Assays for assessing heart failure

Abstract

An immunoassay method for detecting and / or monitoring cardiovascular disease in a patient and / or for assessing the likelihood or severity of cardiovascular disease in a patient, comprising contacting a biological fluid sample from the patient with a monoclonal antibody that specifically binds to a C-terminal epitope of the C5 domain of the α3 chain of type VI collagen and / or contacting a biological fluid sample from the patient with a monoclonal antibody that specifically binds to a C-terminal neo-epitope of the N-terminal propeptide of type III collagen.

Description

[Technical field]

[0001] The present invention relates to an immunoassay for detecting and / or monitoring cardiovascular disease in a patient and / or for assessing the likelihood or severity of cardiovascular disease in a patient. The cardiovascular disease may in particular be heart failure, in particular heart failure with preserved ejection fraction. The immunoassay may be for assessing the likelihood of adverse outcomes of cardiovascular disease. The patient may be a patient undergoing treatment for cardiovascular disease, such as a patient undergoing treatment with an aldosterone antagonist. The present invention also relates to an immunoassay for identifying patients suitable for treatment with an aldosterone antagonist. [Background technology]

[0002] The disease burden of heart failure (HF) has increased dramatically in recent years (citations 1, 2). Approximately half of HF cases are secondary to HF with preserved ejection fraction (HFpEF), which is expected to represent a larger proportion of the total HF disease burden as the population ages (citation 3). Despite numerous phase III randomized controlled trials over recent decades, evidence remains to be provided as to which pharmacological interventions can unequivocally benefit this patient population.

[0003] The heterogeneity of the HFpEF syndrome has been identified as a significant barrier to demonstrating the efficacy of candidate pharmacological interventions. The heterogeneous nature of HFpEF may adversely affect the average response to pharmacological treatments tested in clinical trials due to the differing degrees of contribution from various pathophysiological processes. Thus, the availability of simple, noninvasive biomarkers that can easily identify underlying correlates of specific biological processes that can be targeted using pharmacological interventions presents a promising approach to augment our clinical and therapeutic approaches to HFpEF (citation 4).

[0004] The heterogeneity of HFpEF also has important implications for the differential outcomes of individual patients. The ability to more effectively risk grade HFpEF patients is greatly needed. Novel risk grading markers would be of great value not only to improve our ability to prognose HFpEF patients in clinical practice, but also to inform the enrollment of high-risk individuals in future trials.

[0005] Myocardial fibrosis is believed to play a role in the pathophysiology of HFpEF (citations 5, 6). Increased fibrosis results from excessive formation associated with collagen degradation, ultimately increasing interstitial collagen accumulation in the interstitium. Increased myocardial extracellular matrix accumulation in HFpEF has been demonstrated in autopsy specimens and in vivo studies (citations 6-8), and increased myocardial extracellular matrix accumulation in this condition has been shown to correlate with left ventricular passive stiffness and diastolic dysfunction (citations 5, 8). Myocardial fibrosis may also contribute to reduced coronary flow reserve (citations 6, 9), ventricular dyssynchrony and arrhythmia propensity (citations 10, 11). Given the role of myocardial fibrosis in HFpEF, it would be of high value as a simple fibrosis biomarker that reflects the dynamic process underlying fibrosis progression or regression (citation 10).

[0006] Extracellular volume fraction (ECVF), an index of myocardial fibrosis measured by cardiac magnetic resonance imaging, has been reported to predict adverse outcomes (citations 12, 13) or risk of HFpEF (citation 14) in patients with HFpEF. Although MRI promises to play an important role in the assessment of myocardial fibrosis in preclinical studies, early phase investigations in humans, and in some clinical settings, its cost and availability will likely limit or prevent its use in global Phase III trials and clinical implementation. Furthermore, many patients with HFpEF are not candidates for ECVF measurement due to claustrophobia or advanced renal disease. This leaves the search for widespread biomarkers of tissue fibrosis as an area of ​​great challenge.

[0007] However, the search for suitable biomarkers for HFpEF is complicated by the fact that "fibrosis is not just fibrosis," and that ECM remodeling of different compartments and collagen types may have different biological and prognostic relevance (reference 15). For example, differential associations of collagen neoepitope fragments with liver fibrosis have been reported in chronic hepatitis B versus C. Also, while myocardial fibrosis is thought to be important in HFpEF, extracardiac fibrosis may also play an important role. For example, fibro-fatty infiltration of skeletal muscle has been reported in HFpEF (reference 16). Similarly, fibrosis also occurs in the arterial wall, kidney, and liver dysfunction, all of which may contribute to adverse outcomes in this population.

[0008] There is also a need for biomarkers of collagen turnover that would allow identification of individuals who would benefit from aldosterone antagonists such as spironolactone. This concept has been evaluated in previous studies in other populations (HF with reduced ejection fraction and / or HF after myocardial infarction) (citation 17), but only one study has investigated this issue to date in HFpEF (citation 18). In this previous study, the ratio of serum carboxy-terminal telopeptide of collagen type I to serum matrix metalloproteinase-1 (CITP:MMP-1) appeared to identify patients who showed a reduced echocardiographic mitral inflow to annular tissue velocity ratio (a marker of left ventricular filling pressure) after 12 weeks of treatment with spirolactam (citation 18). However, this study did not examine clinical events.

[0009] Type III collagen is expressed in most collagen-I-containing tissues other than bone and is an important component of connective tissues, muscle tissues, and skin. Collagen type III is essential for collagen type I fibril formation in the cardiovascular system and other organs. During fibril assembly, the N-terminal propeptide of type III procollagen (which consists of three identical α chains with a total molecular weight of 42 kDa) is cleaved by specific N-proteases prior to incorporation of the mature collagen into the extracellular matrix (ECM). The cleaved propeptide may be retained in the ECM or released into the circulation. However, cleavage of the propeptide may be incomplete, leaving the propeptide bound to the molecule. This leads to the formation of thin fibrils with abnormal crosslinks, causing abnormal molecules and a tendency to rapid turnover. PIIINP is the N-terminal propeptide of collagen type III and is removed during synthesis of mature type III collagen. Therefore, the level of the N-terminal propeptide of collagen type III (PIIINP) in a suitable sample can be a marker for the formation and / or degradation of collagen type III.

[0010] PRO-C3 is a biomarker for the formation of collagen type III and contains a C-terminal neo-epitope of the N-terminal propeptide of collagen type III (i.e., the C-terminal neo-epitope of PIIINP), which is formed after cleavage of the propeptide from the pro-collagen by ADAMTS-2. The PRO-C3 biomarker and PRO-C3 assay (specifically, the PRO-C3 ELISA) are described in WO2014 / 170312. The assay utilizes a monoclonal antibody that specifically binds to the C-terminal 10 amino acid sequence of PIIINP to target the free C-terminus of the N-terminal pro-peptide formed after cleavage (reference 19). PRO-C3 is a well-explored biomarker for liver fibrosis and has been associated with both fibrotic burden in the liver and fibrotic progression and adverse outcomes in patients with different liver manifestations (references 20-26).

[0011] Collagen type VI is a unique extracellular collagen that can form independent microfibril networks in the basement membrane of cells. It can interact with other matrix proteins, including collagen, biglycan, and proteoglycans. In muscle, collagen type VI becomes part of the sarcolemma and is involved in anchoring muscle fibers to the intramuscular extracellular matrix, and is involved in force transmission. Furthermore, mutations in collagen type VI can cause Bethlem myopathy and Ullrich congenital muscular dystrophy. It has been reported that the C-terminal amino acid sequence of this collagen type VI α3 chain is cleaved from mature type VI microfibrils after secretion. However, collagen type VI is not only involved in muscle and muscle loss.

[0012] Microfilamentous interstitial collagen type VI, a triple-helical molecule composed of constituent chains α1(VI), α2(VI) and α3(VI), is expressed in most connective tissues and is prominent in adipose tissue, where it anchors cells through interconnections with other ECM proteins. During the formation of microfilaments, the triple-helical core of collagen type VI is proteolytically released from the pro-peptide, and cleavage of the C-terminal pro-peptide of the α3(VI) chain generates the endotrophin, an adipokine.

[0013] PRO-C6 is a biomarker for collagen VI formation and endotrophin release, and contains a C-terminal epitope of the C5 domain of the α3 chain of collagen VI that is cleaved when the new collagen VI molecule is assembled into the extracellular matrix, which is also the C-terminal epitope of the biologically active fragment endotrophin. The PRO-C6 biomarker and PRO-C6 assay (specifically, the PRO-C6 ELISA) are described in WO 2016 / 156526. The assay utilizes a monoclonal antibody that specifically binds to the C-terminal 10 amino acid sequence of the C5 domain of the α3 chain of collagen VI. The role of endotrophin as a profibrotic, proinflammatory and protumorigenic molecule has been observed in preclinical models of breast cancer and liver fibrosis (citations 27-31). PRO-C6 has been established as a predictive biomarker for mortality and disease progression in patients with chronic kidney disease and diabetic kidney disease (citations 32-34), and as a predictive marker for responsiveness to glucose-lowering therapy in diabetic patients (citation 35).

[0014] Collagen IV is a type of collagen found primarily in the basement membrane of blood vessels. The vascular wall is composed of two major types of extracellular matrix: the basement membrane and the interstitial matrix. The basement membrane is composed of two independent polymer networks: one composed of type IV collagen and one composed of laminin, in addition to proteoglycans and various other glycoproteins (references 36, 37). The collagen IV network in the basement membrane is highly cross-linked and is thought to maintain mechanical stability. Basement membranes also contain matrix metalloproteases (MMPs), a widespread group of broad-spectrum proteases that function in the degradation and reconstruction of the basement membrane. By degrading the basement membrane scaffold, MMPs can release cryptic fragments that have signaling functions. For example, cleavage of collagen IV by MMP9 exposes cryptic sites involved in angiogenesis (reference 37).

[0015] C4M is a biomarker for MMP-mediated degradation of type IV collagen, containing an N-terminal neo-epitope of a fragment of type IV collagen formed by cleavage of the α1(IV) chain by MMP12. The C4M biomarker, and the C4M assay (specifically, the C4M ELISA) have been previously described (reference 38). The assay utilizes a monoclonal antibody that specifically binds to the N-terminal neo-epitope. Summary of the Invention

[0016] We investigated the potential of PRO-C6 and PRO-C3 as biomarkers for cardiovascular disease. We investigated the levels of PRO-C6 and PRO-C3 in the circulation of a population of patients with heart failure with preserved ejection fraction (HFpEF). We analyzed baseline PRO-C3 and PRO-C6 levels, investigated the relationship between biomarkers and outcomes, and found that both PRO-C3 and PRO-C6 are valid diagnostic and predictive biomarkers for heart failure.

[0017] In addition, we investigated the potential of C4M as a biomarker for patients with cardiovascular disease who may respond to treatment with aldosterone antagonists. We examined C4M levels in patients with HFpEF and found that C4M identifies patients who are more likely to respond well to treatment with the aldosterone antagonist spironolactone.

[0018] Thus, in a first aspect, the present invention provides an immunoassay method for detecting and / or monitoring cardiovascular disease in a patient and / or for assessing the likelihood or severity of cardiovascular disease in a patient, comprising: (i) contacting a sample of a biological fluid from a patient with a monoclonal antibody that specifically binds to a C-terminal epitope of the C5 domain of the α3 chain of type VI collagen, and / or contacting a sample of a biological fluid from a patient with a monoclonal antibody that specifically binds to a C-terminal neo-epitope of the N-terminal propeptide of type III collagen; (ii) detecting and determining the amount of binding between each monoclonal antibody used in step (i) and the peptide in the sample; and (iii) correlating the amount of binding of each monoclonal antibody determined in step (ii) with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the patient at previous time points and / or with a predefined cut-off value.

[0019] The immunoassay may be, but is not limited to, a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA). Such assays are techniques known to those skilled in the art.

[0020] The cardiovascular disease may in certain embodiments be heart failure, in particular heart failure with preserved ejection fraction (HFpEF). The method may, in one embodiment, be a method for assessing the severity of cardiovascular disease in a patient, including assessing the patient's likelihood of death and / or hospitalization as a result of cardiovascular disease and / or a composite state of adverse cardiovascular events. The patient may be, for example, a patient being treated for cardiovascular disease. The patient biological fluid sample may be, but is not limited to, blood, serum, plasma, urine or amniotic fluid. Preferably, the biological fluid is serum or plasma.

[0021] The term "monoclonal antibody" as used herein refers to both whole antibodies and fragments thereof, such as Fab fragments, F(ab')2 fragments, single chain Fv fragments, or other such fragments known to those skilled in the art, that retain the binding specificity of the whole antibody. As is well known, whole antibodies typically have a "Y-shaped" structure in which two identical polypeptide chains are paired, each member of the pair consisting of one "light" chain and one "heavy" chain. The N-terminal regions of each of the light and heavy chains comprise the variable region, while the C-terminal portions of each of the heavy and light chains constitute the constant region. The variable regions contain three complementarity determining regions (CDRs), which are primarily responsible for antigen recognition. The constant regions allow the antibody to recruit cells and molecules of the immune system. An antibody fragment that retains the binding specificity comprises at least the CDRs and a sufficient portion of the remainder of the variable region to retain this binding specificity.

[0022] In the method of the present invention, monoclonal antibodies containing any constant region known in the art can be used. Human constant light chains are classified as kappa and lambda light chains. Constant heavy chains are classified as mu, delta, gamma, alpha, or epsilon, which define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. The IgG isotype has several subclasses, including, but not limited to, IgG1, IgG2, IgG3, and IgG4. The monoclonal antibody may preferably belong to the IgG isotype, including any one of IgG1, IgG2, IgG3, or IgG4.

[0023] The CDRs of the antibody can be determined using methods known in the art, such as those described by Kabat et al. The antibody can be generated from a B cell clone as described in those examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG or IgA isotype or human IgG1, IgG2, IgG3 or IgG4 subclasses. The amino acid sequence of the generated antibody can be determined using standard techniques. For example, RNA can be isolated from cells and used to generate cDNA by reverse transcription. The cDNA is then PCRed with primers that amplify the heavy and light chains of the antibody. For example, a primer specific to the leader sequence for all VH (variable heavy) sequences can be used along with a primer that binds to a sequence located in the constant region of a predetermined isotype. The light chain can be amplified using a primer that binds to the 3' end of the kappa or lambda chain along with a primer that anneals to the leader sequence of Vkappa or Vlambda. Full-length heavy and light chains can be generated and sequenced.

[0024] In some embodiments of the method according to the first aspect of the invention, the biological fluid sample is contacted with a monoclonal antibody that specifically binds to a C-terminal epitope of the C5 domain of the α3 chain of collagen type VI. Preferably, the monoclonal antibody specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO:1) (also referred to herein as the "PRO-C6 sequence" or simply "PRO-C6"). Preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is KPGVISVMGTA (SEQ ID NO:2) or a truncation of the C-terminal amino acid sequence that is KPGVISVMG (SEQ ID NO:3).

[0025] Preferably, the ratio of the affinity of the antibody for the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO:1) to the extended C-terminal amino acid sequence KPGVISVMGTA (SEQ ID NO:2) and / or the truncated C-terminal amino acid sequence KPGVISVMG (SEQ ID NO:3) is at least 10 to 1, more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0026] The term "C-terminus" as used herein means a C-terminal peptide sequence at the end of a polypeptide, i.e., at the C-terminus of a polypeptide, and should not be construed as referring to its general orientation.

[0027] Monoclonal antibodies that specifically bind to PRO-C6 sequences may preferably comprise one or more complementarity determining regions (CDRs) selected from the following: CDR-L1: RSSQRIVHSNGITFLE (SEQ ID NO: 4) CDR-L2: RVSNRFS (SEQ ID NO: 5) CDR-L3: FQGSHVPLT (SEQ ID NO: 6) CDR-H1: DFNMN (SEQ ID NO: 7) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID NO: 8) CDR-H3: WGNGKNS (SEQ ID NO: 9)

[0028] Preferably, the antibody comprises at least 2, 3, 4, 5 or 6 of the CDR sequences listed above.

[0029] Preferably, the light chain variable region of the monoclonal antibody comprises the following CDR sequences: CDR-L1: RSSQRIVHSNGITFLE (SEQ ID NO: 4) CDR-L2: RVSNRFS (SEQ ID NO: 5), and CDR-L3: FQGSHVPLT (SEQ ID NO: 6)

[0030] Preferably, the light chain of the monoclonal antibody comprises framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences present between the CDRs in the light chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0031] [ka]

[0032] Preferably, the heavy chain variable region of the monoclonal antibody comprises the following CDR sequences: CDR-H1: DFNMN (SEQ ID NO: 7) CDR-H2: AINPHNGATSYNQKFSG (SEQ ID NO: 8), and CDR-H3: WGNGKNS (SEQ ID NO: 9)

[0033] Preferably, the heavy chain of the monoclonal antibody comprises framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences present between the CDRs in the following heavy chain sequence (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0034] [ka]

[0035] As used herein, the amino acid sequences of the framework present between the CDRs of an antibody are substantially identical or substantially similar to the amino acid sequences of the framework present between the CDRs of another antibody if they have at least 70%, 80%, 90%, or at least 95% similarity or identity to the amino acid sequences of the framework present between the CDRs of the other antibody. The similar or identical amino acids may be contiguous or non-contiguous.

[0036] The framework sequence may contain one or more amino acid substitutions, insertions and / or deletions. The amino acid substitutions may be conservative, meaning that the substituted amino acid has similar chemical properties as the original amino acid. A person skilled in the art will understand which amino acids share similar chemical properties. For example, the following groups of amino acids share similar chemical properties in terms of size, charge, polarity, etc.: Group 1 Ala, Ser, Thr, Pro, Gly; Group 2 Asp, Asn, Glu, Gln; Group 3 His, Arg, Lys; Group 4 Met, Leu, Ile, Val, Cys; Group 5 Phe, Thy, Trp.

[0037] Programs such as the CLUSTAL program can be used to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in any appropriate sequence. For optimal alignment, it is possible to calculate the amino acid identity or similarity (conservation of amino acid type in addition to identity). Programs such as BLASTx align the longest stretches of similar sequences and assign values ​​to the matching positions. In this way, a comparison can be obtained and several regions of similarity are found, each with a different score. It is envisaged to use these two types of analysis in the present invention. The identity or similarity is preferably calculated over the entire length of the framework sequences.

[0038] In certain preferred embodiments, a monoclonal antibody that specifically binds to the PRO-C6 sequence has the light chain variable region sequence:

[0039] [ka]

[0040] and / or a heavy chain variable region sequence:

[0041] [ka]

[0042] (CDRs in bold and underlined; framework sequences in italics)

[0043] In some embodiments of the method of the first aspect of the invention, the biological fluid sample is contacted with a monoclonal antibody that specifically binds to a C-terminal neo-epitope of the N-terminal propeptide of collagen type III. Preferably, the monoclonal antibody specifically binds to the C-terminal amino acid sequence CPTGPQNYSP (SEQ ID NO: 14) (also referred to herein as the "PRO-C3 sequence" or simply "PRO-C3"). More preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is CPTGPQNYSPQ (SEQ ID NO: 15) or a truncation of the C-terminal amino acid sequence that is CPTGPQNYS (SEQ ID NO: 16).

[0044] Preferably, the ratio of the affinity of the antibody for the C-terminal amino acid sequence CPTGPQNYSP (SEQ ID NO: 14) to the extended C-terminal amino acid sequence CPTGPQNYSPQ (SEQ ID NO: 15) and / or the truncated C-terminal amino acid sequence CPTGPQNYS (SEQ ID NO: 16) is at least 10 to 1, more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0045] Monoclonal antibodies that specifically bind to PRO-C3 sequences may preferably comprise one or more complementarity determining regions (CDRs) selected from the following: CDR-L1: RSSQNIVYSNGDTYFE (SEQ ID NO: 17) CDR-L2: KVSQRFS (SEQ ID NO: 18) CDR-L3: FQGAHDPPA (SEQ ID NO: 19) CDR-H1: GYTFINYVIH (SEQ ID NO: 20) CDR-H2: YMNPYNDVPKNNAKFRG (SEQ ID NO: 21) CDR-H3: GGFFGPLSY (SEQ ID NO: 22)

[0046] Preferably, the antibody comprises at least 2, 3, 4, 5 or 6 of the CDR sequences listed above.

[0047] Preferably, the light chain variable region of the monoclonal antibody comprises the following CDR sequences: CDR-L1: RSSQNIVYSNGDTYFE (SEQ ID NO: 17) CDR-L2: KVSQRFS (SEQ ID NO: 18), and CDR-L3: FQGAHDPPA (SEQ ID NO: 19)

[0048] Preferably, the light chain of the monoclonal antibody comprises framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences present between the CDRs in the light chain sequence below (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0049] [ka]

[0050] Preferably, the heavy chain variable region of the monoclonal antibody comprises the following CDR sequences: CDR-H1: GYTFINYVIH (SEQ ID NO: 20) CDR-H2: YMNPYNDVPKNNAKFRG (SEQ ID NO: 21), and CDR-H3: GGFFGPLSY (SEQ ID NO: 22)

[0051] Preferably, the heavy chain of the monoclonal antibody comprises framework sequences between the CDRs that are substantially identical to or substantially similar to the framework sequences present between the CDRs in the following heavy chain sequence (CDRs are shown in bold and underlined, framework sequences are shown in italics):

[0052] [ka]

[0053] In some embodiments, a monoclonal antibody that specifically binds to a PRO-C3 sequence has the following light chain variable region sequence:

[0054] [ka]

[0055] and / or the heavy chain variable region sequence:

[0056] [ka]

[0057] (CDRs in bold and underlined; framework sequences in italics)

[0058] In some embodiments of the method according to the first aspect of the present invention, the binding amount of a monoclonal antibody specific for a C-terminal epitope of the C5 domain of the α3 chain of collagen type VI and / or the binding amount of a monoclonal antibody specific for a C-terminal neo-epitope of the N-terminal propeptide of collagen type III (PIIINP) is correlated with values ​​associated with normal healthy individuals and / or with values ​​associated with the severity of a known disease and / or with values ​​obtained from the patient at a previous time point.

[0059] As used herein, the term "values ​​associated with normal healthy individuals and / or values ​​associated with known disease severity" refers to standardized amounts determined by the above methods for subjects considered to be healthy, i.e., free of cardiovascular disease, and / or standardized amounts determined by the above methods for subjects known to have cardiovascular disease with known severity.

[0060] In some embodiments of the method according to the first aspect of the present invention, the binding amount of a monoclonal antibody specific for a C-terminal epitope in the C5 domain of the α3 chain of collagen type VI and / or the binding amount of a monoclonal antibody specific for a C-terminal neo-epitope in the N-terminal propeptide of collagen type III (PIIINP) is compared to one or more predetermined cut-off values.

[0061] As used herein, a "cutoff value" refers to an amount of binding that is statistically determined to indicate a high likelihood of cardiovascular disease in a patient, or cardiovascular disease of a particular level of severity, in this sense, the measured value of biomarker binding in a patient sample is equal to or greater than the statistical cutoff value, which corresponds to at least a 70% probability, preferably at least an 80% probability, preferably at least an 85% probability, more preferably at least a 90% probability, and most preferably at least a 95% probability, of the presence or possibility of cardiovascular disease, or of the severity of the disease of a particular level.

[0062] The predetermined cut-off value for the binding amount of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the α3 chain of collagen type VI is preferably 11.0 ng / mL or more, more preferably 16.0 ng / mL or more. The predetermined cut-off value for the binding amount of the monoclonal antibody specific for the C-terminal neo-epitope of PIIINP is preferably 10.0 ng / mL or more, more preferably 14.0 ng / mL or more. In this regard, it has been found by using a combination of various statistical analyses that when the binding amount of the monoclonal antibody specific for the C-terminal epitope of the C5 domain of the α3 chain of collagen type VI is measured to be at least 11 ng / mL or more, particularly at least 16.0 ng / mL or more, it can be determined that there is a cardiovascular disease and / or the risk of hospitalization or death is increased. By setting the statistical cut-off value to 11.0 ng / mL or more, more preferably 16.0 ng / mL or more, the method of the present invention can be used to provide a prediction with a high level of reliability for the increased risk of cardiovascular disease and / or hospitalization or death. Similarly, it has been found that the presence of cardiovascular disease and / or increased risk of hospitalization or death can be determined when the amount of binding of monoclonal antibodies specific for the C-terminal neo-epitope of PIIINP is measured to be at least 10.0 ng / mL or more, particularly at least 14.0 ng / mL or more, and by using a statistical cut-off value of PRO-C3 of 10.0 ng / mL or more, more preferably 14.0 ng / mL or more, the method of the present invention can be used to provide a prediction with a high level of confidence regarding the increased risk of cardiovascular disease and / or hospitalization or death. The application of such a statistical cut-off value is particularly advantageous as it leads to a single diagnostic assay; i.e., it does not require a direct comparison with healthy individuals and / or patients with known disease severity to reach a diagnostic conclusion.This may also be particularly advantageous when utilizing the assay to evaluate patients who already have medical signs or symptoms (e.g., as determined by physical exam and / or consultation with a medical professional) that are generally indicative of cardiovascular disease, as it may serve as a rapid and definitive tool to confirm an early prognosis, potentially obviating the need for more invasive procedures and allowing an appropriate treatment plan to be initiated quickly. It may also avoid the need for a prolonged hospital stay. In the specific case of cardiovascular disease, a faster definitive diagnosis may lead to detection of the disease at an earlier stage, further improving overall chances of survival and / or reducing the risk of hospitalization.

[0063] In a second aspect, the present invention provides a method for monitoring and / or assessing the severity of cardiovascular disease in a patient undergoing treatment with an aldosterone antagonist, the method comprising: (i) contacting a biological fluid sample from a patient undergoing treatment with an aldosterone antagonist with a monoclonal antibody that specifically binds to a C-terminal epitope of the C5 domain of the α3 chain of type VI collagen, and / or contacting a biological fluid sample from said patient with a monoclonal antibody that specifically binds to a C-terminal neo-epitope of the N-terminal propeptide of type III collagen. (ii) detecting and determining the amount of binding between each monoclonal antibody used in step (i) and the peptide in the sample; and (iii) correlating the amount of binding of each monoclonal antibody determined in step (ii) with values ​​associated with normal healthy individuals and / or with values ​​associated with known disease severity and / or with values ​​obtained from the patient at previous time points and / or with predefined cut-off values.

[0064] Such methods may allow for the identification and monitoring of patients who respond optimally to treatment with aldosterone antagonists. Aldosterone antagonists (also known as anti-mineralcorticoids) include spironolactone, eplerenone, canrenone, finerenone and mexilenone. Preferably, the aldosterone antagonist is spironolactone. Preferred embodiments of the second aspect of the invention are as described above in relation to the first aspect.

[0065] In a third aspect, the present invention provides a method for identifying a patient having cardiovascular disease who is more likely to respond favourably to treatment with an aldosterone antagonist, the method comprising: (i) contacting a biological fluid sample from a patient with a monoclonal antibody that specifically binds to the N-terminal amino acid sequence ILGHVPGMLL (SEQ ID NO:27) (also referred to herein as the "C4M sequence" or simply "C4M"); (ii) detecting and determining the amount of binding between each monoclonal antibody used in step (i) and the peptide in the sample; and (iii) correlating the amount of monoclonal antibody binding determined in step (ii) with a value associated with patients more likely to respond favorably to treatment with an aldosterone antagonist and / or with a value associated with patients less likely to respond favorably to treatment with an aldosterone antagonist and / or with a predetermined cut-off value.

[0066] Preferably, the aldosterone antagonist is spironolactone. The immunoassay may be, but is not limited to, a competitive assay or a sandwich assay. The immunoassay may be, for example, a radioimmunoassay or an enzyme-linked immunosorbent assay (ELISA).

[0067] The cardiovascular disease may in certain embodiments be heart failure, in particular heart failure with preserved ejection fraction (HFpEF). The patient biological fluid sample may be, but is not limited to, blood, serum, plasma, urine or amniotic fluid. Preferably, the biological fluid is serum or plasma.

[0068] Preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the N-terminal amino acid sequence that is EILGHVPGMLL (SEQ ID NO:28) or a truncation of the N-terminal amino acid sequence that is LGHVPGMLL (SEQ ID NO:29).

[0069] Preferably, the ratio of the affinity of the antibody for the N-terminal amino acid sequence ILGHVPGMLL (SEQ ID NO:27) to the extended N-terminal amino acid sequence EILGHVPGMLL (SEQ ID NO:28) and / or the truncated N-terminal amino acid sequence LGHVPGMLL (SEQ ID NO:29) is at least 10 to 1, more preferably at least 50 to 1, at least 100 to 1, at least 500 to 1, at least 1,000 to 1, at least 10,000 to 1, at least 100,000 to 1, or at least 1,000,000 to 1.

[0070] The term "N-terminus" as used herein means an N-terminal peptide sequence at the end of a polypeptide, i.e., at the N-terminus of a polypeptide, and should not be construed as referring to its general orientation. [Brief description of the drawings]

[0071] [Figure 1A] Hazard ratios for the primary outcome corresponding to standard deviation change in fibrosis biomarkers in unadjusted analyses (one model per biomarker) [Figure 1B] Hazard ratios for a combined outcome of death or heart failure hospitalization corresponding to a standard deviation change in fibrosis biomarkers in unadjusted analyses (one model per biomarker) [Diagram 2]Kaplan-Meier survival curves for the primary endpoint between subjects stratified by tertiles of Pro-C6 (left) and Pro-C3 (right). [Diagram 3] Kaplan-Meier survival curves for a combined outcome of death or hospitalization for heart failure among subjects stratified by tertiles of Pro-C6 (left) and Pro-C3 (right). EXAMPLES

[0072] [Example 1: Development of Antibody to Pro-C6] The monoclonal antibody specific for Pro-C6 was generated by immunologically analyzing the last 10 amino acids of the α3 chain of type VI collagen (i.e., the C-terminal sequence 3168' KPGVISVMGT '3177 (SEQ ID NO: 1)) as described in WO2016 / 156526 (Nordic biosciences, incorporated herein by reference). Briefly, 4-6 week old Balb / c mice were subcutaneously immunized with 200 μm emulsified antigen containing 60 μg of immunogenic peptide. Successive immunizations were performed at 2 week intervals with Freund's incomplete adjuvant until a stable serum titer level was reached, and mice were bled from the second immunization. Serum titers were detected at each bleed, and mice with the highest antiserum titers and the most innately reactive were selected for fusion. The selected mice were rested for one month, followed by an intravenous boost with 100 μl of 0.9% sodium chloride solution containing 50 μg of immunogenic peptide 3 days before isolating the spleen for cell fusion.

[0073] Mouse spleen cells were fused with SP2 / 0 myeloma fusion partner cells. The fused cells were grown in 98-well plates and incubated in a CO2-incubator, where standard limiting dilution was used to promote monoclonal expansion. Cell lines specific to the selected peptide and not cross-reacting with either the extended peptide (KPGVISVMGTA (SEQ ID NO: 2), Chinese Peptide Company, China) or the truncated peptide (KPGVISVMG (SEQ ID NO: 3), American Peptide Company, USA) were selected and subcloned. Finally, the antibodies were purified using an IgG column.

[0074] The antibodies produced were sequenced and the CDRs determined. The sequences of the strands are as follows (CDRs are underlined and in bold): [Heavy chain sequence (mouse IgG1 isotype)]

[0075] [ka]

[0076] [ka]

[0077] [Light chain sequence (mouse kappa isotype)]

[0078] [ka]

[0079] [ka]

[0080] [Example 2] Antibody development for Pro-C3 Monoclonal antibodies specific for Pro-C3 were developed as described in WO 2014 / 170312 (Nordic biosciences, incorporated herein by reference) using the sequence 145'-CPTGPQNYSP-'153 (SEQ ID NO: 14) of the α1 chain of PIIINP as the immunogenic peptide. Briefly, monoclonal antibody generation was initiated by subcutaneous immunization of 4-5 week old Balb / C mice with 200 μl of emulsified antigen containing 50 μg of PIIINP neo-epitope C-terminal sequence (OVA-CGG-CPTGPQNYSP (SEQ ID NO: 32)) using Freund's incomplete adjuvant. Immunizations were repeated every 2 weeks until a stable serum titer level was reached. Spleen cells were fused with SP2 / 0 myeloma cells to generate hybridomas and cloned in culture dishes using the semi-solid medium method. Supernatants were screened for reactivity against calibrator peptides and natural products by indirect ELISA using streptavidin-coated plates, using biotin-CGG-CPTGPQNYSP (SEQ ID NO: 33) as screening peptide and, to further test the specificity of the clones, the free peptide CPTGPQNYSP (SEQ ID NO: 14) as calibrator.

[0081] The native reactivity and affinity of the antibodies were evaluated with different biological materials such as urine, serum and amniotic fluid (AF) from humans and rats in a preliminary ELISA using 2 ng / ml of biotinylated peptides on streptavidin-coated microtiter plates and supernatants from growing monoclonal hybridoma cells. Antibody specificity was tested in a preliminary assay using extended peptides for selection exclusion (i.e., a calibrator peptide with 10 amino acid substitutions and one additional amino acid at the cleavage site, respectively). The isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA). The subtype was determined to be IgG2 subtype.

[0082] The antibodies generated were sequenced and the CDRs determined. The sequences of the strands are as follows (CDRs are underlined and in bold): [Heavy chain sequence (mouse IgG2A isotype)]

[0083] [ka]

[0084] [ka]

[0085] [Light chain sequence (mouse kappa isotype)]

[0086] [ka]

[0087] [ka]

[0088] [Example 3: Development of C4M antibodies] Monoclonal antibodies specific for C4M were developed as previously described by Sand et al. (reference 38) (herein incorporated by reference) using as immunogenic peptide the N-terminal neo-epitope sequence 162'-ILGHVPGMLL-'171 (SEQ ID NO: 27) generated by MMP-12 cleavage between amino acids 161 and 162 of the α1 chain of type IV collagen. Briefly, monoclonal antibody generation was initiated by subcutaneous immunization of 4-6 week old Balb / C mice with 200 μl of emulsified antigen containing 50 μg of immunogenic peptide (ILGHVPGMLL-GGC-KLH (SEQ ID NO: 36)) in Freund's incomplete adjuvant. Immunizations were performed every 2 weeks until a stable serum titer level was reached. Mice with the highest serum titers were selected for fusions. Mice were rested for one month and then boosted intravenously with 50 μg of immunogenic peptide in 100 μl of 0.9% sodium chloride solution 3 days before isolating spleens for cell fusion. Mouse spleen cells were fused with SP2 / 0 myeloma fusion partner cells. The resulting hybridoma cells were cloned using a semi-solid medium method and transferred to 96-well microtiter plates for further growth and incubated in a CO2 incubator. Standard limiting dilution was used to promote monoclonal growth.

[0089] The native reactivity and peptide affinity of the monoclonal antibodies was evaluated by substitution of the original samples (human, rat and mouse serum, plasma, urine) in a preliminary indirect ELISA with the biotinylated peptide (ILGHVPGMLL-K-biotin (SEQ ID NO: 37)) on streptavidin-coated microtiter plates and supernatants from growing monoclonal hybridoma cells. The specificity of the clones was tested for the free peptide (ILGHVPGMLL (SEQ ID NO: 27)), the nonsense peptide, and the extended peptide (EILGHVPGMLL (SEQ ID NO: 28)). Isotyping of the monoclonal antibodies was performed using the SBA Clonotyping System-HRP kit. The monoclonal antibodies were purified from the supernatants collected from the selected clones using a HiTrap protein G column and then labeled with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the manufacturer's instructions.

[0090] The monoclonal antibody with the best native reactivity, peptide affinity and stability was selected from the antibody-producing clones generated after fusion of mouse spleen cells with myeloma cells. The selected clone was of IgG1 subtype, and the antibody showed reactivity to healthy human, rat and mouse sera as well as human plasma EDTA, but showed no reactivity to extended or nonsense peptides.

[0091] [Example 4 PRO-C3 Immunoassay] PRO-C3 was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic bioscience, as described in WO2014 / 170312 and in detail in other publications (citation 19). Briefly, these procedures were as follows: A 96-well streptavidin-coated ELISA plate from Roche, cat. 11940279, was coated with the biotinylated peptide, Biotin-CGG-CPTGPQNYSP (SEQ ID NO: 33), dissolved in coater buffer (50 mM PBS-BTE + 10% sorbitol, pH 7.4), incubated for 30 minutes at 20°C in the dark, followed by washing with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). Then, 20 μl of peptide calibrator or sample was added to the appropriate wells, followed by 100 μl of HRP-conjugated monoclonal antibody NB61N-62 dissolved in incubation buffer (50 mM PBS-BTB + 10% Liquid II (Roche), pH 7.4), and the plate was incubated for 20 hours at 4°C and washed. Finally, 100 μl of tetramethylbenzidine (TMB) (Kem-En-Tec cat.:438OH) was added, the plate was incubated for 15 min at 20°C in the dark, 100 μl of stop solution (1% H2SO4) was added to stop the reaction, and the plate was analyzed in an ELISA reader (Molecular Devices, SpectraMax M, CA, USA) at 450 nm with a reference of 650 nm. The calibration curve was plotted using a four-parameter mathematical fitting model.

[0092] [Example 5 PRO-C6 Immunoassay] PRO-C6 was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic bioscience, as described in WO2016 / 156526 and in detail in other publications (citation 39). Briefly, these procedures were as follows: The ELISA plates used for assay development are streptavidin coated from Roche (cat.:11940279). All ELISA plates were analyzed on an ELISA reader SpectraMax M from Molecular Devices (CA, USA). We labeled selected monoclonal antibodies with horseradish peroxidase (HRP) using the Lightning link HRP labeling kit according to the manufacturer's instructions (Innovabioscience, Babraham, Cambridge, UK). 96-well streptavidin plates were coated with biotinylated synthetic peptide, Biotin-KPGVISVMGT (SEQ ID NO:38) (Chinese Peptide Company, China) dissolved in coating buffer (40 mM Na2HPO4, 7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, pH 7.4) and incubated at 20°C for 30 min. 20 μL of standard peptides or samples diluted in incubation buffer (40 mM Na2HPO4, 7 mM KH2PO4, 137 mM NaCl, 2.7 mM KCl, 0.1% Tween 20, 1% BSA, 5% Liquid II, pH 7.4) were added to the appropriate wells, followed by 100 μL of HRP-conjugated monoclonal antibody 10A3 and incubated for 21 h at 4°C. Finally, 100 μl of tetramethylbenzidine (TMB) (Kem-En-Tec cat. 438OH) was added and the plate was incubated for 15 min at 20°C in the dark. All the above incubation steps included shaking at 300 rpm. After each incubation step, the plate was washed five times with washing buffer (20 mM Tris, 50 mM NaCl). The TMB reaction was stopped by adding 100 μL of stop solution (1% H2SO4) and measured at 450 nm with a reference at 650 nm.

[0093] [Example 6 C4M Immunoassay] C4M was measured using an enzyme-linked immunosorbent assay (ELISA) developed at Nordic bioscience as described in Sand et al. (reference 38). Briefly, the procedures were as follows: 96-well streptavidin-coated microtiter plates (cat. no. 11940279, Roche Diagnostics, Hvidovre, Denmark) were coated with 100 μl of biotinylated peptide (ILGHVPGMLL-K-biotin (SEQ ID NO: 37)) dissolved in coating buffer (50 mM Tris, 1% bovine serum albumin, 0.1% Tween-20, and 0.4% bronidox (BTB), pH 8.0) and incubated for 30 min at 20° C. 20 μl of standard peptide or sample dissolved in assay buffer (50 mM Tris-BTB, pH 8.0) was added to the appropriate wells, followed by 100 μl of conjugated monoclonal antibody diluted in assay buffer and incubated for 1 h at 20° C. Finally, 100 μl of tetramethylbenzidine (TMB) (cat. No. 4380H, Kem-En-Tec, Taastrup, Denmark) was added and the plate was incubated for 15 min at 20°C. The TMB reaction was stopped by adding 100 μl of stop solution (1% H2SO4). All incubation steps were performed in the dark with shaking at 300 rpm, followed by five washes in wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). The results were analyzed spectrophotometrically at 450 nm with a reference at 650 nm using an ELISA microplate reader (VersaMax, Molecular Devices, Sunnyvale, CA, USA). A standard curve was generated by performing serial dilutions of the standard peptide and plotting using a four-parameter mathematical fitting model.

[0094] [Example 7 Biomarker analysis in TOPCAT samples] This study evaluated the association of neoepitope biomarkers of collagen types III, IV, and VI formation (Pro-C3, Pro-C4, and Pro-C6, respectively) and collagen types III, IV, and VI degradation (C3M, C4M, and C6M, respectively) with subject outcomes in subjects with HFpEF enrolled in the TOPCAT trial.

[0095] [method] [Research population] This study used data and biospecimens from the TOPCAT study obtained from the National Heart, Lung, and Blood Institute. The parent study data are available to other researchers via the National Institutes of Health biolincc website.

[0096] The design of the TOPCAT trial and general characteristics of the study population have been described in previous publications (citations 40-42). Briefly, TOPCAT is a multicenter, international, randomized, double-blind, placebo-controlled trial of spironolactone that enrolled 3445 adults with HFpEF across more than 270 centers in six countries between August 2006 and January 2012. Primary results of the trial have been published previously (citation 42). All study participants provided signed informed consent.

[0097] Inclusion criteria for TOPCAT were: age ≥50 years; HF diagnosed based on at least one HF symptom at study screening and at least one HF sign within 12 months prior to screening; left ventricular EF ≥45% (per regional read); at least one EF hospitalization within 12 months prior to study screening or BNP (B-type natriuretic peptide) >100 pg / mL or NT-proBNP (N-terminal pro-BNP) >360 pg / mL within 60 days prior to screening (no other explanation for high natriuretic peptide levels); and serum potassium <5.0 mmol / L prior to randomization ( 40 , 42 ).

[0098] Exclusion criteria have been published in detail previously (reference 40) and included severe systemic illness with a life expectancy <3 years, significant chronic pulmonary disease, infiltrative or hypertrophic cardiomyopathy, constrictive pericarditis, history of cardiac transplant or LV assist device, known chronic liver disease, severe chronic kidney disease (glomerular filtration rate [eGFR] <1.73 m 2 Included are: hyperkalemia (defined as an estimated serum creatinine > 30 mL / min per 1000 mL / min or serum creatinine > 2.5 mg / dL), history of significant hyperkalemia, known intolerance to aldosterone antagonists, and recent myocardial infarction, coronary artery bypass graft, or percutaneous coronary intervention.

[0099] The primary goal of the study was to determine whether spironolactone was associated with a reduction in a composite outcome consisting of cardiovascular death, censoring due to cardiac arrest, or hospitalization for heart failure. All HF hospitalizations were mediated by a clinical endpoint committee at Brigham and Women's Hospital according to prespecified criteria, as previously described ( citation 40 ), and were blinded to study-drug assignment. In this analysis, we examined the relationship between biomarkers and tissue fibrosis and: (1) the primary endpoint defined above; (2) a composite outcome consisting of death or hospitalization for heart failure, which is increasingly utilized in HFpEF studies ( citation 43 ). Given the significant geographic variation in study populations ( citation 6 ), analyses in this study were restricted to subjects enrolled in the United States.

[0100] [Biomarker assay] Stored plasma samples were obtained from Biolincc for all participants enrolled in the United States who had stored plasma from baseline testing (n=206). Specific biomarkers of collagen formation (Pro-C3, Pro-C4 and Pro-C6) and degradation (C3M, C4M and C6M) were measured using enzyme-linked immunosorbent assays (ELISAs). ELISAs for Pro-C3, Pro-C6 and C4M were performed as described above (see Examples 4, 5 and 6, respectively). Pro-C4 is a known biomarker of collagen type IV formation, and Pro-C4 ELISA was performed as described in Leeming et al. (reference 44). C3M and C6M are known biomarkers of collagen type III degradation and collagen type VI degradation, respectively, and C3M and C6M ELISA was performed as described in Barascuk et al. (reference 45) and Juhl et al. (reference 46), respectively. NT-proBNP levels were measured using a validated Luminex® bead-based multiplex assay (Bristol Myers-Squibb; Ewing Township, NJ).

[0101] [Statistical analysis] Participant characteristics were summarized using mean (SD) for normally distributed variables and median (interquartile range) for non-normally distributed continuous variables. Categorical variables are expressed as counts (percentages). Subjects enrolled in the United States and with samples available for measurement of biomarkers of interest were compared with subjects enrolled in the United States and without samples available. Two-sample t-tests were used for normally distributed variables, Kruskal-Wallis tests for non-normally distributed variables, and chi-square or Fisher's exact tests, as appropriate, for categorical variables.

[0102] The association between biomarkers and the primary outcome (cardiovascular death, censoring due to cardiac arrest, or hospitalization for heart failure), as well as the composite outcome consisting of HF hospitalization or all-cause death, was assessed using Cox regression. Kaplan-Meier survival curves were constructed between tertiles of each biomarker and compared using the log-rank test. To assess whether the unadjusted groups were independent of confounding factors, adjusted Cox models were constructed accordingly, including (1) the MAGGIC risk score incorporating multiple demographic, clinical, and laboratory variables (Model 1) ( citation 47 ); (2) the addition of NT-proBNP levels to the MAGGIC risk score (Model 2); and (3) empirically selected important individual clinical covariates, including age, sex, diabetes status, estimated glomerular filtration rate, systolic blood pressure (SBP), and history of myocardial infarction (Model 3). Hazard ratios were standardized for all biomarkers (expressed per standard deviation increase or per one-point increase in z-score) to provide an intuitive comparison between biomarkers.

[0103] Finally, interactions between pre-randomization levels of each biomarker and randomized spironolactone treatment were tested as predictors of the above-mentioned outcomes, and if interactions were found, survival analyses graded according to the median values ​​of the biomarkers were performed to assess the effect of spironolactone treatment. Statistical significance was defined as a two-sided p-value <0.05. All probability values ​​presented are two-sided. Statistical analyses were performed using the Matlab statistics and machine learning toolbox (Matlab 2016b, The Mathworks; Natwick, MA) and SPSS for Mac v22 (SPSS Inc., Chicago, IL).

[0104] [result] Table 1 shows a comparison of US-enrolled study participants who had frozen biological samples available for biomarker measurement with US-enrolled study participants who did not. There were no significant differences between subgroups in age or sex. Subjects with available samples represented a slightly greater proportion of white participants (85.44 vs. 77.36%) and a slightly lower proportion of black participants (12.62 vs. 17.7%). The prevalence of NYHA stage III-IV, myocardial infarction, stroke, peripheral arterial disease, or history of diabetes did not differ between subgroups, but the prevalence of COPD was lower and the prevalence of hypertension and atrial fibrillation was greater among participants with available samples. Use of antihypertensives, diuretics, glucose-lowering agents, and ACE inhibitors / ARBs did not differ between groups. Subjects with available samples were more likely to be receiving statins.

[0105] [Table 1]

[0106] [Relationship between baseline biomarkers of tissue fibrosis and outcomes] Figure 1A shows the standardized hazard ratios of all fibrosis biomarkers tested (one model per biomarker) for the primary endpoint in unadjusted analyses. Figure 1B shows the corresponding standardized hazard ratios for death or heart failure hospitalization. In these analyses, Pro-C6 (HR = 1.90; 95% Cl = 1.54-2.34; P < 0.0001) and Pro-C3 (HR = 1.57; 95% Cl = 1.28-1.94; P < 0.0001) strongly predicted the primary endpoint of the study. Similarly, Pro-C6 (HR = 1.94; 95% Cl = 1.60-2.35; P < 0.0001) and Pro-C3 (HR = 1.56; 95% Cl = 1.29-1.89; P < 0.0001) predicted the composite endpoint consisting of death or heart failure hospitalization.

[0107] Figure 2 shows Kaplan-Meier survival curves for the primary endpoint corresponding to tertiles of Pro-C6 (left) and Pro-C3 (right), respectively. Pro-C6 graded subjects across a wide range of absolute risk. From the low tertile of Pro-C6 (Pro-C6 <11.0 ng / ml) to the high tertile (Pro-C6 >16.0 ng / ml), a significant decrease in recurrence-free survival was graded. For Pro-C3, only the high tertile (Pro-C3 >14.0 ng / ml) showed a significant decrease in recurrence-free survival. A similar pattern was found for death or heart failure hospitalization, as shown in Figure 3.

[0108] In models that included both Pro-C6 and Pro-C3, Pro-C6 independently predicted the primary outcome (HR = 1.84; 95% Cl = 1.36-2.47; P < 0.0001) and death / HF hospitalization (HR = 1.92; 95% Cl = 1.46-2.53; P < 0.0001). In contrast, in these models, Pro-C3 was not significantly associated with either the primary outcome (HR = 1.06; 95% Cl = 0.76-1.46; P = 0.74) or death / HF hospitalization (HR = 1.01; 95% Cl = 0.75-1.37; P = 0.93). Similarly, in models including both Pro-C6 (as a continuous variable) and Pro-C3 levels >14 ng / mL (distribution of high tertiles expressed as a binary variable), Pro-C6 status, but not Pro-C3 status, independently predicted the primary outcome and death / HF hospitalization.

[0109] Next, an adjusted analysis was performed for Pro-C6 only (Table 2). In models adjusted for the MAGGIC risk score, ProC6 strongly predicted the primary outcome (HR=1.88; 95% Cl=1.52-2.33; P<0.0001) and death / HF hospitalization (HR=1.91; 95% Cl=1.57-2.33; P<0.0001). The hazard ratio for Pro-C6 was very similar when additionally adjusting for NT-ProBNP (adjusted model 2, Table 2). When adjusted for Pro-C6, NT-ProBNP only weakly predicted outcome, whereas the MAGGIC risk score failed to predict the primary outcome or death / HF hospitalization. Similarly, in models adjusted for age, sex, diabetes status, estimated glomerular filtration rate, SBP, NYHA severity III / IV, and history of myocardial infarction (adjusted model 3, Table 2), Pro-C6 strongly predicted the primary outcome (HR = 1.81; 95% Cl = 1.44-2.27; P < 0.0001) and the outcome of death / HF hospitalization (HR = 1.84; 95% Cl = 1.49-2.26; P < 0.0001).

[0110] As shown in Table 2, for the primary outcome, Harrell's C-statistic for the model including Pro-C6 alone (0.705) was much higher than that for the model including MAGGIC risk score (0.552), the model including BNP plus MAGGIC risk score (0.582), or the model including the combination of clinical variables included in adjusted model 3 (0.64). Similarly, for death / heart failure-related hospitalization, Harrell's C-statistic for the model including Pro-C6 alone (0.707) was much higher than that for the model including MAGGIC risk score (adjusted model 1: 0.571), the model including BNP plus MAGGIC risk score (adjusted model 2: 0.602), or the model including the combination of clinical variables (adjusted model 3: 0.623). Thus, the addition of Pro-C6 to models that already included the MAGGIC risk score, to models that added BNP to the MAGGIC risk score, or to models that already included a combination of clinical variables significantly improved Harrell's C-statistic (Table 2).

[0111] [Table 2]

[0112] *Numbers in brackets represent estimated standard errors. Adjusted model 1: Adjusted for MAGGIC risk score. Adjusted model 2: Adjusted for MAGGIC risk score and NT-proBNP level. Adjusted model 3: adjusted for age, sex, diabetes, estimated glomerular filtration rate, systolic blood pressure (SBP), NYHA severity III / IV and history of myocardial infarction.

[0113] [Interaction with randomized group] Significant interactions between baseline levels of C4M and randomized treatment group were found as predictors of the primary endpoint (P=0.0061 for C4M-treated group) and death / HF hospitalization (P=0.0063 for C4M-treated group), indicating a more favorable response to treatment among participants with lower C4M levels at baseline. No interactions with treatment group were found for other fibrosis biomarkers tested.

[0114] [discussion] The relationship between biomarkers of ECM turnover measured at baseline in participants enrolled in the TOPCAT study was studied. Pro-C6 and Pro-C3, biomarkers of fibrosis assessed for collagen types VI and III formation, respectively, were demonstrated to predict the risk of developing cardiovascular events, as well as a composite endpoint of all-cause mortality / HF-related hospitalization in this population. Notably, Pro-C6 was a strong independent predictor of these outcomes and subjects graded across a wide range of absolute risks. Pro-C6 alone performed better as a predictor of outcome than the MAGGIC risk score, NT-ProBNP, or a combination of clinical variables. The addition of Pro-C6 to the MAGGIC risk score, with or without additional adjustment for NT-proBNP levels, resulted in a significant increase in Harrell's C-statistic, a measure of model fit, and similar discrimination to the receiver-operator characteristic curves. Furthermore, an interaction was found between levels of C4M, a biomarker of collagen type IV degradation (present primarily in vascular basement membranes), and the risk reduction associated with randomization to spironolactone versus placebo. In these post-hoc analyses, subjects with higher C4M levels appeared to derive greater benefit from randomization to spironolactone. These findings support a critical role for tissue fibrosis in HFpEF, and it may be possible to identify biomarkers of ECM turnover that are easily measured and implemented in a variety of settings for risk grading in this population.

[0115] In this study, high levels of Pro-C6 strongly predicted outcome. It is important to note that the remarkable predictive power of this biomarker greatly exceeds that of the MAGGIC risk score, the MAGGIC risk score plus NT-proBNP levels, and the combination of important clinical variables. Moreover, Pro-C6 significantly improves the discrimination of models that already include these predictors; in contrast, the addition of standard predictors to models that already include Pro-C6 resulted in only a small improvement in model fit. Thus, Pro-C6 appears to be a particularly powerful and robust independent predictor of outcome in HFpEF. Thus, Pro-C6 may be useful in the diagnosis of FpEF, to identify good candidates for antifibrotic treatment, and / or to monitor and characterize the efficacy of such treatment.

[0116] A particularly interesting finding of this study is the significantly higher interaction between C4M and risk adjustment associated with randomized treatment with spironolactone. These findings support the notion that biomarkers of collagen turnover can identify individuals who will benefit from spironolactone. This study is the first to report an interaction between a biomarker of collagen turnover and reduced risk of clinical events associated with spironolactone therapy. Lower C4M levels were found to be associated with a greater reduction in risk associated with spironolactone failure. C4M is a marker of collagen degradation; therefore, lower levels indicate reduced degradation and a concomitant increase in collagen accumulation, making them amenable to treatment with spironolactone.

[0117] Collagen IV is present not only in blood vessels but also in the glomerular basement membrane, which prevents leakage of plasma proteins into the urine. Interestingly, however, C4M was not associated with albuminuria in the present study population. Similarly, in contrast to the significant interaction between changes in C4M and spironolactone, no interaction was found between proteinuria and spironolactone treatment in a recent analysis of the TOPCAT trial (citation 48). Thus, the interaction between C4M and spironolactone effects is unlikely to be mediated by degradation of the glomerular basement membrane.

[0118] In summary, fibrogenesis, as assessed by Pro-C6, is a strong and independent predictor of poor prognosis in HFpEF. In contrast, low levels of C4M appear to identify HFpEF patients with a particularly favorable response to aldosterone antagonists (mineralocorticoid receptor antagonists).

[0119] In this specification, unless otherwise specified, the word "or" is used to mean an operator that returns a true value if one or both of the stated conditions are met, as opposed to an "exclusive or" operator that requires only one of several conditions to be met. The word "comprising" is used in the sense of "including" and not "consisting of." All prior teachings acknowledged above are incorporated herein by reference. Any acknowledgment in this specification of a prior-published document should not be taken as an admission or representation that the teaching of that document is the common general knowledge in Australia or anywhere else at the time of this specification.

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Claims

1. 1. An immunoassay method for assessing the severity of cardiovascular disease in a patient, the assessment comprising assessing the likelihood of death and / or hospitalization of the patient as a result of cardiovascular disease, the cardiovascular disease being heart failure with preserved ejection fraction (HFpEF), the method comprising: (i) contacting a biological fluid sample from a patient with heart failure with preserved ejection fraction with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO:1) of the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen; (ii) detecting and determining the amount of binding between the monoclonal antibody used in step (i) and the peptide in the sample; and (iii) correlating the amount of binding of said monoclonal antibody determined in step (ii) with values ​​associated with known disease severity and / or with pre-defined cut-off values.

2. The method of claim 1 , wherein the patient is undergoing treatment for cardiovascular disease.

3. The method of claim 1 or 2, wherein the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence which is KPGVISVMGTA (SEQ ID NO: 2) or a truncation of the C-terminal amino acid sequence which is KPGVISVMG (SEQ ID NO: 3).

4. wherein step (i) further comprises contacting the biological fluid sample from the patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence CPTGPQNYSP (SEQ ID NO: 14) of the C-terminal neo-epitope of the N-terminal propeptide of type III collagen; the step (ii) comprises detecting and determining the amount of binding between the monoclonal antibody that specifically binds to the C-terminal amino acid sequence KPGVISVMGT (SEQ ID NO: 1) of the C-terminal epitope of the C5 domain of the α3 chain of type VI collagen and the monoclonal antibody that specifically binds to the C-terminal amino acid sequence CPTGPQNYSP (SEQ ID NO: 14) of the C-terminal neo-epitope of the N-terminal propeptide of type III collagen and a peptide in the sample; and 4. The method of claim 1, wherein step (iii) comprises correlating the amount of binding of each monoclonal antibody determined in step (ii) with a value associated with known disease severity and / or a predetermined cut-off value.

5. The method described in claim 4, wherein the monoclonal antibody that specifically binds to the C-terminal amino acid sequence CPTGPQNYSP (SEQ ID NO: 14) of the C-terminal neo-epitope of the N-terminal propeptide of type III collagen does not recognize or specifically bind to an extension of the C-terminal amino acid sequence of the C-terminal neo-epitope, which is CPTGPQNYSPQ (SEQ ID NO: 15), or a truncation of the C-terminal amino acid sequence of the C-terminal neo-epitope, which is CPTGPQNYS (SEQ ID NO: 16).

6. The method according to any one of claims 1 to 5, wherein the biological fluid is serum or plasma.

7. The method of claim 1 , wherein the immunoassay is a competitive assay or a sandwich assay.

8. The method of any one of claims 1 to 7, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

9. A method according to any one of claims 1 to 8, wherein the patient is a patient with heart failure with preserved ejection fraction receiving treatment with an aldosterone antagonist.

10. 10. The method of claim 9, wherein the aldosterone antagonist is spironolactone.

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