or biomarkers of acute decompensated heart failure, including but not limited heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, BNP, and iron and/or anaemia status, including ferritin, transferrin, and ferritin/transferrin saturation levels.
  • risk factors or biomarkers of acute decompensated heart failure including but not limited heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, BNP, and iron and/or anaemia status, including ferritin, transferrin, and ferritin/transferrin saturation levels.
  • the methods, assays and test kits require that level of an erythroferrone (ERFE) protein in a biological sample obtained from the patient is measured, typically by detecting binding between a binding agent that selectively binds to the ERFE peptide of interest.
  • the binding agent is selected from an antibody or antigen binding antibody fragment thereof, or a nucleic based aptamer (e.g. deoxyribose or ribose nucleic acid aptamers) which antibody, antigen binding antibody fragment or aptamer specifically bind to the ERFE peptide of interest.
  • the binding agent may include a detectable label to facilitate identification when bound to the ERFE peptide and/or may be immobilised to a solid phase, such as, without limitation, a plate, a porous strip, a bead, a chip, a chromatography column and/or a mass spectrometry chamber.
  • the binding agent is an antibody
  • the antibody may be selected from a polyclonal, monoclonal, chimeric or humanised antibody or antigen-binding antibody fragment thereof.
  • the binding agent is an aptamer
  • the aptamer may be selected from a doxyribose nucleic acid or ribose nucleic acid based aptamers. Further information concerning antibodies, apatmers and their generation for particular antigen peptides (e.g. ERFE) is given in more detail below.
  • the present invention also provides a method for diagnosing atrial fibrillation in a patient, the method comprising the steps of:
  • an increase in the level of the ERFE peptide in the biological sample obtained from the patient compared to the reference interval from a control population is diagnostic of atrial fibrillation in the patient.
  • measurement of the ERFE peptide is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
  • ERFE peptides are measured in a sample, including biological samples, by detecting binding between a ERFE peptide and a binding agent that selectively binds to the ERFE peptide.
  • Binding agents for use in the methods of the present disclosure preferably have low cross-reactivity with other analytes.
  • the binding agents may include antibodies or antigen-binding fragments such as Fab and F(ab) 2 , prepared using antigenic ERFE peptides or fragments thereof as immunising antigens, as well as aptamers or nucleic acid based binding agents including deoxyribose and ribose nucleic acid based aptamers.
  • the polypeptide or fragments may also be coupled or immobilised to a solid support.
  • the binding agent is an antibody.
  • the antibody may be a monoclonal or polyclonal antibody.
  • Methods for producing polyclonal and monoclonal antibodies as well as antigen- binding fragments thereof are well known in the art (for example see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988), but have also been developed in-house by Applicants (e.g. Siriwardena et al. (2010) Circulation 122:255-264).
  • monoclonal antibodies are preferred. It will be appreciated by a person skilled in the art that humanised antibodies are not required for in vitro assays.
  • the binding agent is a dexoyribose or ribose nucleic acid based aptamer.
  • the antibody is raised against an antigenic ERFE peptide or the aptamer is generated using SELEX (systematic evolution of ligands by exponential enrichment).
  • the antigenic ERFE peptide is a human ERFE peptode, and comprises a sequence defined by SEQ ID NO: 1 as follows:
  • Monoclonal antibodies may be produced by methods known in the art. These include the immunological method described by Kohler et al (1975) Nature 256(5517) :495-7 as well as the recombination DNA method described by Huse et al (1989) 246(4935) : 1275-81. The use of recombinant phage antibody systems to produce single chain variable antibody fragments, and subsequent mutation (such as site specific mutagenesis) or chain shifting to produce antibodies to ERFE peptides is also contemplated.
  • Antibodies specific to ERFE can be raised after first conjugating these or similar peptides to a large protein such as limpet hemocyanin (KLH), bovine serum albumin (BSA) or bovine thyroglobulin to make them immunogenic. Coupling can be effected by use of any protein crosslinking agent including for example the common agents glutaraldehyde, carbodiimide or N-(e-maleimido-caproyloxy) succinimide ester (MCS)— providing a cysteine residue is added to the peptide sequence prior to coupling. Injection of these conjugates into rabbits, sheep, mice or other species at monthly intervals followed by collection of blood samples two weeks later will enable production of polyclonal antibodies or monoclonal antibodies from the spleens of mice.
  • KLH limpet hemocyanin
  • BSA bovine serum albumin
  • MCS N-(e-maleimido-caproyloxy) succinimide ester
  • the mouse host described above may be sacrificed and its spleen removed.
  • the messenger RNA (mRNA) are then isolated and cDNA made from the mRNA using specific primers for the heavy and light chains of the variable region of the antibodies and the polymerase chain reaction (PCR) amplification.
  • the DNA sequences for the heavy and light chains are joined with a linker sequence, to ensure the correct reading frame.
  • the DNA construct is inserted into a vector, for example, a plasmid or bacteriophage, or virus, for transformation into a host.
  • the vector is a bacteriophage.
  • Suitable hosts may be selected from prokaryotic, yeast, insect or mammalian cells.
  • a prokaryotic host preferably Escherichia coli is used.
  • the bacteriophage produces a viral coat and the antibody fragments are expressed on the coat, a phage display library.
  • the phage display library can be screened for antibody fragments with the appropriate affinity for the specific antigens.
  • the library can be screened many times and modifications can be made to the antibody construct through protein engineering techniques, such as site directed mutagenesis and chain shuffling all of which are within the capabilities of the person skilled in the art.
  • antibody or antibodies as used herein refers to a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope [34-36].
  • antibody includes antigen binding fragments such as, for example, fragments, subsequences, complementarity determining regions (CDRs) that retain capacity to bind to an antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment [37], which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • Single chain antibodies are also included by reference in the term "antibody.” Further discussion of antibodies and fragments may be found in references (e.g.
  • antiserum obtained by immunizing an animal such as a mouse, rat or rabbit with an antigen, such as for example, BNPsp or BNPsp fragments, as well as antigenic variants thereof.
  • an antigen such as for example, BNPsp or BNPsp fragments
  • methods of preparing polyclonal antibodies are known to the skilled artisan.
  • Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant.
  • the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections.
  • the immunizing agent may include BNPsp or BNPsp fragments, antigenic variants thereof or a fusion protein thereof.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor.
  • adjuvants include Freund's complete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).
  • the immunization protocol may be selected by one skilled in the art without undue experimentation.
  • Monoclonal antibodies may be prepared using hybridoma methods well known in the art [e.g. 45-47].
  • the hybridoma cells may be cultured in a suitable culture medium, alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal.
  • Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the American Type Culture Collection, Virginia, USA.
  • Immunoassays may be used to screen for immortalized cell lines that secrete the antibody of interest. Sequences of BNPsp or BNPsp fragments or antigenic variants thereof may be used in screening .
  • Well known means for establishing binding specificity of monoclonal antibodies produced by the hybridoma cells include immunoprecipitation, radiolinked immunoassay (RIA), enzyme-linked immunoabsorbent assay (ELISA) and Western blot [48].
  • RIA radiolinked immunoassay
  • ELISA enzyme-linked immunoabsorbent assay
  • Western blot [48].
  • the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis [49]. Samples from immunised animals may similarly be screened for the presence of polyclonal antibodies.
  • Monoclonal antibodies can also be obtained from recombinant host cells.
  • DNA encoding the antibody can be obtained from a hybridoma cell line.
  • the DNA is then placed into an expression vector, transfected into host cells (e.g., COS cells, CHO cells, E. coli cells) and the antibody produced in the host cells.
  • the antibody may then be isolated and/or purified using standard techniques.
  • the monoclonal antibodies or fragments may also be produced by recombinant DNA means (e.g. [50]). DNA modifications such as substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [50] are also possible.
  • the antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art (e.g. [51-53]. Production of chimeric [54], bivalent antibodies [55] and multivalent antibodies are also contemplated herein [56].
  • the monoclonal antibodies secreted by the cells may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, reverse phase HPLC, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography [58].
  • Bispecific antibodies may also be useful. These antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Antibodies with greater than two specificities for example trispecific antibodies are also contemplated herein.
  • Antibodies used in the immunoassays described herein specifically bind to BNPsp or BNPsp fragments.
  • the term “specifically binds” is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody “specifically binds” if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s).
  • the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule.
  • antibodies bind with affinities of at least about 10-6M, or 10- 7M, or at least about 10-8M, or 10-9M, or 10-10, or 10-11 or 10-12M.
  • phage display methods A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome that encodes the polypeptide.
  • the establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides.
  • Phage displaying a polypeptide with affinity to a target binds to the target and these phage are enriched by affinity screening to the target.
  • the identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means (e.g. [64]).
  • the antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding.
  • the screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h.
  • the microtiter wells are then washed and a labelled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a colour reaction will appear where antibody to the immobilized polypeptide(s) is present.
  • the antibodies so identified may then be further analysed for affinity and specificity in the assay design selected.
  • the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
  • the present invention also contemplates aptamers that selectively bind to ERFE peptides.
  • Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers offer molecular binding and recognition equivalent to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
  • the aptamer is a monomer (one unit).
  • the aptamer is a multimeric aptamer.
  • the multimeric aptamer may comprise a plurality of aptamer units (mers). Each of the plurality of units of the aptamer may be identical. In such a case the multimeric aptamer is a homomultimer having a single specificity but enhanced avidity (multivalent aptamer).
  • the multimeric aptamer may comprise two or more aptameric monomers, wherein at least two mers of the multimeric aptamer are non-identical in structure, nucleic acid sequence or both.
  • a multimeric aptamer is referred to herein as a heteromultimer.
  • the heteromultimer may be directed to a single binding site i.e., monospecific (such as to avoid steric hindrance).
  • the heteromultimer may be directed to a plurality of binding sites i.e., multispecific.
  • the heteromultimer may be directed to a plurality of binding sites on different analytes, including for example, BNP, BNPsp and fragments thereof. Further description of the multimeric aptamer is provided hereinbelow.
  • a plurality of multimeric aptamers may be conjugated to form a conjugate of multimeric aptamers.
  • the multimeric aptamer may comprise, two (dimer), three (trimer), four (tetramer), five (pentamer), six (hexamer), and even more units.
  • Aptamers of the invention can be synthesized and screened by any suitable methods in the art.
  • aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment).
  • Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX [30-32].
  • aptamers that bind to a cell surface target molecule can be screened by capillary electrophoresis and enriched by SELEX based on the observation that aptamer- target molecule complexes exhibited retarded migration rate in native polyacrylamide gel electrophoresis as compared to unbound aptamers.
  • a random aptamer library can be created that contains monomeric, dimeric, trimeric, tetrameric or other higher multimeric aptamers.
  • a random aptamer library (either ssDNA or RNA) can be modified by including oligonucleotide linkers to link individual aptamer monomers to form multimeric aptamer fusion molecules.
  • a random oligonucleotide library is synthesized with randomized 45 nt sequences flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., known as 5'-arm and 3'-arm, which are used for the amplification of selected aptamers.
  • a linking oligonucleotide i.e., linker
  • linker is designed to contain sequences complementary to both 5'-arm and 3'-arm regions of random aptamers to form dimeric aptamers.
  • a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure is engineered to include sequences complementary to the 3'-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization.
  • 3-5 or 5-10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors.
  • multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
  • Multimeric aptamers may be multivalent but be of single binding specificity (i.e., homomultimeric aptamers).
  • the multimeric aptamer may be multivalent and multi- specific (i.e., heteromultimeric aptamers).
  • each monomer of the homomultimeric aptamer binds the target protein (e.g., BNP, BNPsp or fragments thereof) in an identical manner.
  • target protein e.g., BNP, BNPsp or fragments thereof
  • all monomeric components of the homomultimeric aptamer are identical.
  • a heteromultimeric aptamer comprises a plurality of monomeric aptamers at least two of which bind different sites on a single target protein or bind at least two different target proteins.
  • RNA-aptamers are well-established using protocols described in the scientific literature (e.g. [33]).
  • a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other examples, 12-30, 14-30, 15-30 nt, 30- 100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.
  • the aptamer has affinity at the range of 10-100 nM, which, after binding of the aptamer to a tumor cell surface molecule, permits dissociation of the aptamer from the target molecule (e.g., BNP or BNPsp), which leads to the release and recycle of the aptamer nucleic acid nanostructure to target other tumor cells.
  • T he affinity of individual aptamers can be increased by 4-50 fold by constructing multimeric aptamers linked together by covalent or non-covalent linkages. Methods of multimerizing aptamers are further described hereinbelow.
  • the desirable affinity of an aptamer to an analyte of interets can be fine-tuned by adjusting the multiplexity of the monomeric aptamer.
  • Multimerization can be done at the library level as follows.
  • a linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various examples, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric aptamer in the multimeric structure.
  • the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein.
  • the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers.
  • Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations.
  • Multimers can be linearly linked by continuous linear synthesis of DNA without spacers or with nucleic acid spacers. Aptamer synthesis usually relies on standard solid phase phosphoramitide chemistry.
  • dimers, trimers and tetramers or higher oligomeric structures can be linked by a polymeric spacer.
  • Methods of generating such polymeric structures are provided in (e.g.) [65].
  • the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities.
  • the aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5'-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamer.
  • modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or - fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants.
  • the resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
  • the monomeric or multimeric aptamer of the invention can be further attached or conjugated to a detectable or therapeutic moiety (i.e., a pharmaceutical moiety).
  • a diagnostic or therapeutic moiety can be attached to an aptamer embodied herein to provide additional biological activity, such as for diagnosing, preventing, or treating a condition or disease.
  • a diagnostic moiety such as a detectable moiety e.g., label (e.g., His tag, flag tag), fluorescent, radioactive, biotin/avidin etc., can be bound to the aptamer, and imaging, immunohistochemistry, or other invasive or non-invasive methods used to identify the location(s) and extend of binding of the conjugate to locations within the body.
  • a cytotoxic agent such as a chemotherapeutic agent, radioactive moiety, toxin, antibody, nucleic acid silencing agents e.g., small interfering RNA (siRNA) or other molecule with therapeutic activity when delivered to cells expressing a molecule to which the aptamer is targeted, may be used to enhance the therapeutic activity of the aptamer or provide a biological activity where the aptamer is providing the targeting activity.
  • siRNA small interfering RNA
  • conjugates to the aptamers described herein are contemplated, such as but not limited to scaffolds, sugars, proteins, antibodies, polymers, and nanoparticles, each of which have art- recognized therapeutic or diagnostic utilities and can be targeted to particular sites in vivo using an aptamer embodied herein.
  • the present invention includes use of a detection system involving the binding of ERFE peptides to a binding agent and then detecting the amount of bound peptide.
  • a similar solution is to detect the amount of unbound binding agent in a sample to get an indication of unbound or bound ERFE. It is intended that such alternative methods fall within the scope of the present invention as functional alternatives to directly detecting the amount of bound binding agent.
  • concentration of ERFE in a sample can be readily calculated from the amount of ERFE in a sample when the sample volume is known.
  • the measuring steps comprise detecting binding between an ERFE peptide and a binding agent that binds, selectively or specifically, to the ERFE peptide, and has low cross-reactivity with other markers of biological events.
  • an assay for measuring the level of an Erythroferrone (ERFE) peptide in a biological sample from a subject at risk of acquiring heart failure within 1 year of diagnosis with any medical condition comprising a binding agent that selectively binds to a ERFE peptide and which binding agent can be quantatively measured upon binding to the ERFE peptide.
  • ERFE Erythroferrone
  • the binding agent is an antibody or an antigen-binding fragment thereof.
  • the antibody may be a monoclonal, polyclonal, chimeric or humanized antibody or antigen-binding fragment thereof.
  • the assay, as well as methods involving assays, of the present invention is an immunoassay.
  • the antibodies of the present invention are particularly useful in immunoassays for determining the presence and/or amount of ERFE in a sample. Due to variable binding affinities of different antibodies, the person skilled in the art will appreciate that a standard binding curve of measured values versus amount of ERFE in a sample should be established for a particular antibody to enable the amount of ERFE in a sample to be determined. Such a curve is used to determine the true amount of ERFE in a sample.
  • Sample materials include biological fluids but are not limited thereto. In terms of the present invention, usually a biological fluids are selected from whole blood, plasma or serum.
  • Immunoassays specific for ERFE peptides usually will require the production of antibodies that specifically bind to ERFE peptides.
  • the antibody recognizes a human ERFE peptide defined by SEQ ID NO: l.
  • the antibodies can be used to construct immunoassays with broad specificity, as in competitive binding assays below, or used in conjunction with other antibodies described below in sandwich type assays to produce assays specific to ERFE peptides.
  • the person skilled in the art will appreciate that non- competitive assays are also possible.
  • the latter antibodies for sandwich immunoassays include those specific for amino acid sequences including SEQ ID NO: l.
  • indicators may also be used. Indicators may be employed in ELISA and RIA assay formats.
  • Polyclonal and monoclonal antibodies can be used in competitive binding or sandwich type assays.
  • a liquid sample is contacted with the antibody and simultaneously or sequentially contacted with a labelled ERFE peptide or modified peptide containing the epitope recognised by the antibody.
  • the label can be a radioactive component such as 125 I, 131 I, 3 H, 14 C or a non- radioactive component that can be measured by time resolved fluorescence, fluorescence, fluorescence polarisation, luminescence, chemiluminescence or colorimetric methods. These compounds include europium or other actinide elements, acrinidium esters, fluorescein, or radioactive material such as those above, that can be directly measured by radioactive counting, measuring luminescent or fluorescent light output, light absorbance etc.
  • the label can also be any component that can be indirectly measured such as biotin, digoxin, or enzymes such as horseradish peroxidase, alkaline phosphatase. These labels can be indirectly measured in a multitude of ways.
  • Horseradish peroxidase for example can be incubated with substrates such as o-Phenylenediamine Dihyhdrochloride (OPD) and peroxide to generate a coloured product whose absorbance can be measured, or with luminol and peroxide to give chemiluminescent light which can be measured in a luminometer.
  • Biotin or digoxin can be reacted with binding agents that bind strongly to them; e.g. avidin will bind strongly to biotin.
  • binding agents can in turn be covalently bound or linked to measurable labels such as horseradish peroxidase or other directly or indirectly measured labels as above.
  • These labels and those above may be attached to the peptide or protein : during synthesis, by direct reaction with the label, or through the use of commonly available crosslinking agents such as MCS and carbodiimide, or by addition of chelating agents.
  • the labelled peptide bound to the binding agent is separated from the unbound labelled peptide.
  • the separation may be accomplished by addition of an anti-gamma globulin antibody (second-antibody) coupled to solid phase particles such as cellulose, or magnetic material.
  • second-antibody is raised in a different species to that used for the primary antibody and binds the primary antibody. All primary antibodies are therefore bound to the solid phase via the second antibody. This complex is removed from solution by centrifugation or magnetic attraction and the bound labelled peptide measured using the label bound to it.
  • separating bound from free label include formation of immune complexes, which precipitate from solution, precipitation of the antibodies by polyethyleneglycol or binding free labelled peptide to charcoal and removal from solution by centrifugation of filtration.
  • the label in the separated bound or free phase is measured by an appropriate method such as those presented above.
  • Competitive binding assays can also be configured as solid phase assays that are easier to perform and are therefore preferable to those above.
  • This type of assay use a solid support including plates with wells (commonly known as ELISA or immunoassay plates), solid beads or the surfaces of tubes.
  • the primary antibody is either adsorbed or covalently bound to the surface of the plate, bead or tube, or is bound indirectly through a second anti gamma globulin or anti Fc region antibody adsorbed or covalently bound to the plate.
  • Sample and labelled peptide are added to the plate either together or sequentially and incubated under conditions allowing competition for antibody binding between ERFE in the sample and the labelled peptide.
  • Unbound labelled peptide can subsequently be aspirated off and the plate rinsed leaving the antibody bound labelled peptide attached to the plate.
  • the labelled peptide can then be measured using techniques described above.
  • Sandwich type assays are more preferred for reasons of specificity, speed and greater measuring range.
  • an excess of the primary antibody to ERFE is attached to the well of an ELISA plate, bead or tube via adsorption, covalent coupling, or an anti Fc or gamma globulin antibody, as described above for solid phase competition binding assays. Sample fluid or extract is contacted with the antibody attached to the solid phase. Because the antibody is in excess this binding reaction is usually rapid.
  • a second antibody to an ERFE peptide is also incubated with the sample either simultaneously or sequentially with the primary antibody.
  • This second antibody is chosen to bind to a site on ERFE that is different from the binding site of the primary antibody. These two antibody reactions result in a sandwich with the ERFE from the sample sandwiched between the two antibodies.
  • the second antibody is usually labelled with a readily measurable compound as detailed above for competitive binding assays.
  • a labelled third antibody which binds specifically to the second antibody may be contacted with the sample. After washing the unbound material the bound labelled antibody can be measured by methods outlined for competitive binding assays. After washing away the unbound labelled antibody, the bound label can be quantified as outlined for competitive binding assays.
  • a dipstick type assay may also be used .
  • These assays are well known in the art. They may for example, employ small particles such as gold or coloured latex particles with specific antibodies attached.
  • the liquid sample to be measured may be added to one end of a membrane or paper strip preloaded with the particles and allowed to migrate along the strip. Binding of the antigen in the sample to the particles modifies the ability of the particles to bind to trapping sites, which contain binding agents for the particles such as antigens or antibodies, further along the strip. Accumulation of the coloured particles at these sites results in colour development are dependent on the concentration of competing antigen in the sample.
  • Other dipstick methods may employ antibodies covalently bound to paper or membrane strips to trap antigen in the sample. Subsequent reactions employing second antibodies coupled to enzymes such as horse radish peroxidase and incubation with substrates to produce colour, fluorescent or chemiluminescent light output will enable quantitation of antigen in the sample.
  • the clinical performance of a laboratory test depends on its diagnostic/prognostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups.
  • Prognostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
  • a receiver operating characteristic (ROC) plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 -specificity for the complete range of decision thresholds.
  • sensitivity or the true-positive fraction [defined as (number of true-positive test results)/(number of true-positive+number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup.
  • the false-positive fraction, or 1-specificity defined as (number of false-positive results)/(number of true-negative+number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup.
  • the ROC plot is independent of the prevalence of disease in the sample.
  • Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold.
  • a test with perfect discrimination has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity).
  • the theoretical plot for a test with no discrimination is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes.
  • One convenient objective to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number.
  • kits or articles of manufacture will be formatted for assays known in the art, and in certain examples for RIA or ELISA assays, as are known in the art.
  • kits or articles of manufacture may also include detection or measurement involving one or more additional markers or risk factors for acute decompensated heart failure (e.g.) including heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, and BNP.
  • the kit or article of manufacture may be comprised of one or more containers and may also include collection equipment, for example, bottles, bags (such as intravenous fluids bags), vials, syringes, and test tubes. At least one container will be included and will hold a product which is effective for use in the assays and methods described herein.
  • the product is typically a peptide binding agent, particularly an antibody or antigen-binding fragment of the invention, or a composition comprising any of these.
  • an instruction or label on or associated with the container indicates that the composition is used for predicting, diagnosing, or monitoring heart failure in the subject.
  • Other components may include needles, diluents and buffers.
  • the kit may include at least one container comprising a pharmaceutically acceptable buffer, such as phosphate- buffered saline, Ringer's solution or dextrose solution.
  • Binding agents that selectively bind ERFE peptides or functional derivatives thereof are desirably included in the kit or article of manufacture.
  • the binding agent is an antibody or antigen-binding fragment of the invention.
  • the antibody used in the assays and kits may be monoclonal or polyclonal, for example, and may be prepared in any mammal as described above, and includes antigen binding fragments and antibodies prepared using native and fusion peptides, for example.
  • the ERFE peptide binding agent is immobilized on a solid matrix, for example, a porous strip or chip to form at least one detection site for a ERFE peptide or a fragment(s) thereof.
  • the measurement or detection region of the porous strip may include a plurality of detection sites, such detection sites containing a detection reagent.
  • the sites may be arranged in a bar, cross or dot or other arrangement.
  • a test strip or chip may also contain sites for negative and/or positive controls. The control sites may alternatively be on a different strip or chip.
  • the different detection sites may contain different amounts of immobilized nucleic acids or antibodies, e.g., a higher amount in the first detection site and lower amounts in subsequent sites.
  • the number of sites displaying a detectable signal provides a quantitative indication of the amount of an ERFE peptide or a functional derivative (or at least a derivative that still correlates with heart failure) thereof present in the sample.
  • kits or article of manufacture may be a device for sample analysis comprising a disposable testing cartridge with appropriate components (markers, antibodies and reagents) to carry out sample testing.
  • the device will conveniently include a testing zone and test result window.
  • Immunochromatographic cartridges are examples of such devices. See for example US 6,399,398; US 6,235,241 and US 5,504,013.
  • the device may be an electronic device which allows input, storage and evaluation of levels of the measured marker against control levels and other marker levels.
  • US 2006/0234315 provides examples of such devices.
  • Ciphergen's Protein Chip® which can be used to process SELDI results using Ciphergen's Protein Chip® software package.
  • Erythroferrone is a protein produced in bone marrow erythroblasts that promotes increased intestinal absorption of iron and the release of iron from stores. It does this by suppressing the hepatic synthesis of the master iron regulator known as hepcidin (Ganz et al. Blood 2017; Pautz et al. Nat Genetics 2014). ERFE is also known as myonectin and is produced by the gene known as FAM132B. Iron deficiency is a known co-morbidity in patients suffering heart failure and as such, and Applicants hypothesized that the response or potential marker utility of ERFE in these patients warranted further investigation.
  • ADHF acute decompensated heart failure
  • ERFE ERFE in 44 healthy volunteers.
  • Median values and interquartile range (IQR) were 1.12 (0.64-1.90) ng/mL with a 99 th percentile of 6.21ng/mL.
  • IQR interquartile range
  • Example 2 ERFE as Diagnostic Biomarker of Acute Decompensated Heart Failure
  • Table II Case Processing Summary
  • ERFE is a more powerful biomarker for the diagnosis of acute decompensated heart failure where [Hb] > 129 g/L than high sensitivity Troponin T reveals (i.e. hsTnT_ED) in the same patient cohort.
  • ERFE performs just as well as NT-proBNP in diagnosing acute decompensated heart failure in a patient. Further, these data further confirmed the clinical utility of ERFE as a biomarker for the diagnosis of acute decompensated heart failure as compared to other standard markers routinely used (e.g.) high sensitivity Troponin T (hsTnT) and proadrenomedullin (proADM) . Receiver Operating Curve analysis ( Figure 3) generated and the following information.
  • Example 3 ERFE in Multivariate Diagnosis of Acute Heart Failure
  • Step l a BMI .043 .021 3.973 1 .046 1.044 1.001 1.088
  • Example 4 ERFE as Diagnostic Biomarker of Acute Heart Failure in Patients [NT- proBNP] ⁇ 125 pmol/L
  • Step l a NT-proBNP .785 .322 5.930 1 .015 2.192 1.165 4.122
  • ERFE performed well.
  • Table VI which included the important clinical variables of age, gender, BMI, history (Hx) of myocardial infarction (MI), Hx of acute decompensated heart failure (ADHF), Hx of diabetes and renal function (GFR) as a base, log adjusted levels of ERFE, NTproBNP and highly sensitive cardiac troponin T were assessed.
  • ERFE remained as a significant biomarker based predictor of new acute decompensated heart failure within one year of diagnosis with any medical condition, alongside the clinical variables of previous MI and diabetes (as highlighted). That is to say, ERFE was superior to
  • Table VIII Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Dx of ADHF
  • step 1 Variable(s) entered on step 1: LogERFE, LogNTproBNPED, LoghsTnTED.
  • Table IX Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Px of ADHF
  • Table X Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Px of ADHF
  • step 1 Variable(s) entered on step 1: LogERFE, LogNTproBNPED, LoghsTnTED.
  • Example 8 ERFE as Univariate or Multivariate Predictor of New Heart Failure or Mortality ⁇ 1 Year
  • Step l a Hx_CHF 1.020 .250 16.642 .000 2.774 1.699 4.528
  • Step 2 b Hx_CHF 922 .256 13.020 OOP 2.514 1.524 4.149
  • Example 9 ERFE as a Diagnostic Biomarker of Atrial Fibrillation
  • erythroferrone could diagnose atrial fibrillation in a patient, as confirmed by electro-cardiogram (ECG).
  • ECG electro-cardiogram
  • Figure 5 illustrates a ROC for the diagnosis of acute decompensated heart failure in those patients who present with concomitant atrial fibrillation.
  • regression analysis model includes age, Hx HF, BMI, PND, orthopnea, hsTnT and NT-proBNP .(all clinical things docs use). Significant variables that pass a forward conditional test are (note that NT-proBNP is not one of them) :
  • Table XVIZ Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation
  • Table XVII Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation + ERFE and hsTNT
  • Table XVIII Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation (ALL variables)
  • Step ED_P D 2.001 .534 14.038 1 .000 7.400 2.597 21.083 l a Constan -.182 .303 .363 1 .547 .833
  • ERFE is the best biomarker for the diagnosis of acute decompensated heart failure in those patients presenting with atrial fibrillation, among all variables tested. Further analysis (data not shown) reveals that ERFE is the only significant biomarker which could diagnose acute decompensated heart failure in those patients presenting with atrial fibrillation.

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Abstract

The present invention is concerned with the diagnosis and prediction of acute decompensated heart failure in a patient. In particular, the present invention provides test kits, methods and assays for diagnosing acute decompensated heart failure (ADHF) in a patient, or for predicting occurrence of acute decompensated heart failure in a patient within one year of a diagnosis with any medical condition by interrogating levels of erythroferrone (ERFE) peptide, for example in circulating blood, plasma or serum levels, where an increase in the levels or ERFE is diagnostic of having, or prognostic of developing, acute decompensated heart failure in the patient. Measurement of ERFE peptide may be performed using a binding agent that selectively binds to ERFE peptide in a biological sample from a patient. The performance of ERFE as a biomarker of ADHF may be further enhanced where haemaglobin levels approximate or exceed a normal range in the patient(s) interrogated.

Description

TEST FOR HEART FAILURE
TECHNICAL FIELD
The present invention is concerned with the diagnosis or prediction of acute decompensated heart failure in a patient. In particular, the present invention provides test kits, assays and methods useful for diagnosing acute decompensated heart failure in a patient, or for predicting a patient's risk of acquiring acute decompensated heart failure within one year of diagnosis with any medical condition, including a cardiac disease or disorder.
BACKGROUND OF THE INVENTION
Acute decompensated heart failure (ADHF) is a sudden worsening of the signs and symptoms of heart failure, which typically includes difficulty breathing (dyspnea), leg or feet swelling, and fatigue. ADHF is a common and potentially serious cause of acute respiratory distress. The condition is caused by severe congestion of multiple organs by fluid that is inadequately circulated by the failing heart. An attack of decompensation can be caused by underlying medical illness, such as myocardial infarction, an abnormal heart rhythm, infection, or thyroid disease.
Treatment consists of reducing the fluid level with diuretics and improving heart function with nitrates, or levosimendan; other treatments such as aquapheresis ultra- filtration may also be required.
Identification of ADHF patients has typically been achieved using a jugular venous distension. However, this process can be laborious and requires specialist knowledge to interpret the results. As such there is an ongoing need to develop more useful clinical tools in the diagnosis or prognosis of acute decompensated heart failure in patients presenting to a clinic or emergency department. The present invention seeks to address these requirements.
SUMMARY OF THE INVENTION
The inventions described and claimed herein have many attributes and embodiments including, but not limited to, those set forth or described or referenced in this Summary of the Invention. It is not intended to be all-inclusive and the inventions described and claimed herein are not limited to or by the features or embodiments identified in this Summary of the Invention, which is included for purposes of illustration only and not restriction.
The present invention is predicated on the surprising and unexpected discovery of the correlation between levels of erythroferrone (ERFE) as a biomarker for the prediction or diagnosis of acute decompensated heart failure in a patient. Accordingly, in one aspect of the present invention there is provided a method for diagnosing acute decompensated heart failure in a patient, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
In another aspect of the present invention there is provided a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of a diagnosis with any medical condition, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, the biological sample has been obtained from the patient within one year of diagnosis with any medical condition, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of the diagnosis with any medical condition.
In a further aspect of the present invention there is provided a method for monitoring or assessing a patient diagnosed with acute heart failure comprising :
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval obtained from the same subject at an earlier time, wherein the reference interval is a level of ERFE peptide that distinguishes a subject having heart failure from subjects not having heart failure such that a level of ERFE peptide that is above the reference interval indicates the subject has acute heart failure; and
(iii) setting a treatment regimen or adjusting a treatment regimen for the subject based on comparing the levels of ERFE peptide in the biological sample with the reference interval.
In yet another aspect of the present invention there is provided an assay for measuring the level of an erythroferrone (ERFE) peptide from a patient with, or at risk of acquiring, heart failure, the assay comprising a binding agent that selectively binds to an ERFE peptide, which binding agent can be quantitatively measured upon binding to the ERFE peptide in a biological sample from the subject.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for diagnosing or predicting acute decompensated heart failure in a patient, the test kit or article of manufacture comprising an erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, together with instructions for how to diagnose heart failure in a patient, or instructions for how to predict a patient's risk of acquiring heart failure within one year of a diagnosis with any medical condition.
In a further aspect of the present invention there is provided a method of diagnosing atrial fibrillation in a patient, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of atrial fibrillation in the patient.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between ERFE and acute decompensated heart failure (n=65). In the entire study group (n=200), ERFE could diagnose acute decompensated heart failure (n=65; AUC=0.76±0.04 [95% confidence interval : 0.69-0.83], P<0.001) and levels were significantly higher in acute decompensated heart failure versus all other diagnoses (median [ERFE]=7.11 ng/ml_ in acute decompensated heart failure versus [ERFE] = 1.57 ng/ml_ in all other diagnoses, P=0.0014).
Figure 2 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and acute decompensated heart failure, (ii) NT-proBNP and acute decompensated heart failure, and (iii) ERFE+NT-proBNP and acute decompensated heart failure in patients, where in all patients haemaglobin levels [Hb] > 129 g/L. The ROC curves yielded an AUC: ERFE=0.777±0.040; NT-proBNP=0.841±0.033; and ERFE+NT-proBNP=0.849±0.030. ERFE was elevated in the acute decompensated heart failure patients as compared to ERFE levels from patients in a control population.
Figure 3 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and acute decompensated heart failure, (ii) NT-proBNP and acute decompensated heart failure, (iii) high sensitivity Troponin T (hsTnT) and acute decompensated heart failure, (iv) proadrenomedullin (proADM) and acute decompensated heart failure, and (v) ERFE+NT-proBNP and acute decompensated heart failure in patients, where in all patients haemaglobin levels [Hb] > 140 g/L. The ROC curves yielded an AUC: ERFE=0.810±0.053; NT-proBNP=0.841±0.051; hsTnT=0.616± 0.059, proADM = 0.728±0.053; and ERFE+NT-proBNP=0.835±0.046. ERFE was elevated in the acute decompensated heart failure patients as compared to ERFE levels from patients in a control population.
Figure 4 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between (i) ERFE and atrial fibrillation. In the entire study group (n=200), ERFE could diagnose atrial fibrillation (n=65; AUC=0.69±0.03). ERFE was elevated in atrial fibrillation patients as compared to ERFE levels from patients in a control population.
Figure 5 shows a Receiver Operating Curve (plot of sensitivity versus 1-specificity) demonstrating the correlation between ERFE and acute decompensated heart failure in those patients with atrial fibrillation. In the entire study group (n=91), ERFE could diagnose acute decompensated heart failure in those patients with atrial fibrillation (n=65; AUC=0.69±0.062), although it did not add to the performance of NT-proBNP. ERFE was elevated in atrial fibrillation patients as compared to ERFE levels from patients in a control population.
DETAILED DESCRIPTION
General Definitions
Unless specifically defined otherwise, all technical and scientific terms used herein shall be taken to have the same meaning as commonly understood by one of ordinary skill in the art to which the inventions belong (for example, in immunology, immunohistochemistry, protein chemistry, and biochemistry).
Unless otherwise indicated, the recombinant protein and immunological techniques utilized in the present invention are standard procedures well known to those skilled in the art. Such techniques are described and explained throughout the literature in sources such as, J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning : A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (editor), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M. Glover and B.D. Hames (editors), DNA Cloning : A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M. Ausubel et al., (editors), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley- Interscience (1988, including all updates until present), Ed Harlow and David Lane (editors) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al., (editors) Current Protocols in Immunology, John Wiley 8i Sons (including all updates until present). The term "and/or", e.g., "X and/or Y" shall be understood to mean either "X and Y" or "X or Y" and shall be taken to provide explicit support for both meanings or for either meaning.
Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition of matter, group of steps or group of compositions of matter shall be taken to encompass one and a plurality (i.e. one or more) of those steps, compositions of matter, groups of steps or group of compositions of matter.
It is intended that reference to a range of numbers disclosed herein (for example 1 to 10) also incorporates reference to all related numbers within that range (for example, 1, 1.1, 2, 3, 3.9, 4, 5, 6, 6.5, 7, 8, 9 and 10) and also any range of rational numbers within that range (for example 2 to 8, 1.5 to 5.5 and 3.1 to 4.7) and, therefore, all sub-ranges of all ranges expressly disclosed herein are expressly disclosed. These are only examples of what is specifically intended and all possible combinations of numerical values between the lowest value and the highest value enumerated are to be considered to be expressly stated in this application in a similar manner.
Throughout this specification the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations or any two or more of said steps or features.
The present invention is not to be limited in scope by the specific examples described herein, which are intended for the purpose of exemplification only. Functionally-equivalent products, compositions and methods are clearly within the scope of the invention, as described herein.
Any embodiment herein shall be taken to apply mutatis mutandis to any other embodiment unless specifically stated otherwise.
Selected Definitions
The term "STEMI" as used herein means ST-elevation myocardial infarction.
The term "N-STEMI" as used herein means non ST-elevation myocardial infarction.
The term "UAP" or "UA" as used herein means unstable angina (pectoris). The terms "peptide" and "polypeptide" or "protein" may be used interchangeably throughout this specification, and encompass amino acid chains of any length, including full length sequences in which amino acid residues are linked by covalent peptide bonds. Polypeptides useful in the present invention may be purified natural products, or may be produced partially or wholly using recombinant or synthetic techniques. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or other multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant, or derivative thereof. Polypeptides herein may have chain lengths of at least 4 amino acids, at least 5 amino acids, or at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, at least 21, at least 22, or all 23 amino acids of the full-length EPOsp and/or CNPsp. Reference to other polypeptides of the invention or other polypeptides described herein should be similarly understood.
As used in this specification, the term "fragment" or "functional derivative" in relation to a polypeptide is a subsequence of a polypeptide that may be detected using a binding agent. The term may refer to a polypeptide, an aggregate of a polypeptide such as a dimer or multimer, a fusion polypeptide, a polypeptide fragment, a polypeptide variant or derivative thereof.
The term "isolated" as applied to the polypeptide sequences disclosed herein is used to refer to sequences that are removed from their natural cellular or other naturally-occurring biological environment. An isolated polypeptide may be obtained by any method or combination of methods including biochemical, recombinant, and synthetic techniques. The polypeptide sequences may be prepared by at least one purification step.
The term "purified" as used herein does not require absolute purity. Purified refers in various embodiments, for example, to at least about 80%, 85%, 90%, 95%, 98%, or 99% homogeneity of a polypeptide, for example, in a sample. The term should be similarly understood in relation to other molecules and constructs described herein.
Term "variant" as used herein refers to polypeptide sequences different from the specifically identified sequences, wherein 1 to 6 or more or amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of one, two, three, four, five or six amino acids are contemplated. Variants may be naturally occurring allelic variants, or non-naturally occurring variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, variants of the polypeptides useful in the invention have biological activities including signal peptide activity or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "variant" with reference to polypeptides encompasses all forms of polypeptides as defined herein. Variant polypeptide sequences exhibit at least about 50%, at least about 60%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about
87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to a sequence of the present invention. With regard to polypeptides, identity is found over a comparison window of at least 5 to 7 amino acid positions.
Polypeptide variants also encompass those which exhibit a similarity to one or more of the specifically identified sequences that is likely to preserve the functional equivalence of those sequences, including those which could not reasonably be expected to have occurred by random chance. As discussed above, in the case of ERFE variants function may be as either a polypeptide, or antigenic polypeptide, or both.
Polypeptide sequence identity and similarity can be determined in the following manner. The subject polypeptide sequence is compared to a candidate polypeptide sequence using BLASTP (from the BLAST suite of programs, version 2.2.18 [April 2008]]) in bl2seq, which is publicly available from NCBI (ftp://ftp.ncbi.nih.gov/blast/). The default parameters of bl2seq are utilized except that filtering of low complexity regions should be turned off.
The similarity of polypeptide sequences may be examined using the following UNIX command line parameters: bl2seq -i peptideseql -j peptideseq2 -F F -p blastp. The parameter -F F turns off filtering of low complexity sections. The parameter -p selects the appropriate algorithm for the pair of sequences. This program finds regions of similarity between the sequences and for each such region reports an "E value" which is the expected number of times one could expect to see such a match by chance in a database of a fixed reference size containing random sequences. For small E values, much less than one, this is approximately the probability of such a random match. Variant polypeptide sequences commonly exhibit an E value of less than 1 x 10-5, less than 1 x 10-6, less than 1 x 10-9, less than 1 x 10-12, less than 1 x 10-15, less than 1 x 10-18 or less than 1 x 10-21 when compared with any one of the specifically identified sequences. Polypeptide sequence identity may also be calculated over the entire length of the overlap between a candidate and subject polypeptide sequences using global sequence alignment programs. EMBOSS- needle (available at http:/www. ebi.ac.uk/emboss/align/) and GAP (Huang, X. (1994) On Global Sequence Alignment. Computer Applications in the Biosciences 10, 227-235.) as discussed above are also suitable global sequence alignment programs for calculating polypeptide sequence identity. Use of BLASTP is preferred for use in the determination of polypeptide variants according to the present invention.
The term "biological sample" as used herein includes biological fluids selected from blood including venous blood and arterial blood, plasma, serum, intertistial fluid, or any other body fluid. The term "biological sample" also includes heart tissue sample. The term "biological sample" and "body fluid sample" as used herein refers to a biological sample or a sample of bodily fluid obtained for the purpose of, for example, diagnosis, prognosis, classification or evaluation of a subject of interest, such as a patient. In certain examples, such a sample may be obtained for diagnosing acute decompensated heart failure, for performing risk stratification of acute decompensated heart failure, for making a prognosis of a disease course in a patient with acute decompensated heart failure, for identifying a patient with elevated risk of acute decompensated heart failure, or combinations thereof. In addition, one of skill in the art would realise that certain body fluid samples would be more readily analysed following a fractionation or purification procedure, for example, separation of whole blood into serum or plasma components.
The term "level" as used herein is intended to refer to the amount per weight or weight per weight of erythoferrone (ERFE). It is also intended to encompass "concentration" expressed as amount per volume or weight per volume. The term "circulating level" is intended to refer to the amount per weight or weight per weight or concentration of an ERFE peptide present in the circulating fluid, such as whole blood, serum or plasma.
An "increase" or "decrease" in the level of an ERFE peptide (or any other biomarker for that matter) compared with a control, or a "change" or "deviation" from a control (level) in one example is statistically significant. An increased level, decreased level, deviation from, or change from a control level or mean or historical control level can be considered to exist if the level differs from the control level by about 5% or more, by about 10% or more, by about 20% or more, or by about 50% or more compared to the control level. Statistically significant may alternatively be calculated as P<0.05. Increased levels, decreased levels, deviation, and changes can also be determined by recourse to assay reference limits or reference intervals. These can be calculated from intuitive assessment or non-parametric methods. Overall, these methods may calculate the 0.025, and 0.975 fractiles as 0.025* (n+ 1) and 0.975 (n+ 1). Such methods are well known in the art. Presence of a marker absent in a control may be seen as a higher level, deviation or change. Absence of a marker present in a control may be seen as a lower level, deviation or change.
Specifically, the term "reference interval" as used herein is intended to refer to a figure within a statistical band of a representative concentration or alternatively a figure with an upper or lower concentration. The reference interval will typically be obtained from subjects that do not have any pre-existing conditions that could result in artificially elevating the level of circulating ERFE.
The term "delta %" as used herein is understood to refer to a percentage change in a given variable (i.e. the level or concentration of ERFE peptide). The delta % is determined by taking the final concentration of ERFE peptide in a biological sample, subtracting the initial concentration of ERFE peptide and dividing it by the initial concentration of ERFE peptide where the result is presented as a percentage. Thus, by way of non-limiting example, an increase of 300 pmol/mL from an initial concentration of 800 pmol/mL represents a 37.5% change.
For the sake of clarity, an increase of 31-39 delta % includes for example, 31, 32, 33, 34, 35, 36, 37, 38 or 39 delta % increase, as well as fractions there between.
The term "subject" or "patient" may used interchangeably herein to refer to a human or non-human primate. In one example, the subject is a human.
The term "suitable control population" according to the present invention refers to the mean circulating ERFE peptide level from sex- and age-matched subjects for which their cardiac disease or disorder status is known. The control population is used to provide a suitable reference interval by which a measured ERFE peptide level is compared.
The term "binding agent" as used herein is intended to refer to any molecule that binds (e.g.) an ERFE peptide, including small molecules, antibodies from any species whether polyclonal or monoclonal, antigen-binding fragments such as Fab and Fab2, humanized antibodies, chimeric antibodies, or antibodies modified in other ways including substitution of amino acids, and/or fusion with other peptides or proteins (e.g. PEG). It further includes aptamers (i.e. 40-80 polynucleotide molecules) which have been selectively evolved to have binding affinity for a target antigen, for example, ERFE peptide or fragmen thereof. It also includes receptors or binding proteins from any species or modified forms of them. In one example, the binding agent specifically binds to ERFE.
The terms "specifically binds" or "selectively binds" may be used interchangeably throughout this specification, and shall be taken to mean that the binding agent reacts or associates more frequently, more rapidly, with greater duration and/or with greater affinity to a particular substance than it does with alternative substances. For example, a binding agent that specifically binds to ERFE binds that protein/peptide or an epitope or immunogenic fragment thereof with greater affinity, avidity, more readily, and/or with greater duration than it binds to unrelated protein/peptide and/or epitopes or immunogenic fragments thereof. It is also understood by reading this definition that, for example, a binding agent that specifically binds to a first target (e.g. ERFE) may or may not specifically bind to a second target. As such, "specific binding" does not necessarily require exclusive binding or non-detectable binding of another molecule. Generally, but not necessarily, reference to binding means specific binding. The term "antibody" refers to an immunoglobulin molecule capable of selectively binding to a target, such as ERFE, by virtue of an antigen binding site contained within at least one variable region. This term includes four chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, primatized antibodies, de-immunized antibodies, half antibodies, bispecific antibodies) and single domain antibodies such as domain antibodies and heavy chain only antibodies (e.g., camelid antibodies or cartilaginous fish immunoglobulin new antigen receptors (IgNARs)). An antibody generally comprises constant domains, which can be arranged into a constant region or constant fragment or fragment crystallisable (Fc). Preferred forms of antibodies comprise a four-chain structure as their basic unit. Full-length antibodies comprise two heavy chains (~50-70 kDa) covalently linked and two light chains (~23 kDa each). A light chain generally comprises a variable region and a constant domain and in mammals is either a k light chain or a l light chain. A heavy chain generally comprises a variable region and one or two constant domain(s) linked by a hinge region to additional constant domain(s). Heavy chains of mammals are of one of the following types a, d, e, g, or m. Each light chain is also covalently linked to one of the heavy chains. For example, the two heavy chains and the heavy and light chains are held together by inter- chain disulfide bonds and by non-covalent interactions. The number of inter-chain disulfide bonds can vary among different types of antibodies. Each chain has an N-terminal variable region (VH or VL wherein each are ~110 amino acids in length) and one or more constant domains at the C- terminus. The constant domain of the light chain (CL which is ~110 amino acids in length) is aligned with and disulfide bonded to the first constant domain of the heavy chain (CH which is -330-440 amino acids in length). The light chain variable region is aligned with the variable region of the heavy chain. The antibody heavy chain can comprise 2 or more additional CH domains (such as, CH2, CH3 and the like) and can comprise a hinge region can be identified between the CHI and Cm constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (preferably human) antibody. The term "antibody" encompasses not only intact polyclonal or monoclonal antibodies, but also variants, fusion proteins comprising an antibody portion with an antigen binding site, humanised antibodies, human antibodies, chimeric antibodies, primatised antibodies, de-immunised antibodies or veneered antibodies.
The term "antigen-binding fragment" or "antigen-binding antibody fragment" shall be taken to mean any fragment of an antibody that retains the ability to bind to ERFE and preferably one which specifically binds to ERFE. This term includes a Fab fragment, a Fab' fragment, a F(ab') fragment, a single chain antibody (SCA or SCAB) amongst others. An "Fab fragment" consists of a monovalent antigen-binding fragment of an antibody molecule, and can be produced by digestion of a whole antibody molecule with the enzyme papain, to yield a fragment consisting of an intact light chain and a portion of a heavy chain . An "Fab' fragment" of an antibody molecule can be obtained by treating a whole antibody molecule with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of a heavy chain. Two Fab' fragments are obtained per antibody molecule treated in this manner. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments held together by two disulfide bonds, and is obtained by treating a whole antibody molecule with the enzyme pepsin, without subsequent reduction. An "Fv fragment" is a genetically engineered fragment containing the variable region of a light chain and the variable region of a heavy chain expressed as two chains. A "single chain antibody" (SCA) is a genetically engineered single chain molecule containing the variable region of a light chain and the variable region of a heavy chain, linked by a suitable, flexible polypeptide linker.
The term "'chimeric antibody" refers to antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species (e.g., murine, such as mouse) or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species (e.g., primate, such as human) or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (U.S. Patent No. 4,816,567; and Morrison et al. (1984) Proc. Natl Acad. Sci USA 81 :6851-6855).
The term "humanized antibody" shall be understood to refer to a chimeric molecule, generally prepared using recombinant techniques, having an epitope binding site derived from an immunoglobulin from a non-human species and the remaining immunoglobulin structure of the molecule based upon the structure and/or sequence of a human immunoglobulin. The antigen-binding site preferably comprises the complementarity determining regions (CDRs) from the non-human antibody grafted onto appropriate framework regions in the variable domains of human antibodies and the remaining regions from a human antibody. Epitope binding sites may be wild type or modified by one or more amino acid substitutions. It is known that the variable regions of both heavy and light chains contain three complementarity-determining regions (CDRs) which vary in response to the epitopes in question and determine binding capability, flanked by four framework regions (FRs) which are relatively conserved in a given species and which putatively provide a scaffolding for the CDRs. When nonhuman antibodies are prepared with respect to a particular epitope, the variable regions can be "reshaped" or "humanized" by grafting CDRs derived from nonhuman antibody on the FRs present in the human antibody to be modified.
The term "epitope" includes any antigenic (e.g., a protein) determinant capable of specific binding to an antibody and/or a T cell receptor. That is, a site on an antigen to which B and/or T cells respond. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. An epitope typically includes, for example, at least 3, 5 or 8-10 amino acids. The amino acids may be contiguous, or non-contiguous amino acids juxtaposed by tertiary folding. Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents.
As used herein, the term "antigenic variant" refers to polypeptide sequences different from the specifically identified sequences, wherein one or more amino acid residues are deleted, substituted, or added. Substitutions, additions or deletions of 1, 2, 3 or 4 amino acids are specifically contemplated. Variants may be naturally-occurring allelic antigenic variants, or non-naturally occurring antigenic variants. Variants may be from the same or from other species and may encompass homologues, paralogues and orthologues. In certain embodiments, antigenic variants of the polypeptides useful in the invention have biological activities including hormone function or antigenic-binding properties that are the same or similar to those of the parent polypeptides. The term "antigenic variant" with reference to (poly)peptides encompasses all forms of polypeptides as defined herein. The term "antigenic variant" encompasses naturally occurring, recombinantly and synthetically produced polypeptides.
In addition to computer/database methods known in the art, polypeptide antigenic variants may be identified by physical methods known in the art, for example, by screening expression libraries using antibodies raised against polypeptides of the invention (Sambrook et al., Molecular Cloning : A Laboratory Manual, 2nd Ed. Cold Spring Harbor Press, 1987) by recombinant DNA techniques also described by Sambrook et al. or by identifying polypeptides from natural sources with the aid of such antibodies.
As used herein, the term "marker" or "biomarker" in the context of an analyte means any antigen, molecule or other chemical or biological entity that is specifically found in circulation or associated with a particular tissue (e.g. heart muscle) that it is desired to be identified in or on a particular tissue affected by a disease or disorder, for example heart failure. In specific examples, the marker is a circulating peptide (e.g.) ERFE. In other examples, the marker is a cell surface antigen or a nuclear antigen that is differentially or preferentially expressed by specific cell types. In other examples the marker is an intracellular antigen that is differentially or preferentially expressed by specific cell types.
The term "ROC" means Receiver Operating Curve and a ROC plot depicts the overlap between two distributions by plotting the sensitivity versus 1 -specificity for a complete range of decision thresholds. The term "AUC" means Area Under the Curve which yields information about the strength of a correlation determined by the Receiver Operating Curve analysis. Typical ROC values where the AUC > 0.70 yields a statistically significant correlation.
As used herein, the term "effective amount" refers to the amount of a therapy that is sufficient to result in the prevention of the development, recurrence, or onset of a disease or condition and one or more symptoms thereof, to enhance or improve the prophylactic effect(s) of another therapy, reduce the severity, the duration of disease, ameliorate one or more symptoms of the disease or condition, prevent the advancement of the disease or condition, cause regression of the disease or condition, and/or enhance or improve the therapeutic effect(s) of another therapy.
As used herein, the terms "manage", "managing", and "management" in the context of the administration of a therapy to a subject refer to the beneficial effects that a subject derives from a therapy (e.g., a prophylactic or therapeutic agent) or a combination of therapies, while not resulting in a cure of the disease or condition. In certain examples, a subject is administered one or more therapies (e.g., one or more prophylactic or therapeutic agents) to "manage" the disease or condition so as to prevent the progression or worsening of the disease or condition.
As used herein, the term "therapeutic agent" refers to any molecule, compound, and/or substance that is used for the purpose of treating and/or managing a disease or disorder, such as unstable angina. Examples of therapeutic agents include, but are not limited to, proteins, immunoglobulins (e.g., multi-specific Igs, single chain Igs, Ig fragments, polyclonal antibodies and their fragments, monoclonal antibodies and their fragments), peptides (e.g., peptide receptors, selectins), binding proteins, biologies, proliferation-based therapy agents, hormonal agents, radioimmunotherapies, targeted agents, epigenetic therapies, differentiation therapies, biological agents, and small molecule drugs.
As used herein, the terms "therapies" and "therapy" can refer to any method(s), composition(s), and/or agent(s) that can be used in the prevention, treatment and/or management of a disease or condition or one or more symptoms thereof.
As used herein, the terms "treat", "treatment" and "treating" in the context of the administration of a therapy to a subject refer to the reduction, inhibition, elimination or amelioration of the progression and/or duration of (e.g.) acute decompensated heart failure, the reduction, inhibition, elimination or amelioration of the severity of (e.g.) acute decompensated heart failure, and/or the amelioration of one or more symptoms thereof resulting from the administration of one or more therapies. The term "presentation" and "index presentation" as used herein refers to those patients who have presented to an emergency department, to a hospital, to a medical clinic, to a health or well-being clinic, to a doctor, to a general practitioner, to a surgery etc. Description of the Present Invention
Erythroferrone (ERFE) protein is produced in bone marrow erythroblasts which promote increased intestinal absorption of iron and the release of iron from stores. ERFE performs this function by suppressing the hepatic synthesis of the master iron regulator known as hepcidin (Ganz et al. Blood 2017; Pautz et al. Nat Genetics 2014). ERFE is also known as myonectin and is produced by the gene known as FAM132B. Iron deficiency is a known co-morbidity in patients suffering heart failure and as such, Applicants hypothesized that the response or potential biomarker utility for ERFE in these patients warranted further investigation.
Applicants identified elevated levels of erythroferrone (ERFE) in patients with acute decompensated heart failure. In reference to Figure 1 read in conjunction with Example 2 and Tables III & Ilia, ERFE could diagnose acute decompensated heart failure in those patients with a confirmed clinical diagnosis (n=65/200) using Receiver Operating Curve Analysis (ROC) where the Area Under the Curve (AUC) for ERFE = 0.760±0.040 ([95% confidence interval : 0.69-0.83], P<0.001). Further ERFE levels were significantly higher in acute decompensated heart failure versus all other diagnoses where median levels of ERFE were 7.11 ng/ml_ in acute decompensated heart failure versus 1.57 ng/ml_ for all other diagnoses (P=0.0014).
Applicants have therefore identified a clinical utility for ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient.
Accordingly, in one aspect of the present invention there is provided a method for diagnosing acute decompensated heart failure in a patient, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
In an example according to this aspect of the present invention, measurement of the ERFE levels is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
Interestingly, Applicants observed that the performance of ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient significantly improved where the haemaglobin levels approximated or exceeded what is considered to be 'normal range'. For example, where [Hb] >129 g/L for a male patient and [Hb]>115 g/L for a female patient. Applicants rationalised this observation based on the role of ERFE in iron metabolism, and role of haemaglobin in iron trafficking. To further illustrate this point, and with reference to Figure 2 when read in conjunction with Example 2/Tables IV & IVa, where [Hb] > 129 g/L, ROC analysis revealed for ERFE an AUC = 0.777±0.040 which is statistically more powerful where no account of [Hb] levels was factored in (i.e. AUC = 0.760±0.040; Figure 1 and Example 2/Tables III & Ilia).
When the threshold haemaglobin level was [Hb] > 140 g/L, the performance of ERFE as a biomarker for the diagnosis of acute decompensated heart failure in a patient was further enhanced. For example, and with reference to Figure 3 when read in conjunction with Example 2/Table V, where [Hb] > 140 g/L, ROC analysis revealed for ERFE an AUC = 0.810±0.053.
These observations highlight the importance of the haemaglobin status of the patient, and indicate a potential utility for [Hb] levels (e.g.) as a ratio between [Hb] and [ERFE] in diagnosing acute decompensated heart failure in a patient.
In terms of perceived clinical utility, Applicants further demonstrated that elevated levels of ERFE as a biomarker for acute decompensated heart failure performed better or as well as markers routinely used by clinicians for such diagnoses. For example, where [Hb] > 140 g/L, ROC analysis analysis revealed for ERFE an AUC = 0.810±0.053 as compared to high sensitivity cardaic Troponin T (hsTnT; AUC = 0.616±0.059) and proadrenomedullin (proADM; AUC = 0.728±0.053). These data indicate that ERFE is a superior biomarker for diagnosis or prediction of ADHF in patients that hsTnT and proADM.
Further, the performance of ERFE relative to the current 'gold standard' marker NT- proBNP improved as the [Hb] threshold was increased. For example, where [Hb] > 129 g/L, ROC analysis revealed that ERFE had an AUC = 0.777±0.040 as compared to NT- proBNP which had an AUC = 0.841±0.033. These data would indicate that ERFE did not perform as well as NT-proBNP where threshold [Hb] > 129 g/L. However, when [Hb] > 140 g/L, ERFE performed just as well as NT-proBNP, where the AUC = 0.810±0.053 for ERFE as compared to AUC = 0.813±0.051 for NT-proBNP was very similar.
Again, these observations highlight the importance of the haemaglobin status of the patient, and indicate a potential utility for [Hb] levels (e.g.) as a ratio between [Hb] and [ERFE] in diagnosing acute decompensated heart failure in a patient.
Accordingly, in an example according to this and other aspects of the present invention, the method further comprises measuring the haemaglobin levels of the patient to determine whether the patient should be included or excluded from a diagnostic analysis for acute decompensated heart failure involving ERFE.
Furthermore, the data presented in Example 9, when read in conjunction with Figure 5, demonstrates a utility for ERFE in diagnosing acute decompensated heart failure in patients who have or present with atrial fibrillation. ROC analysis revealed ERFE had an AUC of 0.69±0.062.
Accordingly, in yet another aspect of the present invention there is provided a method for diagnosing acute decompensated heart failure in a patient who has atrial fibrillation, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
Applicants then went on to investigate the role of ERFE in predicting acute decompensated heart failure in a patient. By way of illustration only, refer to Example 5, and Tables VIII-X. Using binary logistic regression analysis, Applicants assessed the performance of ERFE relative to hsTnT and NT-proBNP, which regression analysis also included the important clinical variables of age, gender, BMI, history (Hx) of myocardial infarction (MI), Hx of acute decompensated heart failure (ADHF), Hx of diabetes and renal function (GFR) as a base, log adjusted levels of ERFE, NTproBNP and highly sensitive cardiac troponin T were assessed. Importantly, only ERFE remained as a significant biomarker based predictor of new acute decompensated heart failure within one year of diagnosis with any medical condition, alongside the clinical variables of previous MI and diabetes (as highlighted; Table VIII).
As such, Applicants have made the surprising discovery that ERFE may be used to predict an episode of ADHF in a patient within one year of diagnosis with any medical condition, including cardiac related disease and disorders (as well as recurrent ADHF).
Accordingly, in another aspect of the present invention there is provided a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of diagnosis with any medical condition, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) protein in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, the biological sample has been obtained from the patient within one year of diagnosis with any medical condition, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of the diagnosis with any medical condition. In a further aspect of the present invention there is provided a method for predicting a patient's risk of acquiring acute decompensated heart failure within one year of index presentation, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) protein in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, the biological sample has been obtained from the patient within one year of index presentation, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of index presentation.
In an example according to this aspect of the present invention, measurement of the ERFE peptide is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
In another example according to this aspect of the present invention, the medical condition is selected from a cardiac related disease or disorder, diabetes, smoking related disease, chronic pulmonary artery disease, pulmonary artery disease, coronary artery disease, acute and chronic kidney disease.
In a related example, the cardiac related disease or disorder is selected from myocardial infarction, unstable angina, atrial fibrillation, cardiac hypertrophy, mitral regurgitation, valve disorders, heart failure, pericarditis, disorders of nerve conduction, vasovagal syncope or any combination thereof.
Further ERFE could be used to predict hospital readmission for any reason within 30 or 90 days by determining levels or ERFE, where elevated levels meant readmission (following an episode of ADHF or any medical condition) was more likely. For example, refer to Tables IX and X in Example 6.
The utility of ERFE as a biomarker to monitor disease progression, with or without response to treatment is also contemplated by the present invention. For example, the methods, test kits and assays as described herein could be used to (i) establish the ADHF disease state of an individual where elevated levels relative to a reference interval obtained from a control population is diagnostic (or prognostic) of ADHF in the patient, (ii) monitor or assess disease progression as a function of time by establishing ERFE levels in multiple samples taken from the same patient at different time points, and (iii) monitor or assess whether a treatment regime has been successful (i.e.) a measured decrease in the levels of ERFE following an intervention/treatment would indicate that the patient has responded well to the intervention/treatment; the converse being that no reduction in the ERFE levels (or even an increase) would mean that the intervention/treatment has had little or no beneficial effect on the patient.
Accordingly in yet another aspect of the present invention there is provided a method for monitoring or assessing a patient diagnosed with acute heart failure comprising :
(i) measuring the level of an erythroferrone (ERFE) protein in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval obtained from the same subject at an earlier time, wherein the reference interval is a level of ERFE peptide that distinguishes a subject having heart failure from subjects not having heart failure such that a level of ERFE peptide that is above the reference interval indicates the subject has acute heart failure; and
(iii) setting a treatment regimen or adjusting a treatment regimen for the subject based on comparing the levels of ERFE peptide in the biological sample with the reference interval.
The present invention further contemplates test kits and assays for detecting the level of ERFE in a sample, such as a biological sample obtained from a patient of interest.
Accordingly, in another aspect of the present invention there is provided an assay for measuring the level of an erythroferrone (ERFE) protein from a patient with, or at risk of acquiring, heart failure, the assay comprising a binding agent that selectively binds to an ERFE peptide, which binding agent can be quantitatively measured upon binding to the ERFE peptide in a biological sample from the subject.
In an example according to this aspect of the present invention, the assay may be selected from an immunoassay, an enzyme assay, a flourescence assay and a chemiluminescence assay.
Additional data provided by the Applicants in Example 3/Table VI demonstrates that ERFE is a useful biomarker in a multivariate analysis for the diagnosis of acute decompensated heart failure when using the clinical variables of age, gender, body mass index, history of myocardial infarction, history of diabetes, NT-proBNP among others.
Further, the utility of ERFE to diagnose acute decompensated heart failure in those patients where [NT-proBNP]< 125 pmol/L is demonstrated in Example 4/Table VII. NT- proBNP is the current preferred marker used by clinicians for the diagnosis of ADHF, and these data demonstrate the utility of ERFE is identifying a select patient cohort who may otherwise avoid detection if only NT-proBNP was used.
Accordingly, in yet a further aspect of the present invention there is provided a method for diagnosing acute decompensated heart failure in patients where the concentration of N-Terminal B-Type Natriuretic Peptide (NT-proBNP) is < 125 pmol/L, the method comprising the steps of: (i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic that the patient has acute decompensated heart failure.
Finally, Examples 7 and 8, when read in conjunction with Tables XI-XIV demonstrate a further utility for ERFE as a biomarker in uni- and multi-variate equations to predict mortality within 1 year of index presentation, or to predict a subsequent episode of heart failure within one year of index presentation.
Accordingly, in yet a further aspect of the present invention there is provided a method for predicting mortality in a patient within one year of index presentation, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is predictive of the mortality within one year of index presentation.
In an example according to this aspect of the present invention, index presentation includes, but is not limited to, presentation to an emergency department, presentation to a hospital, presentation to a medical clinic, presentation to a health or well-being clinic, presentation to a doctor, presentation to a general practitioner and presentation to a surgery.
In yet a further aspect of the present invention there is provided a test kit or article of manufacture for diagnosing or predicting acute decompensated heart failure in a patient, the test kit or article of manufacture comprising an erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, together with instructions for how to diagnose heart failure in a patient, or instructions for how to predict a patient's risk of acquiring heart failure within one year of index presentation or diagnosis with any medical condition, or instructions for how to predict mortality within one year of index presentation .
According to the methods, assays and test kits described herein, the ERFE peptide is a human ERFE protein as defined by SEQ ID NO: 1, as follows:
MAPARRPAGARLLLVYAGLLAAAAAGLGSPEPGAPSRSRARREPPPGNELPRGPGESRAGPAARPPEPTAERAHSVD
PRDAWMLFVRQSDKGWGKKRSRGKAKKLKFGLPGPPGPPGPQGPPGPIIPPEALLKEFQLLLKGAVRQRERAEPEP
CTCGPAGPVAASLAPVSATAGEDDDDWGDVLALLAAPLAPGPRAPRVEAAFLCRLRRDALVERRALHELGVYYLPD AEGAFRRGPGLNLTSGQYRAPVAGFYALAATLHVALGEPPRRGPPRPRDHLRLLICIQSRCQRNASLEAIMGLESSS
ELFTISWGVLYLQMGQWTSVFLDNASGCSLTVRSGSHFSAVLLGV
(SEQ ID NO: 1)
In a further example the control population is sex and age-matched subjects who do not have acute decompensated heart failure, or who are not predisposed to acute decompensated heart failure, as measured by one or more risk factors.
In another example, the reference interval is the mean ERFE peptide level from the control population.
In yet another example, the sample is a biological sample, and may be selected from plasma, serum, whole blood, arterial blood, venous blood, saliva, bone marrow tissue, heart tissue, vascular tissue and interstitial fluid. In a related example the biological sample is preferably plasma, serum, whole blood, arterial blood and venous blood.
In yet another example, the methods, assays and test kits described herein may be performed in conjunction with the analysis of one or more other risk factors or biomarkers of acute decompensated heart failure, including but not limited heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, BNP, and iron and/or anaemia status, including ferritin, transferrin, and ferritin/transferrin saturation levels.
The methods, assays and test kits require that level of an erythroferrone (ERFE) protein in a biological sample obtained from the patient is measured, typically by detecting binding between a binding agent that selectively binds to the ERFE peptide of interest. In certain examples according to these aspects of the present invention, the binding agent is selected from an antibody or antigen binding antibody fragment thereof, or a nucleic based aptamer (e.g. deoxyribose or ribose nucleic acid aptamers) which antibody, antigen binding antibody fragment or aptamer specifically bind to the ERFE peptide of interest. In a related example, the binding agent may include a detectable label to facilitate identification when bound to the ERFE peptide and/or may be immobilised to a solid phase, such as, without limitation, a plate, a porous strip, a bead, a chip, a chromatography column and/or a mass spectrometry chamber.
In other examples, where the binding agent is an antibody, the antibody may be selected from a polyclonal, monoclonal, chimeric or humanised antibody or antigen-binding antibody fragment thereof. In another example, where the binding agent is an aptamer, the aptamer may be selected from a doxyribose nucleic acid or ribose nucleic acid based aptamers. Further information concerning antibodies, apatmers and their generation for particular antigen peptides (e.g. ERFE) is given in more detail below.
Applicants also demonstrated a correlation between circulating levels of ERFE in patients with atrial fibrilation. Refer to Figure 4 read in conjunction with Example 5/Table IX. Accordingly, the present invention also provides a method for diagnosing atrial fibrillation in a patient, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide in the biological sample obtained from the patient compared to the reference interval from a control population is diagnostic of atrial fibrillation in the patient.
In an example according to this aspect of the present invention, measurement of the ERFE peptide is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
Binding Agents to ERFE
ERFE peptides are measured in a sample, including biological samples, by detecting binding between a ERFE peptide and a binding agent that selectively binds to the ERFE peptide. Binding agents for use in the methods of the present disclosure preferably have low cross-reactivity with other analytes. The binding agents may include antibodies or antigen-binding fragments such as Fab and F(ab)2, prepared using antigenic ERFE peptides or fragments thereof as immunising antigens, as well as aptamers or nucleic acid based binding agents including deoxyribose and ribose nucleic acid based aptamers. The polypeptide or fragments may also be coupled or immobilised to a solid support. In one example, the binding agent is an antibody. The antibody may be a monoclonal or polyclonal antibody. Methods for producing polyclonal and monoclonal antibodies as well as antigen- binding fragments thereof are well known in the art (for example see Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York 1988), but have also been developed in-house by Applicants (e.g. Siriwardena et al. (2010) Circulation 122:255-264). For reasons of specificity, monoclonal antibodies are preferred. It will be appreciated by a person skilled in the art that humanised antibodies are not required for in vitro assays. In another example, the binding agent is a dexoyribose or ribose nucleic acid based aptamer.
In other examples, the antibody is raised against an antigenic ERFE peptide or the aptamer is generated using SELEX (systematic evolution of ligands by exponential enrichment). In another example, the antigenic ERFE peptide is a human ERFE peptode, and comprises a sequence defined by SEQ ID NO: 1 as follows:
MAPARRPAGARLLLVYAGLLAAAAAGLGSPEPGAPSRSRARREPPPGNELPRGPGESRAGP
AARPPEPTAERAHSVDPRDAWMLFVRQSDKGWGKKRSRGKAKKLKFGLPGPPGPPGPQGP PGPI I PPEALLKEFQLLLKGAVRQRERAEPEPCTCGPAGPVAASLAPVSATAGEDDDDWG
DVLALLAAPLAPGPRAPRVEAAFLCRLRRDALVERRALHELGVYYLPDAEGAFRRGPGLNL
TSGQYRAPVAGFYALAATLHVALGEPPRRGPPRPRDHLRLLI CIQSRCQRNASLEAIMGLE
S S SELFTI SWGVLYLQMGQWTSVFLDNASGCSLTVRSGSHFSAVLLGV
( SEQ I D NO : 1 )
Monoclonal antibodies may be produced by methods known in the art. These include the immunological method described by Kohler et al (1975) Nature 256(5517) :495-7 as well as the recombination DNA method described by Huse et al (1989) 246(4935) : 1275-81. The use of recombinant phage antibody systems to produce single chain variable antibody fragments, and subsequent mutation (such as site specific mutagenesis) or chain shifting to produce antibodies to ERFE peptides is also contemplated.
Conventional procedures for generating polyclonal antibodies are detailed in Harlow and Lane (supra). Briefly, the protocol requires immunisation of a selected animal host such as a rabbit, goat, donkey, sheep, rat or mouse (usually a rabbit), with an isolated ERFE peptide on a number of spaced occasions, with one or more test bleeds preceding exsanguination and blood collection. Serum may be separated from clotted blood by centrifugation. Serum may be tested for the presence of polyclonal antibodies using ELISA or radioimmunoassay competitive assays or art equivalent methods.
Antibodies specific to ERFE can be raised after first conjugating these or similar peptides to a large protein such as limpet hemocyanin (KLH), bovine serum albumin (BSA) or bovine thyroglobulin to make them immunogenic. Coupling can be effected by use of any protein crosslinking agent including for example the common agents glutaraldehyde, carbodiimide or N-(e-maleimido-caproyloxy) succinimide ester (MCS)— providing a cysteine residue is added to the peptide sequence prior to coupling. Injection of these conjugates into rabbits, sheep, mice or other species at monthly intervals followed by collection of blood samples two weeks later will enable production of polyclonal antibodies or monoclonal antibodies from the spleens of mice.
For example, the mouse host described above may be sacrificed and its spleen removed. The messenger RNA (mRNA) are then isolated and cDNA made from the mRNA using specific primers for the heavy and light chains of the variable region of the antibodies and the polymerase chain reaction (PCR) amplification. The DNA sequences for the heavy and light chains are joined with a linker sequence, to ensure the correct reading frame. Then the DNA construct is inserted into a vector, for example, a plasmid or bacteriophage, or virus, for transformation into a host. In one example the vector is a bacteriophage.
Suitable hosts may be selected from prokaryotic, yeast, insect or mammalian cells. In one example, a prokaryotic host, preferably Escherichia coli is used. The bacteriophage produces a viral coat and the antibody fragments are expressed on the coat, a phage display library. The phage display library can be screened for antibody fragments with the appropriate affinity for the specific antigens. The library can be screened many times and modifications can be made to the antibody construct through protein engineering techniques, such as site directed mutagenesis and chain shuffling all of which are within the capabilities of the person skilled in the art.
Antibodies and Antigen Binding Fragments
As noted above, antibody or antibodies as used herein refers to a peptide or polypeptide derived from, modelled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope [34-36]. As foreshadowed in the definition section of this specification, the term antibody includes antigen binding fragments such as, for example, fragments, subsequences, complementarity determining regions (CDRs) that retain capacity to bind to an antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHI domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment [37], which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Single chain antibodies are also included by reference in the term "antibody." Further discussion of antibodies and fragments may be found in references (e.g.) [38-44] all of which are incorporated herein in their entirety.
Also included is antiserum obtained by immunizing an animal such as a mouse, rat or rabbit with an antigen, such as for example, BNPsp or BNPsp fragments, as well as antigenic variants thereof. In brief, methods of preparing polyclonal antibodies are known to the skilled artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if desired, an adjuvant. Typically, the immunizing agent and/or adjuvant will be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include BNPsp or BNPsp fragments, antigenic variants thereof or a fusion protein thereof. It may be useful to conjugate the immunizing agent to a protein known to be immunogenic in the mammal being immunized. Examples of such immunogenic proteins include but are not limited to keyhole limpet hemocyanin, bovine serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Examples of adjuvants that may be employed include Freund's complete adjuvant and MPL TDM adjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate). The immunization protocol may be selected by one skilled in the art without undue experimentation.
Monoclonal antibodies may be prepared using hybridoma methods well known in the art [e.g. 45-47]. The hybridoma cells may be cultured in a suitable culture medium, alternatively, the hybridoma cells may be grown in vivo as ascites in a mammal. Preferred immortalized cell lines are murine myeloma lines, which can be obtained, for example, from the American Type Culture Collection, Virginia, USA. Immunoassays may be used to screen for immortalized cell lines that secrete the antibody of interest. Sequences of BNPsp or BNPsp fragments or antigenic variants thereof may be used in screening .
Well known means for establishing binding specificity of monoclonal antibodies produced by the hybridoma cells include immunoprecipitation, radiolinked immunoassay (RIA), enzyme-linked immunoabsorbent assay (ELISA) and Western blot [48]. For example, as noted above, the binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis [49]. Samples from immunised animals may similarly be screened for the presence of polyclonal antibodies.
Monoclonal antibodies can also be obtained from recombinant host cells. DNA encoding the antibody can be obtained from a hybridoma cell line. The DNA is then placed into an expression vector, transfected into host cells (e.g., COS cells, CHO cells, E. coli cells) and the antibody produced in the host cells. The antibody may then be isolated and/or purified using standard techniques.
The monoclonal antibodies or fragments may also be produced by recombinant DNA means (e.g. [50]). DNA modifications such as substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences [50] are also possible. The antibodies may be monovalent antibodies. Methods for preparing monovalent antibodies are well known in the art (e.g. [51-53]. Production of chimeric [54], bivalent antibodies [55] and multivalent antibodies are also contemplated herein [56].
Other known art techniques for monoclonal antibody production such as from phage libraries, may also be used (e.g. [57]).
The monoclonal antibodies secreted by the cells may be isolated or purified from the culture medium or ascites fluid by conventional immunoglobulin purification procedures such as, for example, reverse phase HPLC, protein A-Sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography [58].
Bispecific antibodies may also be useful. These antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. Antibodies with greater than two specificities for example trispecific antibodies are also contemplated herein.
Antibodies used in the immunoassays described herein specifically bind to BNPsp or BNPsp fragments. The term "specifically binds" is not intended to indicate that an antibody binds exclusively to its intended target since, as noted above, an antibody binds to any polypeptide displaying the epitope(s) to which the antibody binds. Rather, an antibody "specifically binds" if its affinity for its intended target is about 5-fold greater when compared to its affinity for a non-target molecule which does not display the appropriate epitope(s). In certain examples, the affinity of the antibody will be at least about 5 fold, preferably 10 fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for a target molecule than its affinity for a non-target molecule. In other examples, antibodies bind with affinities of at least about 10-6M, or 10- 7M, or at least about 10-8M, or 10-9M, or 10-10, or 10-11 or 10-12M.
Affinity is calculated as Kd = koff/kon (koff is the dissociation rate constant, Kon is the association rate constant and Kd is the equilibrium constant). Affinity can be determined at equilibrium by measuring the fraction bound (r) of labelled ligand at various concentrations (c). The data are graphed using the Scatchard equation : r/c=K(n-r) : where r=moles of bound ligand/mole of receptor at equilibrium; c=free ligand concentration at equilibrium; K=equilibrium association constant; and n = number of ligand binding sites per receptor molecule. By graphical analysis, r/c is plotted on the Y-axis versus r on the X-axis, thus producing a Scatchard plot. Antibody affinity measurement by Scatchard analysis is well known in the art [59].
Numerous publications discuss the use of phage display technology to produce and screen libraries of polypeptides for binding to a selected analyte [60-63]. A basic concept of phage display methods is the establishment of a physical association between DNA encoding a polypeptide to be screened and the polypeptide. This physical association is provided by the phage particle, which displays a polypeptide as part of a capsid enclosing the phage genome that encodes the polypeptide. The establishment of a physical association between polypeptides and their genetic material allows simultaneous mass screening of very large numbers of phage bearing different polypeptides. Phage displaying a polypeptide with affinity to a target binds to the target and these phage are enriched by affinity screening to the target. The identity of polypeptides displayed from these phage can be determined from their respective genomes. Using these methods a polypeptide identified as having a binding affinity for a desired target can then be synthesized in bulk by conventional means (e.g. [64]).
The antibodies that are generated by these methods may then be selected by first screening for affinity and specificity with the purified polypeptide of interest and, if required, comparing the results to the affinity and specificity of the antibodies with polypeptides that are desired to be excluded from binding. The screening procedure can involve immobilization of the purified polypeptides in separate wells of microtiter plates. The solution containing a potential antibody or groups of antibodies is then placed into the respective microtiter wells and incubated for about 30 min to 2 h. The microtiter wells are then washed and a labelled secondary antibody (for example, an anti-mouse antibody conjugated to alkaline phosphatase if the raised antibodies are mouse antibodies) is added to the wells and incubated for about 30 min and then washed. Substrate is added to the wells and a colour reaction will appear where antibody to the immobilized polypeptide(s) is present. The antibodies so identified may then be further analysed for affinity and specificity in the assay design selected. In the development of immunoassays for a target protein, the purified target protein acts as a standard with which to judge the sensitivity and specificity of the immunoassay using the antibodies that have been selected. Because the binding affinity of various antibodies may differ; certain antibody pairs (e.g., in sandwich assays) may interfere with one another sterically, etc., assay performance of an antibody may be a more important measure than absolute affinity and specificity of an antibody.
Aptamers
The present invention also contemplates aptamers that selectively bind to ERFE peptides.
Nucleic acid aptamers are nucleic acid species that have been engineered through repeated rounds of in vitro selection equivalently, SELEX (systematic evolution of ligands by exponential enrichment) to bind to various molecular targets such as small molecules, proteins, nucleic acids, and even cells, tissues and organisms. Aptamers offer molecular binding and recognition equivalent to antibodies. In addition to their discriminate recognition, aptamers offer advantages over antibodies as they can be engineered completely in vitro, are readily produced by chemical synthesis, possess desirable storage properties, and elicit little or no immunogenicity in therapeutic applications.
According to an example of the present invention, the aptamer is a monomer (one unit). According to another example of the invention, the aptamer is a multimeric aptamer. The multimeric aptamer may comprise a plurality of aptamer units (mers). Each of the plurality of units of the aptamer may be identical. In such a case the multimeric aptamer is a homomultimer having a single specificity but enhanced avidity (multivalent aptamer).
Alternatively, the multimeric aptamer may comprise two or more aptameric monomers, wherein at least two mers of the multimeric aptamer are non-identical in structure, nucleic acid sequence or both. Such a multimeric aptamer is referred to herein as a heteromultimer. The heteromultimer may be directed to a single binding site i.e., monospecific (such as to avoid steric hindrance). The heteromultimer may be directed to a plurality of binding sites i.e., multispecific. The heteromultimer may be directed to a plurality of binding sites on different analytes, including for example, BNP, BNPsp and fragments thereof. Further description of the multimeric aptamer is provided hereinbelow.
A plurality of multimeric aptamers may be conjugated to form a conjugate of multimeric aptamers. The multimeric aptamer may comprise, two (dimer), three (trimer), four (tetramer), five (pentamer), six (hexamer), and even more units.
Aptamers of the invention can be synthesized and screened by any suitable methods in the art. For example, aptamers can be screened and identified from a random aptamer library by SELEX (systematic evolution of ligands by exponential enrichment). Aptamers that bind to an antigen of interest can be suitably screened and selected by a modified selection method herein referred to as cell-SELEX or cellular-SELEX [30-32]. In other examples, aptamers that bind to a cell surface target molecule (e.g., BNP or BNPsp) can be screened by capillary electrophoresis and enriched by SELEX based on the observation that aptamer- target molecule complexes exhibited retarded migration rate in native polyacrylamide gel electrophoresis as compared to unbound aptamers.
A random aptamer library can be created that contains monomeric, dimeric, trimeric, tetrameric or other higher multimeric aptamers. A random aptamer library (either ssDNA or RNA) can be modified by including oligonucleotide linkers to link individual aptamer monomers to form multimeric aptamer fusion molecules. In other examples, a random oligonucleotide library is synthesized with randomized 45 nt sequences flanked by defined 20 nt sequences both upstream and downstream of the random sequence, i.e., known as 5'-arm and 3'-arm, which are used for the amplification of selected aptamers. A linking oligonucleotide (i.e., linker) is designed to contain sequences complementary to both 5'-arm and 3'-arm regions of random aptamers to form dimeric aptamers. For trimeric or tetrameric aptamers, a small trimeric or tetrameric (i.e., a Holiday junction-like) DNA nanostructure is engineered to include sequences complementary to the 3'-arm region of the random aptamers, therefore creating multimeric aptamer fusion through hybridization. In addition, 3-5 or 5-10 dT rich nucleotides can be engineered into the linker polynucleotides as a single stranded region between the aptamer-binding motifs, which offers flexibility and freedom of multiple aptamers to coordinate and synergize multivalent interactions with cellular ligands or receptors. Alternatively, multimeric aptamers can also be formed by mixing biotinylated aptamers with streptavidin.
A modified cellular SELEX procedure can be employed to select target-binding aptamers. Multimeric aptamers may be multivalent but be of single binding specificity (i.e., homomultimeric aptamers). Alternatively, the multimeric aptamer may be multivalent and multi- specific (i.e., heteromultimeric aptamers).
Thus, each monomer of the homomultimeric aptamer binds the target protein (e.g., BNP, BNPsp or fragments thereof) in an identical manner. Thus according to an example of the invention, all monomeric components of the homomultimeric aptamer are identical.
Conversely, a heteromultimeric aptamer comprises a plurality of monomeric aptamers at least two of which bind different sites on a single target protein or bind at least two different target proteins.
Selection of RNA-aptamers is well-established using protocols described in the scientific literature (e.g. [33]). In certain examples, a suitable nucleotide length for an aptamer ranges from about 15 to about 100 nucleotide (nt), and in various other examples, 12-30, 14-30, 15-30 nt, 30- 100 nt, 30-60 nt, 25-70 nt, 25-60 nt, 40-60 nt, or 40-70 nt in length.
In other examples, the aptamer has affinity at the range of 10-100 nM, which, after binding of the aptamer to a tumor cell surface molecule, permits dissociation of the aptamer from the target molecule (e.g., BNP or BNPsp), which leads to the release and recycle of the aptamer nucleic acid nanostructure to target other tumor cells. T he affinity of individual aptamers can be increased by 4-50 fold by constructing multimeric aptamers linked together by covalent or non-covalent linkages. Methods of multimerizing aptamers are further described hereinbelow.
Thus, in certain examples, the desirable affinity of an aptamer to an analyte of interets (e.g. BNP or BNPsp) can be fine-tuned by adjusting the multiplexity of the monomeric aptamer.
Multimerization can be done at the library level as follows.
In certain examples, a linker polynucleotide has a length between about 5 nucleotides (nt) and about 100 nt; in various examples, 10-30 nt, 20-30 nt, 25-35 nt, 30-50 nt, 40-50 nt, 50-60 nt, 55-65 nt, 50-80 nt, or 80-100 nt. It is within the ability of one of skill in the art to adjust the length of the linker polynucleotide to accommodate each monomeric aptamer in the multimeric structure.
In certain examples, the multimeric aptamers can be identified and screened from a random multimeric aptamer library as described herein. In other examples, the monomeric aptamers are linked to each other by one or a plurality of linker polynucleotides to form multimeric aptamers. Monomeric aptamers can be linked to form multimeric aptamers by any suitable means and in any configurations.
It will be appreciated that the monomeric structures of the invention can be further multimerized by post SELEX procedures.
Multimers can be linearly linked by continuous linear synthesis of DNA without spacers or with nucleic acid spacers. Aptamer synthesis usually relies on standard solid phase phosphoramitide chemistry.
Thus, dimers, trimers and tetramers or higher oligomeric structures (e.g., pentamers, hexamers, heptamers, octamers etc.) can be linked by a polymeric spacer. Methods of generating such polymeric structures are provided in (e.g.) [65].
In certain examples, the aptamers are further modified to protect the aptamers from nuclease and other enzymatic activities. The aptamer sequence can be modified by any suitable methods known in the art. For example, phosphorothioate can be incorporated into the backbone, and 5'-modified pyrimidine can be included in 5' end of ssDNA for DNA aptamer. For RNA aptamers, modified nucleotides such as substitutions of the 2'-OH groups of the ribose backbone, e.g., with 2'-deoxy-NTP or - fluoro-NTP, can be incorporated into the RNA molecule using T7 RNA polymerase mutants. The resistance of these modified aptamers to nuclease can be tested by incubating them with either purified nucleases or nuclease from mouse serum, and the integrity of aptamers can be analyzed by gel electrophoresis.
The monomeric or multimeric aptamer of the invention can be further attached or conjugated to a detectable or therapeutic moiety (i.e., a pharmaceutical moiety).
Thus, as noted above, a diagnostic or therapeutic moiety can be attached to an aptamer embodied herein to provide additional biological activity, such as for diagnosing, preventing, or treating a condition or disease. In one example a diagnostic moiety such as a detectable moiety e.g., label (e.g., His tag, flag tag), fluorescent, radioactive, biotin/avidin etc., can be bound to the aptamer, and imaging, immunohistochemistry, or other invasive or non-invasive methods used to identify the location(s) and extend of binding of the conjugate to locations within the body. For therapeutic uses, a cytotoxic agent such as a chemotherapeutic agent, radioactive moiety, toxin, antibody, nucleic acid silencing agents e.g., small interfering RNA (siRNA) or other molecule with therapeutic activity when delivered to cells expressing a molecule to which the aptamer is targeted, may be used to enhance the therapeutic activity of the aptamer or provide a biological activity where the aptamer is providing the targeting activity. Moreover, other conjugates to the aptamers described herein are contemplated, such as but not limited to scaffolds, sugars, proteins, antibodies, polymers, and nanoparticles, each of which have art- recognized therapeutic or diagnostic utilities and can be targeted to particular sites in vivo using an aptamer embodied herein.
Detection of Binding Agents Including Peptide Binding Assays
The present invention includes use of a detection system involving the binding of ERFE peptides to a binding agent and then detecting the amount of bound peptide. A similar solution is to detect the amount of unbound binding agent in a sample to get an indication of unbound or bound ERFE. It is intended that such alternative methods fall within the scope of the present invention as functional alternatives to directly detecting the amount of bound binding agent. Persons skilled in the art will appreciate that the concentration of ERFE in a sample can be readily calculated from the amount of ERFE in a sample when the sample volume is known.
In the assays, methods and kits according to the present invention, the measuring steps comprise detecting binding between an ERFE peptide and a binding agent that binds, selectively or specifically, to the ERFE peptide, and has low cross-reactivity with other markers of biological events.
Accordingly, in yet another aspect of the present invention there is provided an assay for measuring the level of an Erythroferrone (ERFE) peptide in a biological sample from a subject at risk of acquiring heart failure within 1 year of diagnosis with any medical condition, comprising a binding agent that selectively binds to a ERFE peptide and which binding agent can be quantatively measured upon binding to the ERFE peptide.
In certain examples, the binding agent is an antibody or an antigen-binding fragment thereof. The antibody may be a monoclonal, polyclonal, chimeric or humanized antibody or antigen-binding fragment thereof. As such, in one example the assay, as well as methods involving assays, of the present invention is an immunoassay.
The antibodies of the present invention are particularly useful in immunoassays for determining the presence and/or amount of ERFE in a sample. Due to variable binding affinities of different antibodies, the person skilled in the art will appreciate that a standard binding curve of measured values versus amount of ERFE in a sample should be established for a particular antibody to enable the amount of ERFE in a sample to be determined. Such a curve is used to determine the true amount of ERFE in a sample.
Sample materials include biological fluids but are not limited thereto. In terms of the present invention, usually a biological fluids are selected from whole blood, plasma or serum.
Immunoassays specific for ERFE peptides usually will require the production of antibodies that specifically bind to ERFE peptides. In one example, the antibody recognizes a human ERFE peptide defined by SEQ ID NO: l. The antibodies can be used to construct immunoassays with broad specificity, as in competitive binding assays below, or used in conjunction with other antibodies described below in sandwich type assays to produce assays specific to ERFE peptides. The person skilled in the art will appreciate that non- competitive assays are also possible. The latter antibodies for sandwich immunoassays include those specific for amino acid sequences including SEQ ID NO: l.
In another example, indicators may also be used. Indicators may be employed in ELISA and RIA assay formats.
Polyclonal and monoclonal antibodies can be used in competitive binding or sandwich type assays. In one example of this method a liquid sample is contacted with the antibody and simultaneously or sequentially contacted with a labelled ERFE peptide or modified peptide containing the epitope recognised by the antibody.
The label can be a radioactive component such as 125I, 131I, 3H, 14C or a non- radioactive component that can be measured by time resolved fluorescence, fluorescence, fluorescence polarisation, luminescence, chemiluminescence or colorimetric methods. These compounds include europium or other actinide elements, acrinidium esters, fluorescein, or radioactive material such as those above, that can be directly measured by radioactive counting, measuring luminescent or fluorescent light output, light absorbance etc. The label can also be any component that can be indirectly measured such as biotin, digoxin, or enzymes such as horseradish peroxidase, alkaline phosphatase. These labels can be indirectly measured in a multitude of ways. Horseradish peroxidase for example can be incubated with substrates such as o-Phenylenediamine Dihyhdrochloride (OPD) and peroxide to generate a coloured product whose absorbance can be measured, or with luminol and peroxide to give chemiluminescent light which can be measured in a luminometer. Biotin or digoxin can be reacted with binding agents that bind strongly to them; e.g. avidin will bind strongly to biotin. These binding agents can in turn be covalently bound or linked to measurable labels such as horseradish peroxidase or other directly or indirectly measured labels as above. These labels and those above may be attached to the peptide or protein : during synthesis, by direct reaction with the label, or through the use of commonly available crosslinking agents such as MCS and carbodiimide, or by addition of chelating agents.
Following contact with the antibody, usually for 18 to 25 hours at 4° C, or 1 to 240 minutes at 30° C to 40° C, the labelled peptide bound to the binding agent (antibody) is separated from the unbound labelled peptide. In solution phase assays, the separation may be accomplished by addition of an anti-gamma globulin antibody (second-antibody) coupled to solid phase particles such as cellulose, or magnetic material. The second-antibody is raised in a different species to that used for the primary antibody and binds the primary antibody. All primary antibodies are therefore bound to the solid phase via the second antibody. This complex is removed from solution by centrifugation or magnetic attraction and the bound labelled peptide measured using the label bound to it. Other options for separating bound from free label include formation of immune complexes, which precipitate from solution, precipitation of the antibodies by polyethyleneglycol or binding free labelled peptide to charcoal and removal from solution by centrifugation of filtration. The label in the separated bound or free phase is measured by an appropriate method such as those presented above.
Competitive binding assays can also be configured as solid phase assays that are easier to perform and are therefore preferable to those above. This type of assay use a solid support including plates with wells (commonly known as ELISA or immunoassay plates), solid beads or the surfaces of tubes. The primary antibody is either adsorbed or covalently bound to the surface of the plate, bead or tube, or is bound indirectly through a second anti gamma globulin or anti Fc region antibody adsorbed or covalently bound to the plate. Sample and labelled peptide (as above) are added to the plate either together or sequentially and incubated under conditions allowing competition for antibody binding between ERFE in the sample and the labelled peptide. Unbound labelled peptide can subsequently be aspirated off and the plate rinsed leaving the antibody bound labelled peptide attached to the plate. The labelled peptide can then be measured using techniques described above. Sandwich type assays are more preferred for reasons of specificity, speed and greater measuring range. In this type of assay an excess of the primary antibody to ERFE is attached to the well of an ELISA plate, bead or tube via adsorption, covalent coupling, or an anti Fc or gamma globulin antibody, as described above for solid phase competition binding assays. Sample fluid or extract is contacted with the antibody attached to the solid phase. Because the antibody is in excess this binding reaction is usually rapid. A second antibody to an ERFE peptide is also incubated with the sample either simultaneously or sequentially with the primary antibody. This second antibody is chosen to bind to a site on ERFE that is different from the binding site of the primary antibody. These two antibody reactions result in a sandwich with the ERFE from the sample sandwiched between the two antibodies. The second antibody is usually labelled with a readily measurable compound as detailed above for competitive binding assays. Alternatively a labelled third antibody which binds specifically to the second antibody may be contacted with the sample. After washing the unbound material the bound labelled antibody can be measured by methods outlined for competitive binding assays. After washing away the unbound labelled antibody, the bound label can be quantified as outlined for competitive binding assays.
A dipstick type assay may also be used . These assays are well known in the art. They may for example, employ small particles such as gold or coloured latex particles with specific antibodies attached. The liquid sample to be measured may be added to one end of a membrane or paper strip preloaded with the particles and allowed to migrate along the strip. Binding of the antigen in the sample to the particles modifies the ability of the particles to bind to trapping sites, which contain binding agents for the particles such as antigens or antibodies, further along the strip. Accumulation of the coloured particles at these sites results in colour development are dependent on the concentration of competing antigen in the sample. Other dipstick methods may employ antibodies covalently bound to paper or membrane strips to trap antigen in the sample. Subsequent reactions employing second antibodies coupled to enzymes such as horse radish peroxidase and incubation with substrates to produce colour, fluorescent or chemiluminescent light output will enable quantitation of antigen in the sample.
Receiver Operating Characteristic (ROC) Analysis
The clinical performance of a laboratory test depends on its diagnostic/prognostic accuracy, or the ability to correctly classify subjects into clinically relevant subgroups. Prognostic accuracy measures the test's ability to correctly distinguish two different conditions of the subjects investigated. Such conditions are for example health and disease or benign versus malignant disease.
In each case, a receiver operating characteristic (ROC) plot depicts the overlap between the two distributions by plotting the sensitivity versus 1 -specificity for the complete range of decision thresholds. On the y-axis is sensitivity, or the true-positive fraction [defined as (number of true-positive test results)/(number of true-positive+number of false-negative test results)]. This has also been referred to as positivity in the presence of a disease or condition. It is calculated solely from the affected subgroup. On the x-axis is the false-positive fraction, or 1-specificity [defined as (number of false-positive results)/(number of true-negative+number of false-positive results)]. It is an index of specificity and is calculated entirely from the unaffected subgroup. Because the true- and false-positive fractions are calculated entirely separately, by using the test results from two different subgroups, the ROC plot is independent of the prevalence of disease in the sample. Each point on the ROC plot represents a sensitivity/-specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap in the two distributions of results) has an ROC plot that passes through the upper left corner, where the true-positive fraction is 1.0, or 100% (perfect sensitivity), and the false-positive fraction is 0 (perfect specificity). The theoretical plot for a test with no discrimination (identical distributions of results for the two groups) is a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall in between these two extremes. If the ROC plot falls completely below the 45° diagonal, this is easily remedied by reversing the criterion for "positivity" from "greater than" to "less than" or vice versa. Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test.
One convenient objective to quantify the diagnostic accuracy of a laboratory test is to express its performance by a single number. The most common global measure is the area under the ROC plot. By convention, this area is always ³ 0.5 (if it is not, one can reverse the decision rule to make it so). Values range between 1.0 (perfect separation of the test values of the two groups) and 0.5 (no apparent distributional difference between the two groups of test values). The area does not depend only on a particular portion of the plot such as the point closest to the diagonal or the sensitivity at 90% specificity, but on the entire plot. This is a quantitative, descriptive expression of how close the ROC plot is to the perfect one (area = 1.0).
Kits & Articles of Manufacture
Typically, kits or articles of manufacture will be formatted for assays known in the art, and in certain examples for RIA or ELISA assays, as are known in the art.
The kits or articles of manufacture may also include detection or measurement involving one or more additional markers or risk factors for acute decompensated heart failure (e.g.) including heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, and BNP. The kit or article of manufacture may be comprised of one or more containers and may also include collection equipment, for example, bottles, bags (such as intravenous fluids bags), vials, syringes, and test tubes. At least one container will be included and will hold a product which is effective for use in the assays and methods described herein. The product is typically a peptide binding agent, particularly an antibody or antigen-binding fragment of the invention, or a composition comprising any of these. In one example, an instruction or label on or associated with the container indicates that the composition is used for predicting, diagnosing, or monitoring heart failure in the subject. Other components may include needles, diluents and buffers. Usefully, the kit may include at least one container comprising a pharmaceutically acceptable buffer, such as phosphate- buffered saline, Ringer's solution or dextrose solution.
Binding agents that selectively bind ERFE peptides or functional derivatives thereof are desirably included in the kit or article of manufacture. In one example, the binding agent is an antibody or antigen-binding fragment of the invention. The antibody used in the assays and kits may be monoclonal or polyclonal, for example, and may be prepared in any mammal as described above, and includes antigen binding fragments and antibodies prepared using native and fusion peptides, for example.
In one example of the kit or article of manufacture according to the present invention, the ERFE peptide binding agent is immobilized on a solid matrix, for example, a porous strip or chip to form at least one detection site for a ERFE peptide or a fragment(s) thereof. The measurement or detection region of the porous strip may include a plurality of detection sites, such detection sites containing a detection reagent. The sites may be arranged in a bar, cross or dot or other arrangement. A test strip or chip may also contain sites for negative and/or positive controls. The control sites may alternatively be on a different strip or chip. The different detection sites may contain different amounts of immobilized nucleic acids or antibodies, e.g., a higher amount in the first detection site and lower amounts in subsequent sites. Upon the addition of a test biological sample the number of sites displaying a detectable signal provides a quantitative indication of the amount of an ERFE peptide or a functional derivative (or at least a derivative that still correlates with heart failure) thereof present in the sample.
Also included in the kit or article of manufacture may be a device for sample analysis comprising a disposable testing cartridge with appropriate components (markers, antibodies and reagents) to carry out sample testing. The device will conveniently include a testing zone and test result window. Immunochromatographic cartridges are examples of such devices. See for example US 6,399,398; US 6,235,241 and US 5,504,013.
Alternatively, the device may be an electronic device which allows input, storage and evaluation of levels of the measured marker against control levels and other marker levels. US 2006/0234315 provides examples of such devices. Also useful in the invention are Ciphergen's Protein Chip® which can be used to process SELDI results using Ciphergen's Protein Chip® software package.
The invention is further described with reference to the following examples. It will be appreciated that the invention as claimed is not intended to be limited in any way by these examples.
EXAMPLES
Example 1: ERFE Measurements
Erythroferrone (ERFE) is a protein produced in bone marrow erythroblasts that promotes increased intestinal absorption of iron and the release of iron from stores. It does this by suppressing the hepatic synthesis of the master iron regulator known as hepcidin (Ganz et al. Blood 2017; Pautz et al. Nat Genetics 2014). ERFE is also known as myonectin and is produced by the gene known as FAM132B. Iron deficiency is a known co-morbidity in patients suffering heart failure and as such, and Applicants hypothesized that the response or potential marker utility of ERFE in these patients warranted further investigation.
Applicants used the Intrinsic LifeSciences ELISA to measure ERFE in healthy volunteers and in breathless patients presenting to hospital with symptoms suggestive of acute decompensated heart failure (ADHF).
Applicants measured ERFE in 44 healthy volunteers. Median values and interquartile range (IQR) were 1.12 (0.64-1.90) ng/mL with a 99th percentile of 6.21ng/mL. ERFE levels significantly correlated with the following parameters, as determined by Spearman's rank order correlation:
Age: 0.520, P<0.001
Estimated GFR -0.444, P=0.014
Iron measurements or related parameters were not made in these individuals.
Total of 200 patients had ERFE measured at Emergency Department presentation. 65 had Dx of acute HF.
ERFE significantly correlated with the following parameters: Table I: ERFE Correlation Index Parameters
Figure imgf000038_0001
Hx: history; ED: Emergency Department (i.e. presentation)
Example 2: ERFE as Diagnostic Biomarker of Acute Decompensated Heart Failure In the entire study group (n=200) ERFE could diagnose acute decompensated heart failure (n=65, ROC AUC = 0.76 (95% Cl: 0.69-0.83), P<0.001) and levels were significantly higher in acute decompensated heart failure versus all other diagnoses (median ADHF: 7.11 ng/ml_ vs all other Dx 1.57ng/ml_, P=0.014). Table II: Case Processing Summary
Figure imgf000039_0001
In patients where no account is taken of the haemaglobin concentration (i.e. [Hb]=ALL, Receiver Operating Curve analysis (Figure 1) generated and the following information.
Table III: ERFE Dx for ADHF [Hb]=ALL
Test Result Variable(s) : ERFE_ED
Figure imgf000039_0002
a . under non-parametric assumption; b. null hypothesis true area = 0.50
Indeed, when the patient cohort was expanded to include a total of 451 patients who presented to the Emergency Department with a complaint of breathlessness, including 161 with an adjudicated diagnosis of acute decompensated heart failure, validation of the initial correlations stood, as reflected in Table Ilia as follows.
Table Ilia: ERFE Dx for ADHF (expanded patient cohort)
Figure imgf000039_0003
a . under non-parametric assumption; b. null hypothesis true area = 0.50 These data demonstrate a significant statistical correlation between levels of ERFE in those patients who had a clinical diagnosis of acute decompensated heart failure.
When the concentration of haemaglobin ([Hb]) is > 129g/L, ie. within what is considered to be a 'normal range' (male patient [Hb]~130-175 g/L). Receiver Operating Curve analysis (Figure 2) generated and the following information.
Table IV: ERFE Dx for ADHF [Hb] > 129 g/L
Figure imgf000040_0001
a . under non-parametric assumption; b. null hypothesis true area = 0.50 These data show the clinical utility of ERFE where the [Hb] is in a 'normal range' (i.e.
[Hb] > 129 g/L) for diagnosing acute decompensated heart failure. In other words, the utility of ERFE is clinically more significant for the diagnosis of acute decompensated heart failure where the levels of heamaglobin are considered to be in a normal range. Although ERFE did not perform as well as NT-proBNP in diagnosing acute decompensated heart failure (i.e. AUC [ERFE=0.777±0.040] versus [NT-proBNP=0.841±0.033]), interestingly, it did add slightly to the performance of NT-proBNP in daignosing acute decompensated heart failure [ERFE+NT-proBNP=0.849±0.030] (n=48/174 patients in the cohort interrogated).
Again, in the expanded patient cohort, the initial correlations were further validated as demonstrated by the data presented in Table IVa as follows:
Table IVa: ERFE Dx for ADHF [Hb] > 129 g/L (expanded patient cohort)
Figure imgf000040_0002
Figure imgf000041_0002
a . under non-parametric assumption; b. null hypothesis true area = 0.50
In addition, these data also reveal that ERFE is a more powerful biomarker for the diagnosis of acute decompensated heart failure where [Hb] > 129 g/L than high sensitivity Troponin T reveals (i.e. hsTnT_ED) in the same patient cohort.
However, where the [Hb] > 140 g/L, then ERFE performs just as well as NT-proBNP in diagnosing acute decompensated heart failure in a patient. Further, these data further confirmed the clinical utility of ERFE as a biomarker for the diagnosis of acute decompensated heart failure as compared to other standard markers routinely used (e.g.) high sensitivity Troponin T (hsTnT) and proadrenomedullin (proADM) . Receiver Operating Curve analysis (Figure 3) generated and the following information.
Figure imgf000041_0001
a . under non-parametric assumption; b. null hypothesis true area = 0.50
Example 3: ERFE in Multivariate Diagnosis of Acute Heart Failure
Interestingly, when logistic regression is used to assess some of the important clinical and biomarker factors in diagnosis of acute decompensated heart failure, the whole cohort analysis results are (cf. n = 161 ADHF out of 451). In the base model all the clinical variables of age, gender, BMI, Hx MI, Hx HF, Hx Diabetes, eGFR, PND, orthopnea were included as well as NT-proBNP. The additive block included hsTnT and ERFE on top of the significant variables identified. Only ERFE added to NT-proBNP and the others, ie. it is a diagnostic predictor of ADHF, independent of NT-proBNP. hsTnT was not. Table VI: ERFE in multivariate Dx of ADHF
95% C.I. for
EXP(B) ·
B S.E. Wald df Slg. Exp(B) Lower Upper
Step la BMI .043 .021 3.973 1 .046 1.044 1.001 1.088
NT- .996 .121 67.318 1 .000 2.707 2.134 3.434 proBNP
PND .930 .281 10.935 1 .001 2.534 1.460 4.398
ERFE .285 . .110 6.686 1 .010 1.330 1.071 1.651
Constant -7.522 1.010 55.455 1 .000 .001
Example 4: ERFE as Diagnostic Biomarker of Acute Heart Failure in Patients [NT- proBNP]< 125 pmol/L
When the ADHF cases with ED NT-proBNP <125pmol/L (1050pg/mL) are considered (19/199), the additive value of ERFE still holds, and indicates the utility of ERFE to assist with clinical guideline uncertainty below this NT-proBNP level.
Table VII: ERFE to Dx ADHF in NT-proBNP cases < 125pfnol/L
95% C.I. for
EXP(B)
B S.E. Wald df Sig. Exp(B) Lower Upper
Step la NT-proBNP .785 .322 5.930 1 .015 2.192 1.165 4.122
BMI .. . .056 _ .036 2.434 1 .119 1.058 .986 1.136
Hx_Arrhyth 1.412 .552 6.550 1 .010 : 4.102 1.392 12.092 m _ _ _ _ _ _ _
Figure imgf000042_0001
ERFE .422 .184 5.236 1 .022 1.524 1.062 2.188
Constant -7.446 1.724 18.662 1 .000 .001
Example 5: ERFE as Prognostic Biomarker of Acute Heart Failure
In prognosis of acute decompensated heart failure, ERFE performed well. In a binary logistic regression model (Table VI), which included the important clinical variables of age, gender, BMI, history (Hx) of myocardial infarction (MI), Hx of acute decompensated heart failure (ADHF), Hx of diabetes and renal function (GFR) as a base, log adjusted levels of ERFE, NTproBNP and highly sensitive cardiac troponin T were assessed. Importantly, only ERFE remained as a significant biomarker based predictor of new acute decompensated heart failure within one year of diagnosis with any medical condition, alongside the clinical variables of previous MI and diabetes (as highlighted). That is to say, ERFE was superior to
SUBSTITUTE SHEETS (Rule 26) NTproBNP and hsTnT for prediction of new ADHF within one year of diagnosis with any medical condition, including a cardiac disease or disorder (n=33 cases).
Table VIII: Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Dx of ADHF
Figure imgf000043_0001
a. Variable(s) entered on step 1: LogERFE, LogNTproBNPED, LoghsTnTED.
SUBSTITUTE SHEETS (Rule 26) Example 6: ERFE as Prognostic Biomarker of Hospital Re-Admission
ERFE was the only significant biomarker that could predict new hospital admission within 30 days for any reason, alongside Hx of CHF (n=35).
Table IX: Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Px of ADHF
Figure imgf000044_0001
a. Variable(s) entered on step 1 : LogERFE, LogNTproBNPED, LoghsTnTED. ERFE was also the only predictor of new inpatient admission within 90 days for any reason : even over clinical variables (n=63).
Table X: Binary Logistic Regression Model for ERFE, NT-proBNP, hsTnT for Px of ADHF
Figure imgf000044_0002
a. Variable(s) entered on step 1: LogERFE, LogNTproBNPED, LoghsTnTED.
SUBSTITUTE SHEETS (Rule 26) Example 7: ERFE as Independent Predictor of Mortality < 1 Year
In the expanded patient cohort prognosis, inpatient ERFE was an independent predictor of death within lyr (n=85) in univariate and multivariable regression models, which included the important clinical variables of age, gender, BMI, orthopnea, PND, log Hb, Hx of MI, Hx of heart failure, Hx of diabetes and renal function (eGFR) as a base. Log adjusted levels of ERFE, NTproBNP and highly sensitive cardiac troponin T were assessed as potential additive factors. All clinical variables were inserted on the first block, forward conditional. The significant clinical variables (BMI, Hx CHF, EDGFR and Log Hb) were then carried forward into block 2 (forward conditional) to see if any of NJ-proBNP, hsTnT or ERFE could add to this. Only, BMI, hsTnT and ERFE retained significance for prediction of mortality in the multivariate model.
Table XI: ERFE in univariate model equation for mortality at lyr
95% C.I. for
EXP(B)
Figure imgf000045_0001
Constant 4.341 4.537 .916 1 .339 76.812
SUBSTITUTE SHEETS (Rule 26) Step 2a BMI _ -.076 .023 11.269 . 1 .001 .927 .887 .969
Hx_CHF _ .469 .286 2.683 1 OI 1.598 .912 2.799
GFR _ -.004 .008 .271 1 .603 .996 .980 1.012
Hb -1.095 .908 1.457 1 .227 .334 .056 1.981
Figure imgf000046_0001
Example 8: ERFE as Univariate or Multivariate Predictor of New Heart Failure or Mortality < 1 Year
ERFE does not predict new HF within lyr (n=93), but does act· as a univariate and multivariate predictor of new HF or death within lyr (n = 146).
Table XIII: Univariate ERFE IP to predict composite of new HF/death within lyr
95% C.I. for EXP(B)
B S.E. Waid df Sig. Exp(B) Lower Upper
Figure imgf000046_0002
Step la Hx_CHF 1.020 .250 16.642 .000 2.774 1.699 4.528
eGFR -.005
Figure imgf000046_0004
.007 .558
Figure imgf000046_0003
.455 .995 .980 1.009
Age .030 .012 6.815 .009 1.031 1.008 1.054 Hx MI .493 .261 3.569
Figure imgf000046_0005
.059 1.637 .982 2.730 hsTnT IP .556 , 137 16.591
Figure imgf000046_0006
.000 1.744 1.335 2.280
SUBSTITUTE SHEETS (Rule 26)
Figure imgf000047_0001
Step 2b Hx_CHF 922 .256 13.020
Figure imgf000047_0002
OOP 2.514 1.524 4.149
eGFR -.003 .007 .158
Figure imgf000047_0003
.691 .997 .983 1.012
Age _ .029 .012 6.263 1 .012 1.030 1.006 1.053
Hx_MI _ .477 .263 3.282 1 ,070 1,611 .962 2,697
ERFE IP .159 .081 3.861 1 .049 1.172 1.000 1.374 hsTnT IP .561 .137 16.640 1 .000 1.752 1.338 2.293
Constant -5.142 1.152 19.919 1 .000 .006
Example 9: ERFE as a Diagnostic Biomarker of Atrial Fibrillation
Applicants also surprisingly identified that erythroferrone could diagnose atrial fibrillation in a patient, as confirmed by electro-cardiogram (ECG).
In the entire study group (n=200) ERFE could diagnose atrial fibrillation in the 52 patients with diagnosed AF as confirmed by ECG. Receiver Operating Curve analysis (Figure 4) generated and the following information.
Table XV: ERFE Dx for Atrial Fibrillation
Test Result Variable(s) : ERFE_ED
Figure imgf000047_0004
a. under non-parametric assumption; b. null hypothesis true area = 0.50
As such, Applicants have further identified a clinical utility for ERFE for the diagnosis of atrial fibrillation in a patient suspected of having AF. EXAMPLE 9: Utility of ERFE to Diagnose Acute Decompensated Heart Failure in Patients with Concomitant Atrial Fibrillation
The following data, read in conjunction with Figure 5, documents the utility of ERFE in diagnosing acute decompensated heart failure in those patients who have concomitant atrial fibrillation.
SUBSTITUTE SHEETS (Rule 26) ADHF_Y_ Valid N
N (listwise)
Positive3 _ 59
Negative _ 32
Figure 5 illustrates a ROC for the diagnosis of acute decompensated heart failure in those patients who present with concomitant atrial fibrillation.
Table XVI: Area Under the Curve
Asymptotic 95% Confidence
Test Result Asymptotic Interval
Variable(s)
Figure imgf000048_0002
Area Std. Error3
Figure imgf000048_0001
Lower Bound Upper Bound
NT-proBNP _ .713 .058 001 .600 .827
ERFE _ .688 _ .062 .003 _ .567 _ .809
ERFE + NT-proBNP .723 .059 .000 .607 .839
Logistic regression model of ERFE to Dx ADHF in those with AF (n=87 (57 with ADHF) due to data dropouts compared with ROC curve was then performed.
In first pass, regression analysis model includes age, Hx HF, BMI, PND, orthopnea, hsTnT and NT-proBNP .(all clinical things docs use). Significant variables that pass a forward conditional test are (note that NT-proBNP is not one of them) :
Table XVIZ: Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation
Figure imgf000048_0003
Constan -4.886 2.184 5.003 1 .025 .008
t
SUBSTITUTE SHEETS (Rule 26) Then on second pass, which tries to add ERFE and hsTnT, only ERFE emerges as the ONLY significantly useful biomarker that adds to age and PND (age becomes not significant) :
Table XVII: Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation + ERFE and hsTNT
95% C.I.for
EXP(B)
B S.E. Wald df Siq. Exp(B) Lower Upper
Step PND _ 2.029 .591 11.772 _ 1 .001 7.605 2.387 24.235 la _A¾e _ .051 .031 2.787 _ 1 .095 1.052 .991 1.117
ERFE ,579 .219 6.978 _ 1 .008 1,785 1.161 2.743
Constan -4.776 2.307 4.287 1 .038 .008
Figure imgf000049_0001
To check, Applicants placed all variables into one pass model :
Table XVIII: Logistic regression model for diagnosis of acute decompensated heart failure in patients with atrial fibrillation (ALL variables)
95% C.I.for EXP(B)
B S.E. Wald df Siq. Exp(B) Lower Upper
Step ED_P D 2.001 .534 14.038 1 .000 7.400 2.597 21.083 la Constan -.182 .303 .363 1 .547 .833
_ t _ _ _
Step ED_PND 2.072 .583 12.647 1 .000 7.937 2.534 24.858
2b ERFE _ .652 .223 8.568 _ 1 .003 1.919 1.240 2.968
Constan -1.053 .452 5.430 1 .020 .349
Figure imgf000049_0002
Accordingly, these data demonstrate that ERFE is the best biomarker for the diagnosis of acute decompensated heart failure in those patients presenting with atrial fibrillation, among all variables tested. Further analysis (data not shown) reveals that ERFE is the only significant biomarker which could diagnose acute decompensated heart failure in those patients presenting with atrial fibrillation.
SUBSTITUTE SHEETS (Rule 26) Although the invention has been described by way of example, it should be appreciated that variations and modifications may be made without departing from the scope of the invention as defined in the claims. Furthermore, where known equivalents exist to specific features, such equivalents are incorporated as if specifically referred in this specification.

Claims

1. A method of diagnosing acute decompensated heart failure in a patient, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, an increase in the level of the ERFE peptide obtained from the biological sample compared to the reference interval from a control population is diagnostic of acute decompensated heart failure in the patient from which the biological sample was obtained.
2. A method of predicting a patient's risk of acquiring acute decompensated heart failure within one year of diagnosis with any medical condition, the method comprising the steps of:
(i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval from a control population,
wherein, the biological sample has been obtained from the patient within one year of diagnosis with any medical condition, and wherein an increase in the level of the ERFE peptide obtained from the biological sample compared with the reference interval from a control population is predictive of the patient acquiring acute decompensated heart failure within one year of the diagnosis with any medical condition.
3. A method according to claim 2, wherein the medical condition is selected from the group consisting of a cardiac related disease or disorder, diabetes, smoking related disease, chronic pulmonary artery disease, pulmonary artery disease, coronary artery disease, acute and chronic kidney disease.
4. A method according to claim 3, wherein the cardiac related disease or disorder is selected from myocardial infarction, unstable angina, atrial fibrillation, cardiac hypertrophy, mitral regurgitation, valve disorders, heart failure, pericarditis, disorders of nerve conduction, vasovagal syncope or any combination thereof.
5. A method for monitoring a patient diagnosed with acute heart failure comprising : (i) measuring the level of an erythroferrone (ERFE) peptide in a biological sample obtained from the patient; and
(ii) comparing the measured level of the ERFE peptide against a reference interval obtained from the same subject at an earlier time, wherein the reference interval is a level of ERFE peptide that distinguishes a subject having heart failure from subjects not having heart failure such that a level of ERFE peptide that is above the reference interval indicates the subject has acute heart failure; and
(iii) setting a treatment regimen or adjusting a treatment regimen for the subject based on comparing the levels of ERFE peptide in the biological sample with the reference interval.
6. A method according to any one of claims 1 to 5, wherein the haemaglobin levels in the patient [Hb] are [Hb] > 129 g/L for a male patient, and [Hb] > 115 g/L for a female patient.
7. A method according to any one of claims 1 to 6, wherein :
(i) the control population is sex and age-matched subjects who do not have heart failure, or who are not predisposed to heart failure, as measured by one or more risk factors;
(ii) the reference interval is the mean ERFE peptide level from the control population;
(iii) the method is performed in conjunction with the analysis of one or more risk factors or biomarkers of acute decompensated heart failure including heart rate, haemoglobin concentration, blood pressure, age, sex, weight, level of physical activity, family history of events including obesity, diabetes and cardiac events, and levels of circulating Troponin T, Troponin I, NT-proBNP, BNP, and iron and/or anaemia status, including ferritin, transferrin, and ferritin/transferrin saturation levels; and/or
(iv) the biological sample is selected from plasma, serum, whole blood, arterial blood, venous blood, saliva, bone marrow tissue, heart tissue, vascular tissue and interstitial fluid.
8. A method according to any one of claims 1 to 10, wherein measuring the level of ERFE peptide in a biological sample obtained from the patient is performed by contacting the biological sample with a binding agent that selectively binds to an ERFE peptide, and detecting binding between the ERFE peptide and the binding agent.
9. A method according to claim 8, wherein the binding agent:
(i) comprises a detectable label and/or is immobilised to a solid phase;
(ii) is selected from an antibody, an antigen-binding antibody fragment and an aptamer, and preferably is a monoclonal antibody or antigen-binding antibody fragment thereof.
10. A method according to any one of claims 1 to 9, wherein the ERFE peptide is a human ERFE peptide defined by SEQ ID NO: 1.
11. An assay for measuring the level of an erythroferrone (ERFE) protein from a patient with, or at risk of acquiring, heart failure, the assay comprising a binding agent that selectively binds to an ERFE peptide, which binding agent can be quantitatively measured upon binding to the ERFE peptide in a biological sample from the subject.
12. An assay according to claim 11, which is an immunoassay, an enzyme assay, a fluorescence assay or chemiluminescence assay.
13. An assay according to claim 11 or claim 12, wherein the binding agent is a polyclonal, monoclonal, chimeric or humanised antibody or antigen-binding antibody fragment thereof, or wherein the bindng agent is an aptamer.
14. An erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, for use in diagnosing heart failure in a patient or for predicting a patient's risk of acquiring heart failure within one year of diagnosis with any medical condition.
15. A test kit or article of manufacture for diagnosing or predicting acute decompensated heart failure in a patient, the test kit or article of manufacture comprising an erythroferrone (ERFE) peptide binding agent that selectively binds to an ERFE peptide, together with instructions for how to diagnose heart failure in the patient, or instructions for how to predict the patient's risk of acquiring heart failure within one year of diagnosis with any medical condition.
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