Immunoassay for detecting collagen i fragment

Abstract

The present invention relates to methods of immunoassay for detecting a biomarker which is an MMP-cleaved fragment of collagen type-I and its use in detecting and / or monitoring diseases such as kidney disease and diseases of the cardiovascular system including the heart.

Description

[0001] Immunoassay for detecting Collagen I fragment

[0002] Field of invention

[0003] The present invention relates to methods of immunoassay for detecting a biomarker which is an M MP- cleaved fragment of collagen type-l and its use in detecting and / or monitoring diseases such as kidney disease and diseases of the cardiovascular system including the heart.

[0004] Background

[0005] Type-l collagen is a richly abundant fibrillar collagen and is the major component of the extracellular matrix (ECM). Triple helical collagen type-l heterotrimers are arranged in an a1a1a2 chain format and are highly crosslinked, forming tightly wound fibrils that are interlinked by minor collagens1. The collagen type-l scaffold is localized to the interstitial matrix providing structural support and tensile strength and is integral to organ structure and function2 3. A central fragment of type-l collagen was shown to be released and have signalling properties, termed a matricryptin, in patients with ST-elevated myocardial infarction. The fragment, that has been named C1SIG, was first reported as increased in an MMP-9 null mouse model with an induced myocardial infarction4. The location of the C1SIG fragment was later confirmed with mass-spectrometry and shown to be cleaved by MMP-9 and MMP-2 at the site between amino acids 1158 and 1159 on the mouse collagen type-l alpha-1 chain5. Previous studies established C1 SIG as a matricryptin released from cardiac tissue and as a potential therapeutic due to its ability to reduce left ventricular remodelling after acute myocardial injury when delivered exogenously56.

[0006] Based on the matricryptin identified, it was hypothesised that this epitope could function as a specific biomarker for patients with cardiovascular indications. The inventors have developed and validated a competitive enzyme-linked immunosorbent assay (ELISA) targeting the neoepitope site of this matricryptin, and further evaluated the association of the novel biomarker with cardiovascular disease outcome, and kidney disease.

[0007] Summary of the Invention

[0008] The present inventors have now explored the biomarker potential of measuring the levels of the N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha- 1 chain, specifically with the N-terminal sequence RTGDAGPVGP (SEQ ID NO: 1) (also referred to herein as “C1SIG”, the “target sequence” or the “C1SIG target sequence”). The inventors have devised an immunoassay which has been used to demonstrate that the levels of C1SIG in patient samples (and in particular patient serum and urine samples) are elevated and so can be used to identify patients with diseases including those associated with P22790WQ

[0009] 2 cardiovascular disease and the heart such as cardiovascular events, as well as kidney disease.

[0010] Accordingly, in a first aspect, the present invention provides a monoclonal antibody that specifically binds to the N-terminal neoepitope derived from the collagen type-1 alpha-1 chain. Preferably the neoepitope is generated following cleavage with MMP-2 or MMP-9, in particular at the site between amino acids 1169 and 1170 on the human collagen type-l alpha-1 chain. Preferably, the monoclonal antibody specifically binds to the N-terminal epitope comprising, consisting essentially or consisting of the amino acid sequence RTGDAGPVGP (SEQ.ID NO: 1). Preferably said monoclonal antibody does not bind with the same affinity to an elongated version of said N-terminus amino acid sequence which is preferably GRTGDAGPVGP (SEQ.ID NO: 2), and / or to a truncated version of said N- terminus amino acid sequence which is preferably TGDAGPVGP (SEQ.ID NO: 3).

[0011] In a preferred embodiment, the monoclonal antibody does not bind with the same affinity to a peptide having the N-terminus amino acid sequence GRTGDAGPVGP (SEQ.ID NO: 2) (i.e. an elongated version of the C1SIG target sequence extended at its N-terminus by the addition of a glycine residue). The monoclonal antibody has a reduced binding affinity to said elongated version of the target sequence compared to the binding affinity to said target peptide. Preferably, the monoclonal antibody does not specifically bind to said elongated version of the target sequence. Preferably, the ratio of the affinity of said antibody for the C1SIG target sequence to the affinity of said antibody for said elongated version of the target sequence is at least 5 to 1 , and more preferably is at least 10 to 1, at least 20 to 1, at least 30 to 1 , at least 40 to 1 or at least 50 to 1.

[0012] In a preferred embodiment, the monoclonal antibody does not specifically bind to a peptide having the N-terminus amino acid sequence TGDAGPVGP (SEQ.ID NO: 3) (i.e. a truncated version of the C1SIG target sequence truncated at its N-terminus by the removal of the first arginine residue). Preferably, the ratio of the affinity of said antibody for the C1SIG target sequence to the affinity of said antibody for said truncated version of the target sequence is at least 10 to 1 , and more preferably is at least 20 to 1 , at least 30 to 1 , at least 40 to 1 , at least 50 to 1 or at least 100 to 1.

[0013] In a preferred embodiment, the monoclonal antibody is raised against a synthetic peptide having the N-terminus amino acid sequence RTGDAGPVGP (SEQ.ID NO: 1). For example, the monoclonal antibodies may be raised by: (a) immunizing a rodent (or other suitable mammal) with a synthetic peptide comprising the N-terminus amino acid sequence RTGDAGPVGP (SEQ.ID NO: 1), which peptide may optionally be linked, preferably at or near its C-terminus, to an immunogenic carrier protein (such as keyhole limpet hemocyanin (“KLH”)); (b) isolating and cloning a single antibody producing cell; and (c) assaying the resulting monoclonal antibodies to ensure that they have the desired specificity. An exemplary protocol of the development, production and characterization of suitable monoclonal antibodies is described in the Examples section, infra.

[0014] As used herein the term “N-terminus” refers to an N-terminal peptide sequence at the extremity of a polypeptide, i.e. at the N-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof.

[0015] As used herein the term “C-terminus” refers to a C-terminal peptide sequence at the extremity of a polypeptide, i.e. at the C-terminal end of the polypeptide, and is not to be construed as meaning in the general direction thereof.

[0016] As used herein, the terms “peptide” and “polypeptide” are used synonymously.

[0017] As used herein the term “monoclonal antibody” refers to both whole antibodies and to fragments thereof that retain the binding specificity of the whole antibody, such as for example a Fab fragment, F(ab’)2 fragment, single chain Fv fragment, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure of two identical pairs of polypeptide chains, each pair made up of one "light" and one "heavy" chain. The N-terminal regions of each light chain and heavy chain contain the variable region, while the C-terminal portions of each of the heavy and light chains make up the constant region. The variable region comprises three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant region allows the antibody to recruit cells and molecules of the immune system. Antibody fragments retaining binding specificity comprise at least the CDRs and sufficient parts of the rest of the variable region to retain said binding specificity.

[0018] In the present invention, a monoclonal antibody comprising any constant region known in the art can be used. In the case of mouse antibodies and human antibodies, the constant light chains are classified as either kappa or lambda light chains. Heavy constant chains are classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to IgGI, I gG2, I gG3, and lgG4 in the case of humans and IgGI, lgG2a, lgG2b, P22790WQ

[0019] 4 lgG2c and lgG3 in the case of mice. The monoclonal antibody may preferably be of the IgG isotype, including any one of the IgG subclasses.

[0020] The CDR of an antibody can be determined using methods known in the art such as that described by Kabat et al. Antibodies can be generated from B cell clones. The isotype of the antibody can be determined by ELISA specific for human or murine IgM, IgG or IgA isotype, or human lgG1, lgG2, lgG3 or lgG4 subclasses or murine IgGI, lgG2a, lgG2b, lgG2c and lgG3. The amino acid sequence of the antibodies generated can be determined using standard techniques. For example, RNA can be isolated from the cells and used to generate cDNA by reverse transcription. The cDNA is then subjected to PCR using primers which amplify the heavy and light chains of the antibody. For example, primers specific for the leader sequence for all VH (variable heavy chain) sequences can be used together with primers that bind to a sequence located in the constant region of the isotype which has been previously determined. The light chain can be amplified using primers which bind to the 3’ end of the Kappa or Lambda chain together with primers which anneal to the V kappa or V lambda leader sequence. The full length heavy and light chains can be generated and sequenced.

[0021] Monoclonal antibodies that specifically bind to the N-terminus amino acid sequence RTGDAGPVGP (SEQ. ID NO: 1), can be generated via any suitable techniques known in the art. For example, the monoclonal antibody may be raised against a synthetic peptide comprising or consisting of the amino acid sequence RTGDAGPVGP (SEQ. ID NO: 1), such as for example by: immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence RTGDAGPVGP (SEQ. ID NO: 1), which optionally may linked to an immunogenic carrier protein (such as keyhole limpet hemocyanin), isolating and cloning a single antibody producing cell, and assaying the resulting monoclonal antibodies to ensure that they have the desired specificity.

[0022] The monoclonal antibody that specifically binds to the C1SIG target sequence (i.e. RTGDAGPVGP (SEQ. ID NO: 1)) may preferably comprise one or more complementaritydetermining regions (CDRs) selected from:

[0023] CDR-H1: SYWMH (SEQ ID. NO:4)

[0024] CDR-H2: EIHPSNGRTNYNEKFKS (SEQ ID. NO:5)

[0025] CDR-H3: YYYGSGYAWFAY (SEQ ID. N0:6) P22790WQ

[0026] 5

[0027] CDR-L1 : TLSSQHSTYTIE (SEQ ID. NO:7)

[0028] CDR-L2: GSHSTGD (SEQ ID. NO:8)

[0029] CDR-L3: GVGDTIKEQFVYV (SEQ ID. NO:9)

[0030] Preferably the monoclonal antibody may comprise at least 2, 3,4, 5 or all 6 of the above listed CDR sequences.

[0031] Preferably the monoclonal antibody has a light chain variable region comprising the CDR sequences:

[0032] CDR-L1 : TLSSQHSTYTIE (SEQ ID. NO:7)

[0033] CDR-L2: GSHSTGD (SEQ ID. NO:8)

[0034] CDR-L3: GVGDTIKEQFVYV (SEQ ID. NO:9)

[0035] Preferably the monoclonal antibody may have a light chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the light chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics)

[0036] Preferably the monoclonal antibody has a heavy chain variable region comprising the CDR sequences:

[0037] CDR-H1 : SYWMH (SEQ ID. NO:4)

[0038] CDR-H2: EIHPSNGRTNYNEKFKS (SEQ ID. NO:5) and

[0039] CDR-H3: YYYGSGYAWFAY (SEQ ID. NO:6)

[0040] Preferably the monoclonal antibody may have a heavy chain that comprises framework sequences between the CDRs, wherein said framework sequences are substantially identical or substantially similar to the framework sequences between the CDRs in the heavy chain sequence below (in which the CDRs are shown in bold and underlined, and the framework sequences are shown in italics) P22790WQ

[0041] 6

[0042] SYWMH W QRPGQGLEI / V7GEIHPSNGRTNYNEKFKSKA TLTVDKSSSTA YMQLSSLTSED SAVYYCAGPYYYGSGYAWFAY (SEQ ID. NO: 11)

[0043] As used herein, the framework amino acid sequences between the CDRs of an antibody are substantially identical or substantially similar to the framework amino acid sequences between the CDRs of another antibody if they have at least 70%, 80%, 90% or at least 95% similarity or identity. The similar or identical amino acids may be contiguous or non-contiguous.

[0044] The framework sequences may contain one or more amino acid substitutions, insertions and / or deletions. Amino acid substitutions may be conservative, by which it is meant the substituted amino acid has similar chemical properties to the original amino acid. A skilled person would understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties such as size, charge and polarity: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gin; Group 3 His, Arg, Lys; Group 4 Met, Leu, lie, Vai, Cys; Group 5 Phe Thy Trp.

[0045] A program such as the CLUSTAL program to can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity plus conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention. Identity or similarity is preferably calculated over the entire length of the framework sequences.

[0046] In certain preferred embodiments, the monoclonal antibody that specifically binds to the C1 SIG target sequence may comprise the light chain variable region sequence:

[0047] QLVLTQSSSASFSLGASAKLTCTLSSQHSTYT\EWYQQQPLKPPKYVMELKKDGSHSTGDG IPDRFSGSSSGADRYLSISNIQPEDEAIYICG\ / GDT\KEQF\JY\ / FGGGTKVTVL (SEQ ID. NO:12) and / or the heavy chain variable region sequence:

[0048] Q VQLQQPGAEL VKPGASVKLSCKA SG YTFTSYWM H WVKQRPGQGLEWIGE\H PSNGRTN YNEKFKSKA TLTVDKSSSTA YMQLSSLTSEDSA VYYCA GPYYYGSGYAWFAYI / I / GQGTL V

[0049] TVSA (SEQ ID. NO: 13)

[0050] (CDRs bold and underlined; Framework sequences in italics) The term "specifically bind" as used herein means that the antibody binding is selective for the antigen and that this binding can be distinguished from unwanted or non-specific interactions. The ability of a monoclonal antibody to bind to a specific epitope or peptide sequence can be measured either through an enzyme-linked immunosorbent assay (ELISA) as described herein or other techniques familiar to one of skill in the art, e.g. surface plasmon resonance (SPR) technique (analyzed e.g. on a BIAcore instrument) and traditional binding assays. The extent of binding of a monoclonal antibody to an unrelated protein is less than about 10% of the binding of the monoclonal antibody to the epitope or peptide as measured, e.g., by ELISA. “Affinity” refers to the strength of the sum total of non-covalent interactions between a single binding site of a molecule (e.g., an epitope binding region of an antibody) and its binding partner (e.g., an epitope or antigen). Unless indicated otherwise, as used herein, “binding affinity” refers to intrinsic binding affinity which reflects a 1 :1 interaction between members of a binding pair (e.g., an antigen binding moiety and an antigen). The affinity of a molecule for its partner can generally be represented by the dissociation constant (Kd), which is the ratio of dissociation and association rate constants (kOff and kon, respectively). Thus, equivalent affinities may comprise different rate constants, as long as the ratio of the rate constants remains the same. The dissociation constant represents the concentration of the antigen at which half of the binding sites on the antibody are occupied. A lower Kd indicates a higher binding affinity between the antibody and antigen, while a higher Kd reflects weaker binding. Several methods are available to measure the Kd of an antibody, including surface plasmon resonance (SPR), isothermal titration calorimetry (ITC), and fluorescence-based assays. In certain aspects, a monoclonal antibody that binds to the epitope or peptide has a dissociation constant (KD) of < 1 pM, < 100 nM, < 10 nM, < 1 nM, < 0.1 nM, < 0.01 nM, or < 0.001 nM (e.g. 108M or less, e.g. from 108M to 1013M, e.g. from 109M to 1013M).

[0051] In a second aspect, the present invention provides a method of immunoassay for detecting in a sample a N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain said method comprising: i) contacting a sample comprising a N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody which specifically binds to said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain, ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample. In a preferred embodiment the monoclonal antibody is an antibody of the first aspect of the invention, and so preferred embodiments of the first aspect apply to the second aspect mutatis mutandis.

[0052] In preferred embodiments, the patient sample is a human patient sample. In preferred embodiments, the patient sample is a biofluid sample. The biofluid may be any bodily fluid, and in particular it may be selected from a blood-based sample, urine, saliva, central spinal fluid (CSF) or synovial fluid. Preferably, the sample is urine or a blood-based sample, such as blood (whole blood), plasma or serum. In a particularly preferred embodiment, the bloodbased sample is a serum sample.

[0053] In preferred embodiments the immunoassay is a competition assay or a sandwich assay. The immunoassay may, for example, be a radio-immunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to the person skilled in the art.

[0054] As used herein the term “amount of binding” refers to the quantification of binding between the antibody and peptides in the patient sample. Said quantification may for example be determined by comparing the measured values of binding in the patient sample against a calibration curve produced using measured values of binding in standard samples containing known concentrations of a peptide to which the antibody specifically binds, in order to determine the quantity of peptide to which the antibody specifically binds in the patient sample. Any suitable analytical method can be used for measuring the amount of binding. For example, an ELISA method can be used in which spectrophotometric analysis is used to measure the amount of binding both in the patient samples and when producing the calibration curve.

[0055] In a third aspect, the present invention provides a method of immunoassay for detecting and / or monitoring a disease in a patient, the method comprising: i) contacting a sample comprising a N-terminal amino acid sequence of the neoepitope derived from the collagen type-1 alpha-1 chain with a monoclonal antibody which specifically binds to said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain, ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample, iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

[0056] In a preferred embodiment the monoclonal antibody is an antibody of the first aspect of the invention, and so preferred embodiments of the first aspect apply to the third aspect mutatis mutandis. In a preferred embodiment the immunoassay is an immunoassay of the second aspect of the invention, and so preferred embodiments of the second aspect apply to the third aspect mutatis mutandis.

[0057] An elevated level of binding and so increased amount of the C1SIG biomarker in a patient sample may be indicative of the presence of a disease in the patient and / or a patient with increased disease severity. An elevated level of binding and so increased amount of the C1 SIG biomarker in a patient sample may also be indicative of a patient with a more progressive form of a disease.

[0058] An elevated level of binding and so increased amount of the 01 SIG biomarker in a patient sample may also be indicative of progression of the disease, especially if the results are compared to those from a sample taken from the same patient at an earlier time point. The method can also be used to monitor if a treatment is effective if the results from samples obtained before and after treatment has been initiated are compared. A change in the level of binding may be indicative of a treatment being effective.

[0059] The disease may be a disease associated with the cardiovascular system including the heart. The disease may be a cardiovascular disease. The cardiovascular disease may in certain embodiments be atherosclerosis, and heart failure. “Heart failure” as used herein includes heart failure with reduced ejection fraction (HFrEF), dilated cardiomyopathy, and myocardial infarction in particular patients with suspected ST-elevated myocardial infarction (STEMI). Patients with HFrEF may have suffered a myocardial infarction or have dilated cardiomyopathy. The disease may be a disease associated with the kidney. The disease may be a chronic kidney disease. The kidney disease may in certain embodiments be nephritis, Alport syndrome, or Immunoglobulin A (IgA) nephropathy.

[0060] As used herein the term “predetermined cut-off value” means an amount of binding that is determined statistically to be indicative of a high likelihood of the disease in question (for example cardiovascular disease in general, or kidney disease) or a particular severity thereof in a patient, in that a measured value of the target peptide(s) in a patient sample that is at or above the statistical cut-off value corresponds to at least a 70% probability, preferably at least an 75% probability, more preferably at least an 80% probability, more preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability of the presence of said disease or particular severity thereof.

[0061] As used herein, the term “values associated with normal healthy subjects” means standardised quantities of binding determined by the methods described supra for samples from subjects considered to be healthy, i.e. without disease (e.g. without cardiovascular disease, in particular HFrEF, STEMI or dilated cardiomyopathy; or without kidney disease especially Alport syndrome or Immunoglobulin A (IgA) nephropathy); and the term “values associated with known disease severity” means standardised quantities of binding determined by the methods described supra for samples from patients known to have disease (e.g. cardiovascular disease in general or in particular HFrEF, STEMI or dilated cardiomyopathy; or kidney disease in general or in particular Alport syndrome or Immunoglobulin A (IgA) nephropathy) of a known severity.

[0062] In a fourth aspect, the present invention provides a method of treating a disease in a patient in need thereof, the method comprising:

[0063] (a) carrying out a method of immunoassay in accordance with the third aspect of the present invention on a sample from a patient in order to detect whether said patient has a disease and

[0064] (b) administering to the patient a therapy for the treatment of the disease if it is determined in step (a) that the patient has said disease.

[0065] For example, step (a) may comprise identifying that the patient has a disease, and so requires treatment. Step (a) may also comprise identifying a patient with a particular severity or at a certain stage of a disease, and so a suitable treatment regime can be planned. For example, a patient with a more severe form the disease, or a more progressive form of the disease can be treated more aggressively with a higher and / or more frequent dosage of a suitable medicament.

[0066] For example, step (b) may comprise administering a therapy for the treatment of a disease associated with the cardiovascular system including the heart if it is determined in step (a) that the patient has a disease associated with the cardiovascular system including the heart.

[0067] The disease may be a disease associated with the cardiovascular system including the heart. The disease may be a cardiovascular disease. The cardiovascular disease may in certain embodiments be atherosclerosis, and heart failure. “Heart failure” as used herein includes heart failure with reduced ejection fraction (HFrEF), dilated cardiomyopathy, myocardial infarction in particular patients with suspected ST-elevated myocardial infarction. Patient with HFrEF may have suffered a myocardial infarction or have dilated cardiomyopathy.

[0068] For example, step (b) may comprise administering a therapy for the treatment of a kidney disease if it is determined in step (a) that the patient has a kidney disease.

[0069] The disease may be a disease associated with the kidney. The disease may be chronic kidney disease. The kidney disease may in certain embodiments be nephritis, Alport syndrome, or Immunoglobulin A (IgA) nephropathy.

[0070] An elevated level of binding and so increased amount of the C1SIG biomarker in a patient sample is also indicative of increased chance of death as compared to a patient with a normal level of C1 SIG biomarker.

[0071] The present invention provides a method of identifying a patient at increased risk of death, the method comprising: i) contacting a sample comprising a N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody which specifically binds to said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain, ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample, iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known risk of death and / or with a predetermined cut-off value. As used herein, the term “values associated with a known risk of death” means standardised quantities of binding determined by the methods described supra for samples from patients known to have died within a set period after the sample had been taken. The set period may be 30 days; 3 months; 6 months or 12 months. For example, the death occurred within 30 days or 1 year from the date the sample was taken.

[0072] The increased risk of death may be due to any cause. In particular the increased risk of death is due to a cardiovascular disease or event, i.e. cardiovascular mortality. Thus, the method may be used to identify a patient at increased risk of death due to a cardiovascular disease or event.

[0073] Preferably the “values associated with a known risk of death” can be associated with a risk of death due to a specific disease or cause using standardised quantities of binding determined by the methods described supra for samples from patients known to have died from the disease or cause within a set period after the sample had been taken. The disease or cause may be any disease or cause described herein, for example a disease associated with the cardiovascular system including the heart.

[0074] For example, “values associated with a known risk of death from a disease associated with the cardiovascular system” means standardised quantities of binding determined by the methods described supra for samples from patients known to have died from a disease associated with the cardiovascular system within a set period after the sample had been taken. In particular, the sample may be taken from patients known to HRrEF, myocardial infarction and / or dilated cardiomyopathy and died within a set period after the sample had been taken. The set period may be 30 days; 3 months; 6 months or 12 months. For example, the death occurred within 30 days or 1 year from the date the sample was taken.

[0075] The risk of death may be increased probability of death within a certain time frame. For example, the risk of death may be an increased likelihood of the patient dying within 10 years from when the sample is taken as compared to a patient with a normal level of C1SIG. Preferably, the risk of death may be an increased likelihood of the patient dying within 7 years, more preferably within 5 years, even more preferably within 2.5 years, most preferably within 1 year from when the sample is taken.

[0076] An elevated level of binding and so increased amount of the C1SIG biomarker in a patient sample is also indicative of increased chance of death, in particular death due to a cardiovascular disease or event, i.e. cardiovascular mortality. Therefore, therapy may include increased monitoring of symptoms which may be indicative of a cardiovascular event.

[0077] In a preferred embodiment, the present invention provides a method of identifying a patient at increased risk of death due to a cardiovascular disease or event, the method comprising:

[0078] (a) carrying out a method of immunoassay in accordance with the second aspect of the present invention on a sample from a patient;

[0079] (b) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known cardiovascular disease risk and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value and

[0080] (c) administering to the patient a therapy for the prevention or treatment of said cardiovascular disease if it is determined in step (a) that the patient has increased risk of said cardiovascular disease.

[0081] The term “values associated with known cardiovascular disease risk” means standardised quantities of binding determined by the methods described supra for samples from patients known to have suffered a cardiovascular event or died within a set period after the sample had been taken. For example, the cardiovascular event or death occurred within 30 days or 12 months from the date the sample was taken.

[0082] Patients identified as being at a high risk of cardiovascular disease or death can be monitored or sent for further diagnostic testing. Patients may also be provided with prophylactic treatment to try to reduce the risk.

[0083] In a preferred embodiment, the present invention provides a method of treating cardiovascular disease in a patient in need thereof, the method comprising:

[0084] (a) carrying out a method of immunoassay for detecting cardiovascular disease in accordance with the third aspect of the present invention on a sample from a patient; and

[0085] (b) administering to the patient a therapy for the treatment of said cardiovascular disease if it is determined in step (a) that the patient has said cardiovascular disease.

[0086] The cardiovascular disease may in certain embodiments be atherosclerosis, in particular carotid artery disease or stenosis. The cardiovascular disease may in certain embodiments be heart failure. “Heart failure” as used herein includes heart failure with reduced ejection fraction (HFrEF), dilated cardiomyopathy, myocardial infarction in particular patients with suspected ST-elevated myocardial infarction. Patient with HFrEF may have suffered a myocardial infarction, suspected ST-elevated myocardial infarction or have dilated cardiomyopathy.

[0087] The therapy may be any therapy suitable for reducing the risk of or treating the cardiovascular disease in question. The therapy may for example comprise or consist of one or more surgeries, one or more medicaments or a combination thereof.

[0088] Suitable treatments for cardiovascular disease include ACE inhibitors (e.g. ramipril, captopril, enalapril, lisinopril and perindopril), angiotensin-2 receptor blockers (ARBs or AIIRAs) (e.g. candesartan, losartan, telmisartan and valsartan) , beta blockers (e.g. bisoprolol, carvedilol and nebivolol) , mineralocorticoid receptor antagonists (e.g. spironolactone and eplerenone), diuretics (e.g. furosemide and bumetanide), ivabradine, sacubitril valsartan, hydralazine with nitrate, digoxin, or SGLT2 inhibitors (e.g. Empagliflozin and dapagliflozin.)

[0089] Suitable medicaments for treating atherosclerosis include ACE inhibitors (e.g. ramipril, captopril, enalapril, lisinopril and perindopril), anti-platelet or anticlotting treatments (heparin, warfarin, rivaroxaban, dabigatran, apixaban, endoxaban, enoxaparin, fondaparinux, clopidogrel, ticagrelor, prasugrel, dipyridamole, aspirin, ticlopidine and eptifibatide); medicines to control blood sugar (e.g. empagliflozin, canagliflozin, and liraglutide), metformin, nitrates e.g. nitroglycerin, ranolazine, statins, cholesterol lowering medicines (e.g. ezetimibe, PCSK9 inhibitor, bempedoic acid, and omega-3 fatty acids) and thrombolytic or clot buster medicine (urokinase, reteplase, tenecteplase, alteplase, streptokinase.) Other treatments for cardiovascular diseases, including myocardial infarction, by surgery include percutaneous coronary intervention (PCI), coronary artery bypass grafting (CABG), transmyocardial laser revascularization or coronary endarterectomy, carotid endarterectomy, weight-loss surgery and angioplasty.

[0090] Patients identified as being at a high risk of kidney disease can be monitored or sent for further diagnostic testing. Patients may also be provided with prophylactic treatment to try to reduce the risk.

[0091] In a preferred embodiment, the present invention provides a method of treating kidney disease in a patient in need thereof, the method comprising:

[0092] (a) carrying out a method of immunoassay for detecting kidney disease in accordance with the third aspect of the present invention on a sample from a patient; and

[0093] (b) administering to the patient a therapy for the treatment of said kidney disease if it is determined in step (a) that the patient has said kidney disease. The disease may be a disease associated with the kidney. The disease may be chronic kidney disease. The kidney disease may in certain embodiments be nephritis, Alport syndrome, or Immunoglobulin A (IgA) nephropathy.

[0094] The therapy may be any therapy suitable for reducing the risk of or treating the kidney disease in question. The therapy may for example comprise or consist of one or more surgeries, one or more medicaments or a combination thereof.

[0095] Suitable treatments for kidney disease include dialysis such as haemodialysis or peritoneal dialysis or a transplant. Suitable medications for treating kidney disease include blood thinners (e.g. aspirin or clopidogrel), statins or other medication ot reduce cholesterol in the blood (e.g. statins for example atorvastatin, simvastatin, pravastatin or rosuvastatin) antacids (e.g. Proton pump inhibitors (PPIs), including omeprazole, lansoprazole; and H2-antagonists such as nizatidine or cimetidine), antihistamines or other medications (such as Gabapentin, pregabalin and difelikefalin) to relieve itching; medication to reduce blood pressure, such as angiotensin-converting enzyme (ACE) inhibitors (e.g. ramipril, captopril, enalapril, lisinopril and perindopril), or angiotensin receptor blockers (ARBs) (e.g. Azilsartan, Candesartan, Eprosartan, Irbesartan, Losartan, Olmesartan, Telmisartan, and Valsartan) and SGLT2 (sodium-glucose co-transporter 2) inhibitors (e.g. dapagliflozin, empagliflozin, canagliflozin and ertugliflozin).

[0096] Suitable treatments for treating Alport syndrome or Immunoglobulin A (IgA) nephropathy include medication to reduce blood pressure, such as angiotensin-converting enzyme (ACE) inhibitors (e.g. ramipril, captopril, enalapril, lisinopril and perindopril), or angiotensin receptor blockers (ARBs) (e.g. Azilsartan, Candesartan, Eprosartan, Irbesartan, Losartan, Olmesartan, Telmisartan, and Valsartan) and SGLT2 (sodium-glucose co-transporter 2) inhibitors (e.g. dapagliflozin, empagliflozin, canagliflozin and ertugliflozin), or immunosuppressants such as steroids and budesonide.

[0097] Figures

[0098] The invention will now be described in the examples below which refer to the following figures:

[0099] Figure 1. Sequence alignment of the human C1 SIG target peptide with mouse and rat species. The specific cleavage site producing the neo-epitope is indicated by the downwards arrow. P22790WQ

[0100] 16

[0101] There are 2 amino acid mismatches in the mouse and rat sequences compared to the human sequence at the 5thand 8thamino acid and highlighted in red.

[0102] Figure 2. Specificity of the mAb produced for implementation into the assay. The mAb is specific to the selection peptide (synthetically produced target sequence), shown by the high affinity of the mAb to this peptide and no affinity to the range of deselection peptides tested. The peptides used are as follows: selection peptide is RTGDAGPVGP (SEQ. ID NO:1), elongated is GRTGDAGPVGP (SEQ. ID NO:2), truncated is TGDAGPVGP (SEQ. ID NO:3), nonsense peptide is AGPAGAPGPA (SEQ. ID NO: 16), nonsense coater is AGPAGAPGPA- K-Biotin SEQ. ID NO: 17).

[0103] Figure 3. C1SIG biomarker levels are increased in patients with heart failure with reduced ejection fraction (HFrEF) compared to healthy controls (Ctrl). C1SIG was not able to distinguish between HFrEF patients having suffered a myocardial infarction (Ml) and with dilated cardiomyopathy (DCM). The Mann-Whitney test and Kruskal-Wallis tests were used to determine statistically significant changes in each graph, respectively (Ctrl vs HFrEF: p < 0.0001 , Ctrl vs Ml: p < 0.0001 , Ctrl vs DCM: p = 0.0041).

[0104] Figure 4. C1SIG and C1M increase at 6-12 hours post-admission to hospital with ST- elevation myocardial infarction (STEMI) compared to admission biomarker levels. The Wilcoxon matched pairs signed rank test was used to determine any statistical significance (C1SIG: p < 0.0001 , C1M: p < 0.0001 , n = 171 pairs).

[0105] Figure 5. High C1SIG levels are associated with increased probability of all-cause mortality within 30-days and 1-year after an Ml. Dashed vertical line indicates 30-day outcome. Logrank tests show significant difference of high and low C1SIG levels when associated to allcause mortality (30 days: p < 0.0001 , 1 year: p < 0.0001).

[0106] Figure 6. C1SIG and C1M are both predictive of all-cause mortality risk in univariate and multivariate analysis. Model 1 is based on the Framingham Score (includes age, sex, diastolic blood pressure (BP), systolic BP, hypercholesterolemia status, diabetes status, and smoking status), whereas Model 2 is Model 1 with some clinically relevant biomarkers (mid- regional pro-adrenomedullin, copeptin, pro-atrial natriuretic peptide, ST2 and C-reactive protein). Hazard ratios (HR) are presented with 95% confidence intervals (Cl) and calculated as per biomarker doubling. Figure 7 shows serum levels of C1SIG in healthy individuals and Alport syndrome patients (A). In Figure 7B, Alport syndrome patients are split by a urine protein-to-creatinine ratio (uPCR) greater than or less than 30, indicative of increased kidney dysfunction in uPCR > 30.

[0107] Figure 8 Urinary C1 SIG levels adjusted for urinary creatinine (uC1SIG / uCr) are correlated with estimated glomerular filtration rate (eGFR) (A) and fibrosis score (%) (B). Using Spearman’s correlation on Iog2-transformed biomarker data, there is a positive correlation with eGFR (cor = 0.78, p < 0.0001) and a negative correlation with fibrosis score obtained from biopsy (cor = -0.50, p = 0.0008)

[0108] Figure 9 shows reduction in urinary C1 SIG adjusted for urinary creatinine is associated with increasing chronic kidney disease (CKD) stage. Between stages 1 and 4, there is a significant reduction in uC1SIG / uCr (p = 0.0012), and, similarly, between stages 2 and 4 (p = 0.0008). The number of individuals at each CKD stage is stated in the figure.

[0109] Examples

[0110] The presently disclosed embodiments are described in the following Examples, which are set forth to aid in the understanding of the disclosure, and should not be construed to limit in any way the scope of the disclosure as defined in the claims which follow thereafter. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the described embodiments, and are not intended to limit the scope of the present disclosure nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

[0111] Materials and methods

[0112] Identification of target

[0113] The target was identified in mouse by previous studies78and matched to human type-l collagen protein found to be cleaved between amino acids 1169-1170. The first 10 amino acids of the human fragment from the N-terminal of the cleavage site were considered as the biomarker target. The specific amino acid sequence was analysed for uniqueness and crossreactivity with other proteins using protein blast ( )9. Sequence P22790WQ

[0114] 18 homology to other species was investigated using the sequence alignment tool on UniProt Monoclonal antibody production was initiated based on the human sequence and development of the immunoassay began.

[0115] Monoclonal antibody production and selection

[0116] The monoclonal antibody (mAb) was raised against the COL1A1 sequence1170RTGDAGPVGP1179(SEQ. ID NO:1) using the following method. Six female Balb / C mice aged 6-7 weeks were immunised subcutaneously with emulsified antigen and immunogenic peptide linked to keyhole limpet hemocyanin (RTGDAGPVGP-GGC-KLH) (SEQ. ID NO: 18) every 4 weeks until stable serum titres were reached. Based on the serum titre, mice were selected for spleen harvesting and splenic cells were fused with SP2 / 0 myeloma cells to produce hybridoma cells (described in11). Screening of the clones began 14 days after hybridoma culture and clones positive for peptide inhibition were identified. Clones were subcloned using the limiting dilution method and true monoclonal-producing cells were expanded. Approximately 500ml of supernatant was collected and the IgG monoclonal antibody was purified using a 5ml HiTrap Protein G column (cat. 17-0405-03, Cytiva, Uppsala, Sweden) on an AKTA Start system (GEH29022094EA, Cytiva). Once purified, the mAb was labelled with horseradish peroxidase (HRP) using the Roche HRP-labelling kit (cat. 11829696001 , Roche Diagnostics, Copenhagen, Denmark). The antibodies generated were sequenced and the CDRs determined.

[0117] The sequence of the chains are as follows (CDRs underlined and in bold); framework sequences in italics; constant region normal font:

[0118] Heavy chain: Amino acid sequence (458 aa)

[0119] Q VQLQQPGAEL VKPGASVKLSCKA SG YTFTSYWM H WVKQRPGQGLEWIGE\H PSNGRTN YNEKFKSKA TLTVDKSSSTA YMQLSSLTSEDSA VYYCA GPYYYGSGYAWFAYI / I / GQGTL V WSAAKTTPPSVYPLAPGCGDTTGSSVTLGCLVKGYFPESVTVTWNSGSLSSSVHTFPALL QSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPISTINPCPPCKECHKC PAPNLEGGPSVFIFPPNIKDVLMISLTPKVTCVWDVSEDDPDVQISWFVNNVEVHTAQTQT HREDYNSTIRVVSTLPIQHQDWMSGKEFKCKVNNKDLPSPIERTISKIKGLVRAPQVYILPPP AEQLSRKDVSLTCLWGFNPGDISVEWTSNGHTEENYKDTAPVLDSDGSYFIYSKLNMKTS KWEKTDSFSCNVRHEGLKNYYLKKTISRSPGK (SEQ. ID NO:14)

[0120] Light chain: Amino acid sequence (220 aa)

[0121] QLVLTQSSSASFSLGASAKLTC LSSQHSTY7\EWYQQQPLKPPKYVMELKKDGSHSTGDG

[0122] IPDRFSGSSSGADRYLSISNIQPEDEAIYICGVGDT KEQFVYVFGGGTKVTVLGQPKS P L P22790WQ

[0123] 19

[0124] TVFPPSSEELKENKATLVCLISNFSPSGVTVAWKANGTPITQGVDTSNPTKEGNKFMASSFL

[0125] HLTSDQWRSHNSFTCQVTHEGDTVEKSLSPAECL (SEQ. ID N0:15)

[0126] Development of the assay

[0127] The final assay parameters were determined after many optimising experiments, testing various reagents, concentrations, incubation times and temperatures, and confirming specificity of the mAb to the target peptide using a series of deselection peptides. The final competitive ELISA used colorimetric substrate for quantification and protocol was set as follows: A 96-well streptavidin coated plate (cat. 11940279103, Roche) was coated with 100ul / well of 5 ng / ml coating peptide (RTGDAGPVGP-K-Biotin) (SEQ. ID NO: 19) diluted in coating buffer (50mM PBS-BTB, 8g NaCI / L, pH 7.4) and incubated for 30 minutes at 18-22°C on a shaker at 300 rpm. A standard curve was prepared using a 30 amino acid long selection peptide (RTGDAGPVGPPGPPGPPGPPGPPSAGFDFS) (SEQ. ID NQ:20) which was diluted 2-fold in incubation buffer (50mM PBS-BTB, 8g NaCI / L, pH 7.4, 5% Liq-ll (cat. 11941836001 , Roche)) over 12-points with the last point remaining blank (only incubation buffer) and from a starting concentration of 300 ng / ml. After incubation, the 96-well plate was washed 5-times using washing buffer (25 mM Tris, 50 mM NaCI, 0.1 % (v / v) Tween-20, pH 7.2) and 20uL / well of the standard curve, controls and native samples of interest were added in duplicate. Immediately after, 100uL / well of incubation buffer containing 50 ng / ml of HRP-labelled mAb was added to the wells and incubated for 20 hours (±1 hour) at 4°C on a 300-rpm shaker. Next, the plate was washed 5-times with washing buffer. The colorimetric substrate 3, 3’, 5,5’- tetramethylbenzidine (TMB) One (cat. 4380H, Kem-En-Tec, Taastrup, Denmark) was added 100uL / well and incubated for 30 minutes at 18-22°C on a 300-rpm shaker. After and without washing, the colorimetric reaction was stopped using 100uL / well stopping buffer (0.1% sulphuric acid). The absorbance was measured at wavelength 450 nm with 650 nm as reference using a plate reader (VersaMax microplate reader, Molecular Devices, CA, USA). Data were recorded using SoftMax Pro version 7.1.0 software (Molecular Devices).

[0128] Technical evaluation

[0129] The antibody specificity to the target sequence was evaluated using synthetic peptides of selection and deselection peptides (elongated, truncated, nonsense), and further checked with a nonsense coating peptide in replacement to the original assay’s coating peptide. Specificity was determined as a percentage of signal inhibition over a two-fold dilution of the peptides included. The minimum required dilution (MRD) of serum samples and assay linearity was established using three independent runs of four human serum samples in two-fold dilution. The linearity of the samples was calculated as the recovery percentage of the sample diluted at the determined MRD, where the accepted range of recovery was 100±20%. The MRD was used throughout the rest of the technical evaluation. The limit of blank (LOB) was determined by finding the mean concentration of 60 samples of assay incubation buffer (i.e. blank). The mean IC50 (concentration of half maximal inhibition) and upper limit of quantification (IILOQ) were established with ten independent runs, where the IILOQ was deemed the highest standard point with a recovery percentage of 100±20% of the theoretical concentration. Five independent runs of 4 samples measured in triplicate at the lower end of the measurement range determined the lower limit of quantification (LLOQ) where the coefficient of variation percentage (CV%) was above the 20% criterion in at least one sample. A power regression model fitted the CV% of each sample with the measured concentration and the LLOQ was deemed as the lowest concentration where the CV% was equal to 20%. The inter- and intraassay variation was established by running 8 serum samples and 2 assay controls in duplicate in ten independent runs. The accuracy for detecting the analyte was tested by spiking 3 serum samples of known concentration with a 2-fold dilution of the 30 amino acid selection peptide and measuring the recovery percentage with the nominal concentration as reference. Furthermore, low analyte concentration serum samples were spiked with high analyte concentration serum samples, both as a 2-fold dilution, and the recovery percentage was determined as previously for peptide in serum. A percentage recovery of 100±20% was accepted.

[0130] The stability of the analyte in human serum sample was estimated by repeated freeze-thaw cycles (up to 5 cycles) of five samples and stress testing three samples at 4°C, 20°C and 37°C for 4 and 24 hours. Similarly, assay reagent stability was evaluated by measuring five fresh samples in stressed assay kits, which included kits of up to 3 freeze-thaw cycles and incubation of kits at 4°C, 20°C for 4 and 24 hours. The recovery percentage of the stressed samples were calculated from an unstressed reference sample, and the stressed kits against an unstressed reference assay. Analytical interference of haemoglobin, biotin and lipids in the assay was determined by spiking three serum samples with low / medium / high concentrations of biotin (5 / 40 / 100 ng / ml), low / high concentrations of haemoglobin (2.5 / 5.0 mg / ml) and low / high concentrations of lipids (1.5 / 5.0 mg / ml). The recovery percentage was calculated for the spiked samples against the known concentration of the same sample before spiking. All runs were completed with samples in double determination unless otherwise specified.

[0131] Cardiovascular Disease Studies

[0132] Study population The newly developed assay was used to quantify the matricryptin of interest, named C1SIG, in three cardiovascular studies. In all studies serum or plasma was collected based on standard operating procedures and stored at -80°C. Patients were treated in line with standard of care procedures.

[0133] The first study consisted of patients with heart failure with reduced ejection fraction (HFrEF) (n=50) and healthy control individuals (n=18), obtained from a collaborator. Simple clinical information was available including age and sex.

[0134] The second study included patients with suspected ST-elevated myocardial infarction (STEM I) (n=143), the most common type of heart attack, and plasma samples were taken on admission to hospital and at 6-12 hours post-admission. Extensive clinical data was available, including age, sex, blood pressure, smoking status, and prior disease diagnosis. This study is hereon referred to as STEM I pilot.

[0135] Like the second study, the third study (named PREDICT-CS) included patients that had been admitted to hospital with suspected STEMI (n=1452), but plasma was only withdrawn at admission. Clinical and survival data was available up to 1-year post-STEMI for this cohort. The PREDICT-CS study and the STEMI pilot study were provided by collaborators from Rigshospitalet (Copenhagen, Denmark) and Odense University Hospital (Odense, Denmark).

[0136] Biomarker measurements

[0137] Two collagen type I biomarkers were targeted, C1M and C1SIG, using competitive ELISAs namely the commercially available nordicCI M™ assay and the C1SIG immunoassay described above respectively. C1M is an MMP-generated internal neo-epitope fragment of collagen type I with the amino acid sequence GSPGKDGVRG (SEQ. ID NO:21), described in WO 2010 / 115749. C1SIG is a second MMP-generated neo-epitope fragment but shown to have signalling properties.

[0138] Statistical analysis

[0139] Patient demographics and biomarker data are presented as number with percentage if variable is categorical and mean with standard deviation (SD) if variable is continuous. Statistical analysis included Mann-Whitney U tests, Kruskal-Wallis tests, and Wilcoxon rank tests to determine differences in biomarker concentrations. Survival analysis was conducted using Kaplan-Meier survival curves, log-rank tests and univariate and multivariate cox proportional hazard regression analysis. Analyses were completed using GraphPad Prism software (ver. 10.3.0) and RStudio software (ver. 4.4.3). A p-value below 0.05 was considered statistically significant in all analysis.

[0140] Kidney Disease Studies

[0141] Study populations

[0142] The enzyme-linked immunosorbent assay described above was used to investigate changes of the targeted matricryptin (C1SIG) in individuals with various kidney diseases. The changes of C1 SIG and any associations of kidney function parameters in the kidney cohorts were evaluated. All serum / plasma and / or urine samples were collected under standard operating procedures and stored at -80°C until measurement. Patients were treated in line with standard of care procedures.

[0143] The first study consisted of patients with Alport syndrome (n=71), a genetically driven syndrome with kidney manifestations including nephritis, and healthy control individuals (n=32). The cohort was obtained from the University of Manchester (Manchester, United Kingdom), and simple clinical data were available, including age and sex.

[0144] The second study included patients with Immunoglobulin A (IgA) nephropathy (n=44), which is a kidney disease characterised by the accumulation of IgA antibodies in the glomeruli of the kidneys, leading to inflammation and reduced filtration. The cohort was obtained through collaboration with the General University Hospital in Prague (Prague, Czech Republic), and the clinical data provided included basic clinical variables, kidney function parameters and information on fibrosis of the kidney quantified by biopsy staining.

[0145] Biomarker measurements

[0146] The levels of C1SIG in human plasma / serum and / or urine were measured by the enzyme- linked immunosorbent assay previously above. The urine measurements were run in the assay using a dilution factor of 2 (1 unit urine + 1 unit assay buffer), and all other protocol steps remained the same. In these cohorts, 102 and 42 samples were measured in the Alport and IgAN cohorts, respectively.

[0147] Statistical analysis

[0148] In the Alport study, a Mann-Whitney test was used to compare healthy individuals to those with Alport syndrome (all) and a Kruskal-Wallis test was used to compare healthy individuals to those with Alport syndrome, split by uPCR above and below 30. In the IgAN study, the raw values of urinary C1SIG were adjusted for total protein concentration of the urine using urinary creatinine before statistical analysis and graphical plotting. Spearman's correlation analysis was completed on Iog2-transformed biomarker data, but the results were plotted linearly. The Kruskal-Wallis test was used to compare CKD stages, including Dunn’s multiple comparisons test.

[0149] Results

[0150] Technical assessment and characterization of the C1SIG immunoassay

[0151] The sequence for C1SIG was found to be unique within type-l collagen and showed no crossreactivity with other extracellular matrix proteins in protein blast analysis. The sequence was not conserved in mouse or rat species (Figure 1). The mAb generated towards the identified 01 SIG fragment and contained in the immunoassay has high affinity for the selection peptide (RTGDAGPVGP SEQ.ID N0:1) and lacks affinity for the truncated, elongated and nonsense peptides, indicating the high specificity of the mAb to the target sequence (Figure 2).

[0152] The technical evaluation of the immunoassay is summarised in table 1. The MRD of serum sample was found to be 1+0 (undiluted sample) and linearity testing shows accepted dilution at 1+1 (114%) and within the acceptable mean recovery percentage range (100 ± 20%). The intra- and inter-assay variation was low at 7.6% and 11.3% respectively, with a single sample in each test outside the accepted 15% variation. The LOB was determined at 2.3 ng / ml and the analytical measurement range was found to be 4.7 - 300 ng / ml (LLOQ - IILOQ). The IC50 was established as 37.06 ng / ml, marking the concentration where there is half of maximal inhibitory effect of the antibody. Analyte recovery demonstrated good accuracy of the assay, when adding synthetic peptide into serum (99.9%) and serum into serum (91.5%). Lipid and biotin interference was not detected, however a small amount of interference with haemoglobin was observed. Serum samples recover after stressing up to 5 freeze-thaw cycles (100.4%) and at 37°C for 24 hours (99.4%). The immunoassay was deemed stable after 3 freeze-thaw cycles (104.1 %) and after stressing for 24 hours at 20°C (97.9%).

[0153] Table 1: Technical summary of the C1SIG assay.

[0154]

[0155] Biological evaluation of the immunoassay

[0156] Cardiovascular Disease Studies Serum C1 SIG levels were increased in patients with HFrEF

[0157] The HFrEF cohort included patients who had previously suffered a myocardial infarction (Ml) and patients with dilated cardiomyopathy (DCM), both of which precede HFrEF development. This cohort was predominantly male (74%) and had an average age of 66.7 years, whilst the healthy control cohort was 33% male and 72% of the individuals were above 60 years (Table 2). C1SIG biomarker levels were significantly increased in individuals with HFrEF compared to healthy controls (Fig. 3, p < 0.0001). When splitting patients into prior disease indication, both Ml and DCM patients had increased C1SIG levels compared to healthy controls (Ctrl vs

[0158] Ml: p < 0.0001 , Ctrl vs DCM: p = 0.0041), but C1SIG was unable to distinguish between Ml and DCM patients.

[0159] Table 2: Patients demographics of HFrEF cohort and healthy controls

[0160] HFrEF (n = 51) Healthy controls (n = 18)

[0161] Sex (male) 37 (74%) 6 (33%)

[0162] Age (years) 66.7 (13.0)

[0163] Age - 40-49 years: 5 (28%)

[0164] >60 years: 13 (72%)

[0165] C1 SIG (ng / ml) 581 .0 (196.8) 537.0 (224.6)

[0166] Binary variables are presented as n, percentage (%), and continuous variables are presented as mean, standard deviation (SD).

[0167] The STEMI pilot study (n=143) was conducted to assess whether levels of C1SIG increased immediately after STEMI occurrence. The first plasma sample was taken on admission to hospital with suspected STEMI and the second plasma sample was taken 6-12 hours after admission. This cohort was predominantly male (76.2%) and a mean age of 63.1 years (Table 3). From admission to 6-12 hours post-admission, C1SIG and C1 M levels were significantly increased (Fig 4, both: p < 0.0001).

[0168] Table 3: Patient demographics of STEMI pilot cohort. STEMI pilot (n = 143)

[0169] Sex (male) 109 (76.2%)

[0170] Age (years) 63.1 (13.8)

[0171] Systolic blood pressure (mmHg) 127 (23.7)

[0172] Diastolic blood pressure (mmHg) 73.5 (12.9)

[0173] Hypercholesterolemic 53 (37.1%)

[0174] Diabetic 23 (16.1%)

[0175] Smoker 100 (69.9%)

[0176] C1 SIG at admission (ng / ml) 31 .4 (8.3)

[0177] C1 M at admission (ng / ml) 25.1 (16.3)

[0178] Binary variables are presented as n, percentage (%), and continuous variables are presented as mean, standard deviation (SD).

[0179] Plasma C1SIG levels are associated with cardiovascular outcomes

[0180] In the large PREDICT-CS cohort (n=1 ,452), plasma was taken on admission to hospital with suspected STEM I and their survival outcome was measured after 30-days and 1-year post- admission. The survival rate after 1 year was 8.5%. Commonly in cardiovascular cohorts, the cohort was mainly male (73%), many patients were smokers (72%), some were hypercholesterolemic (34%) and the mean age was 63.5 years (Table 4). Noticeably, patients who died within a year of suffering a STEMI were older and had increased C1SIG and C1 M levels (Table 4). When C1SIG is split by median, high C1SIG is highly associated to all-cause mortality within 30-days and 1-year post-MI in Kaplan-Meier survival analysis (Fig 5, both: p < 0.0001). Cox proportional hazard regression demonstrates the predictive ability of C1SIG and C1 M in predicting all-cause mortality after 1 year in univariate and multivariate analysis. When adjusted for multiple confounders based on the Framingham Score (Model 1), including age, sex, systolic and diastolic blood pressure, hypercholesterolemia status, diabetes and smoking status, C1SIG and C1M remained predictive of all-cause mortality. However, when adding clinically relevant biomarkers (mid-regional pro-adrenomedullin, copeptin, pro-atrial natriuretic peptide, ST2 and C-reactive protein) to Model 1, only C1SIG retained its significant predictive ability (Fig 6).

[0181] Table 4: Patient demographics of PREDICT-CS cohort.

[0182] Survived (n = 1329) Mortality (n = 123) Overall (n = 1452)

[0183] Sex (male) 980 (73.7%) 75 (61.0%) 1055 (72.7%)

[0184] Age (years) 62.7 (12.6) 71.8 (12.7) 63.5 (12.8)

[0185] Systolic blood 129 (26.5) 116 (29.3) 128 (26.9) pressure (mmHg)

[0186] Diastolic blood 74.3 (14.8) 68.4 (17.8) 73.8 (15.2) pressure (mmHg)

[0187] Hypercholesterolemic 448 (33.7%) 51 (41.5%) 499 (34.4%)

[0188] Diabetic 166 (12.5%) 22 (17.9%) 188 (12.9%)

[0189] Smoker 967 (72.8%) 78 (63.4%) 1045 (72.0%)

[0190] C1SIG (ng / ml) 31.6 (11.9) 35.9 (13.4) 32.0 (12.1)

[0191] C1 M (ng / ml) 24.8 (16.6) 36.7 (30.4) 25.8 (18.5)

[0192] Binary variables are presented as n, percentage (%), and continuous variables are presented as mean, standard deviation (SD).

[0193] Kidney Disease Studies

[0194] Serum C1SIG levels are increased in Alport syndrome patients

[0195] In Alport syndrome individuals, serum 01 SIG levels were increased compared to healthy controls (p < 0.0001) (Figure 7A). When the Alport syndrome individuals were split by urinary protein-to-creatinine ratio (uPCR) above and below 30, the serum C1 SIG levels remained significantly higher compared to healthy controls (Healthy vs uPCR < 30, p = 0.0003; healthy vs uPCR > 30, p = 0.0111) but did not separate the individuals with more severe kidney dysfunction (Figure 7B). Urinary C1SIG levels are significantly associated with kidney function and fibrosis score in IgAN individuals

[0196] Urinary levels of 01 SIG adjusted for urinary creatinine (uC1SIG / uCr) are highly correlated with estimated glomerular filtration rate (eGFR), a key clinical parameter for kidney function (r = 0.78, p < 0.0001, Figure 8A), and fibrosis score (%) (r = -0.50, p = 0.0008, Figure 8B). The positive correlation to eGFR and negative correlation to fibrosis score indicate that low levels of urinary 01 SIG show a more severe kidney phenotype. This is supported by the reduction in uC1SIG / uCr with increasing stage of chronic kidney disease in this IgAN cohort (Figure 9).

[0197] Conclusion

[0198] A novel immunoassay was developed and characterised to detect C1 SIG, a biologically active fragment of type I collagen cleaved by MMP-2 and MMP-9. The ELISA was determined as technically robust and highly specific to the fragment of interest.

[0199] C1SIG was increased in HFrEF patients compared to healthy controls. The C1SIG biomarker increased immediately post-MI and was associated to short-term and long-term mortality in STEM I patients. C1 SIG was highly predictive of all-cause mortality within a year of suffering an Ml in univariate and multivariate models.

[0200] Higher levels of serum 01 SIG are found in individuals with Alport syndrome, and urinary 01 SIG levels are strongly correlated with kidney function in IgA nephropathy. Therefore, 01 SIG is a serological and urinary biomarker in kidney disease, specifically Alport Syndrome and IgA nephropathy. Urinary C1 SIG can also be used as a marker for increasing severity of chronic kidney disease.

[0201] In this specification, unless expressly otherwise indicated, the word ‘or’ is used in the sense of an operator that returns a true value when either or both of the stated conditions is met, as opposed to the operator ‘exclusive or’ which requires that only one of the conditions is met. The word ‘comprising’ is used to mean ‘including or consisting of’. All prior teachings acknowledged above are hereby incorporated by reference. No acknowledgement of any prior published document herein should be taken to be an admission or representation that the teaching thereof was common general knowledge in Australia or elsewhere at the date hereof.

[0202] References:

[0203] 1. Sharma, U. et al. Structural basis of homo- and heterotrimerization of collagen I. Nat Commun 8, 14671 (2017). 2. Singh, D., Rai, V. & K Agrawal, D. Regulation of Collagen I and Collagen III in Tissue Injury and Regeneration. Cardiol Cardiovasc Med 07, (2023).

[0204] 3. Dupuy, A. M. et al. Exploring collagen remodeling and regulation as prognosis biomarkers in stable heart failure. Clinica Chimica Acta 490, 167-171 (2019).

[0205] 4. Zamilpa, R. et al. Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction. Proteomics 10, 2214-2223 (2010).

[0206] 5. Lindsey, M. L. et al. A Novel Collagen Matricryptin Reduces Left Ventricular Dilation Post-Myocardial Infarction by Promoting Scar Formation and Angiogenesis. J Am Coll Cardiol 66, 1364-1374 (2015).

[0207] 6. Pozzo, C. F. S. D., Sielski, M. S., de Campos Vidal, B., Werneck, C. C. & Vicente, C. P. A collagen I derived matricryptin increases aorta vascular wall remodeling after induced thrombosis in mouse. Thromb Res 209, 59-68 (2022).

[0208] 7. Zamilpa, R. et al. Proteomic analysis identifies in vivo candidate matrix metalloproteinase-9 substrates in the left ventricle post-myocardial infarction. Proteomics 10, 2214-2223 (2010).

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Claims

Claims1. A monoclonal antibody that specifically binds to the N- terminal neoepitope derived from the collagen type-l alpha-1 chain generated by treatment with MMP- 2, and / or MM P-9.

2. The monoclonal antibody of claim 1, wherein the antibody specifically binds to the N-terminal epitope comprising or consisting of the amino acid sequence RTGDAGPVGP.

3. The monoclonal antibody of claim 1 or claim 2, wherein monoclonal antibody does not bind with the same affinity to an elongated version of said N-terminus amino acid sequence which is GRTGDAGPVGP, and / or to a truncated version of said N-terminus amino acid sequence which is TGDAGPVGP.

4. The monoclonal antibody of any preceding claim, wherein the monoclonal antibody is raised against a synthetic peptide having the N-terminus amino acid sequence RTGDAGPVGP.

5. A method of immunoassay for detecting in a sample a N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain said method comprising:(i) contacting a sample comprising said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody of any one of claims 1 to 4; and(ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample.

6. A method of immunoassay for detecting and / or monitoring a disease in a patient, the method comprising:(i) contacting a sample comprising said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody of any one of claims 1 to 4,(ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample,(iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value.

7. A method of claim 6 wherein said immunoassay method further comprises(iv) administering to the patient a therapy for the treatment of the disease if it is determined in step (iii) that the patient has said disease.

8. A method of treating a disease in a patient in need thereof, the method comprising:(i) contacting a sample comprising an N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody of any one of claims 1 to 4,(ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample,(iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known disease severity and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value;(iv) administering to the patient a therapy for the treatment of the disease if it is determined in step (iii) that the patient has said disease.

9. The method of any one of claims 6 to 8 wherein the disease is associated with the cardiovascular system including the heart.

10. A method of claim 9 wherein the disease associated with or the cardiovascular system including the heart is atherosclerosis or heart failure.

11. The method of any one of claims 6 to 8 wherein the disease is associated with the kidney.

12. A method of claim 12 wherein the disease associated with the kidney is nephritis, Alport syndrome, or Immunoglobulin A (IgA) nephropathy.

13. A method of identifying a patient at increased risk of death due to a cardiovascular disease or event, the method comprising:(i) contacting a sample comprising said N-terminal amino acid sequence of the neoepitope derived from the collagen type-l alpha-1 chain with a monoclonal antibody of any one of claims 1 to 4,(ii) detecting and determining the amount of binding between said monoclonal antibody and peptides in the sample,(iii) correlating said amount of binding with values associated with normal healthy subjects and / or values associated with known cardiovascular disease risk and / or values obtained from said patient at a previous time point and / or with a predetermined cut-off value; and optionally(iv) administering to the patient a therapy for the prevention or treatment of said cardiovascular disease if it is determined in step (a) that the patient has increased risk of said cardiovascular disease.

14. A method of any one of claims 5 to 13 wherein the sample is a biofluid sample.

15. A method of claim 14 wherein the biofluid sample is a urine or a blood-based sample.

16. A method of claim 9 or claim 10, wherein the sample is a blood-based sample.

17. A method of claim 11 or claim 12, wherein the sample is a urine sample.

18. A method of any one of claims 5 to 12 wherein said immunoassay is a competition assay.

19. A method of any one of claims 5 to 13 wherein said immunoassay is an ELISA.