Assays for assessing heart failure

Abstract

The present disclosure relates to immunoassays, particularly immunoassays for detecting and / or monitoring cardiovascular diseases such as heart failure. The present disclosure also relates to monoclonal antibodies and kits for use in the assays. The assays, antibodies, and kits target a C-terminal epitope of type XXVIII collagen.

Description

[Technical Field]

[0001] The present invention relates to immunoassays, particularly to immunoassays for detecting and / or monitoring cardiovascular disease in patients, and to monoclonal antibodies for use in such assays. The cardiovascular disease may be, in particular, heart failure, particularly heart failure with preserved ejection fraction. The immunoassay may be for assessing the likelihood of an adverse outcome of cardiovascular disease.

[0002] The disease burden of heart failure (HF) has increased dramatically in recent years (References 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 (Reference 3). Despite numerous phase III randomized controlled trials over recent decades, evidence remains to be provided as to which pharmacological interventions can clearly benefit this patient population.

[0003] The heterogeneity of HFpEF syndrome has been identified as a significant barrier to demonstrating the efficacy of candidate pharmacological interventions. The heterogeneous nature of HFpEF, with varying degrees of contribution from various pathophysiological processes, may adversely affect the average response to pharmacological treatments tested in clinical trials. Therefore, 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 enhancing our clinical and therapeutic approaches to HFpEF (Reference 4).

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

[0005] Myocardial fibrosis is thought to play a role in the pathophysiology of HFpEF (references 5, 6). Increased fibrosis results from excessive collagen 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 (references 6-8), and increased myocardial extracellular matrix accumulation in this condition has been shown to correlate with left ventricular passive stiffness and diastolic dysfunction (references 5, 8). Myocardial fibrosis may also contribute to reduced coronary flow reserve (references 6, 9), ventricular dyssynchrony, and arrhythmia propensity (references 10, 11). Given the role of myocardial fibrosis in HFpEF, simple fibrosis biomarkers that reflect the dynamic processes underlying fibrosis progression or regression would be of great value (reference 10).

[0006] Extracellular volume fraction (ECVF), an index of myocardial fibrosis measured by cardiac magnetic resonance imaging, has been reported to predict adverse outcomes (12, 13) or the risk of HFpEF (14) in patients with HFpEF. While MRI promises to play an important role in assessing myocardial fibrosis in preclinical studies, early-phase human investigations, and some clinical settings, its cost and availability likely limit or preclude 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 a significant challenge.

[0007] However, the search for appropriate biomarkers for HFpEF is complicated by the fact that "fibrosis is not simply fibrosis," and that ECM remodeling in different compartments and collagen types may have different biological and prognostic relevance (reference 15). For example, differences in the association of collagen neoepitope fragments with liver fibrosis have been reported in chronic hepatitis B versus hepatitis C. Furthermore, 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 occurs in the arterial wall, kidney, and liver dysfunction, all of which may contribute to adverse outcomes in this population.

[0008] Although type XXVIII collagen has been poorly described in the literature, research is beginning to identify its physiological role. Type XXVIII collagen is primarily found in peripheral nerves and dorsal root ganglia, but is also found in skin (references 17, 18). Type XXVIII collagen is a beaded collagen with two von Willebrand factor A domains flanking a 528-amino acid collagen domain, structurally similar to type VI collagen (reference 19). Type XXVIII collagen is found at extremely low levels in healthy lung tissue, but is overexpressed in bleomycin-induced lung injury (reference 20). This may indicate that cells expressing type XXVIII collagen may be involved in tissue repair processes. Type XXVIII collagen has also previously been shown to be upregulated in mouse hepatocellular carcinoma. Summary of the Invention

[0009] The present inventors have now identified that the formation of type XXVIII collagen is upregulated in HFpEF and developed a novel competitive ELISA using a monoclonal antibody targeting the C-terminus of type XXVIII collagen.

[0010] Thus, in a first aspect, the present invention provides an immunoassay method, the method comprising: (i) contacting a biological fluid sample from a patient with a monoclonal antibody that specifically binds to a C-terminal epitope of type XXVIII collagen; and (ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample.

[0011] The method can be used to quantify peptides having the C-terminal epitope of type XVIII collagen present in a biological fluid sample, and can be used, for example, to assess the level of type XXVIII collagen formation.

[0012] In a preferred embodiment, the method is 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, the immunoassay method comprising: (i) contacting a biological fluid sample from a patient with a monoclonal antibody that specifically binds to a C-terminal epitope of type XXVIII collagen; (ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample; and (iii) correlating the amount of binding of the monoclonal antibody determined in step (ii) with a value associated with a normal healthy individual and / or a value associated with a known severity of the disease and / or a value obtained from the patient at a previous time point and / or a predetermined cut-off value.

[0013] 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.

[0014] In some embodiments, the cardiovascular disease may be heart failure. In particular, the cardiovascular disease may be heart failure with preserved ejection fraction (HFpEF). The method may, in some embodiments, be a method for assessing the severity of cardiovascular disease in a patient, including assessing the likelihood of death and / or hospitalization of the patient as a result of cardiovascular disease and / or a composite of adverse cardiovascular events.

[0015] In certain embodiments, the patient may be, for example, a patient undergoing treatment for cardiovascular disease, and the method may include monitoring the cardiovascular disease in the patient. The patient biological fluid sample may be, but is not limited to, blood, serum, plasma, urine, or supernatant obtained from cell or tissue culture. Preferably, the biological fluid is serum or plasma, and most preferably, serum.

[0016] As used herein, the term "monoclonal antibody" refers to both whole antibodies and fragments thereof that retain the binding specificity of whole antibodies, such as Fab fragments, F(ab')2 fragments, single-chain Fv fragments, or other such fragments known to those skilled in the art. As is well known, whole antibodies typically have a "Y-shaped" structure consisting of two identical paired polypeptide chains, each of which consists of one "light" 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 region enables antibodies to recruit cells and molecules of the immune system. Antibody fragments that retain binding specificity contain at least the CDRs and a sufficient portion of the remainder of the variable region to retain this binding specificity.

[0017] The methods of the present invention can use monoclonal antibodies containing any constant region known in the art. 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 isotype as IgM, IgD, IgG, IgA, or 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.

[0018] The CDRs of an antibody can be determined using methods known in the art, such as those described by Kabat et al. Antibodies can be generated from B cell clones as described in those examples. The isotype of the antibody can be determined by ELISA specific for human IgM, IgG, or IgA isotypes 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 can then be subjected to PCR using primers that amplify the heavy and light chains of the antibody. For example, primers specific to the leader sequences of all VH (variable heavy) sequences can be used together with primers that bind to sequences 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 together with a primer that anneals to the leader sequence of Vkappa or Vlamda. Full-length heavy and light chains can be generated and sequenced.

[0019] In some embodiments of the method of the first aspect of the present invention, the biological fluid sample is contacted with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) (also referred to herein as "PRO-C28"). Preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is QETCIQGA (SEQ ID NO: 2). Preferably, the monoclonal antibody does not recognize or specifically bind to a truncation of the C-terminal amino acid sequence that is QETCIQ (SEQ ID NO: 3).

[0020] Preferably, the ratio of the affinity of the antibody for the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) to the extended C-terminal amino acid sequence QETCIQGA (SEQ ID NO: 2) 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.

[0021] Preferably, the ratio of the affinity of the antibody for the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) to the affinity of the antibody for the truncated C-terminal amino acid sequence QETCIQ (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.

[0022] The term "C-terminus" as used herein refers to a C-terminal peptide sequence at the end of a polypeptide, i.e., at the C-end of a polypeptide, and should not be construed as referring to its general orientation.

[0023] Monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) can be produced by any suitable technique known in the art. For example, monoclonal antibodies can be produced against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1), such as by immunizing a rodent (or other suitable mammal) with a synthetic peptide consisting of the sequence QETCIQG (SEQ ID NO: 1), optionally linked to an immunogenic carrier protein (such as keyhole limpet hemokine), isolating and cloning cells producing a single antibody, and assaying the resulting monoclonal antibodies to determine whether they have the desired specificity. An exemplary protocol for producing monoclonal antibodies that specifically bind to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) is described below.

[0024] In some embodiments of the method according to the first aspect of the present invention, the amount of binding of the monoclonal antibody specific for a C-terminal epitope of type XXVIII collagen is correlated 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 a previous time point.

[0025] 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 in subjects who are considered to be healthy, i.e., do not have cardiovascular disease, and / or standardized amounts determined by the above methods in subjects who are known to have cardiovascular disease with known severity.

[0026] In some embodiments of the method according to the first aspect of the present invention, the amount of binding of a monoclonal antibody specific for a C-terminal epitope of type XXVIII collagen is correlated with one or more predetermined cut-off values.

[0027] As used herein, "cutoff value" refers to an amount of binding that is statistically determined to indicate a high probability 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 the probability of disease of a particular level of severity.

[0028] The predetermined cutoff value for the binding amount of the monoclonal antibody specific to the C-terminal epitope of type XXVIII collagen is preferably 100 ng / mL or more. In this regard, it has been found by using statistical analysis that if the binding amount of the monoclonal antibody specific to the C-terminal epitope of type XXVIII collagen is measured to be at least 100 ng / mL or more, the presence of cardiovascular disease can be determined. By setting the statistical cutoff value at 100 ng / mL or more, the method of the present invention can be used to provide a high level of confidence in the diagnosis of cardiovascular disease. The application of such a statistical cutoff value is particularly advantageous because it leads to a single diagnostic assay; that is, it eliminates the need for direct comparison with healthy individuals and / or patients with known disease severity to reach a diagnostic conclusion. This can also be particularly advantageous when utilizing the assay to evaluate patients who already have medical signs or symptoms (e.g., as determined by physical examination and / or consultation with a medical professional) that are generally indicative of cardiovascular disease, as it can 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 promptly. It can also avoid the need for a prolonged hospital stay. In the specific case of cardiovascular disease, a faster definitive diagnosis can lead to detection of the disease at an earlier stage, further improving overall chances of survival and / or reducing the risk of hospitalization.

[0029] In a second aspect, the present invention provides a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide biotin-L-QETCIQG (SEQ ID NO: 4), where L is an optional linking group; - Secondary antibodies used in sandwich immunoassays; - a calibration peptide comprising the sequence QETCIQG (SEQ ID NO: 1); - Antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay Visualization Kit An immunoassay kit is provided, comprising:

[0030] The immunoassay kit is suitable for carrying out the method according to the first aspect. Accordingly, preferred embodiments of the second aspect will be apparent from the above discussion of preferred embodiments of the first aspect. For example, the kit is preferred for detecting and / or monitoring cardiovascular disease in a patient and / or assessing the likelihood or severity of cardiovascular disease in a patient. The monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) is preferably a monoclonal antibody raised against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1). Preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is QETCIQGA (SEQ ID NO: 2). Preferably, the monoclonal antibody does not recognize or specifically bind to a truncation of the C-terminal amino acid sequence that is QETCIQ (SEQ ID NO: 3).

[0031] In a third aspect, the present invention provides a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1). Preferred embodiments of the third aspect will also be apparent from the above discussion of the preferred embodiments of the first aspect. For example, the monoclonal antibody is preferably a monoclonal antibody raised against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1). Preferably, the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is QETCIQGA (SEQ ID NO: 2). Preferably, the monoclonal antibody does not recognize or specifically bind to a truncation of the C-terminal amino acid sequence that is QETCIQ (SEQ ID NO: 3). [Brief explanation of the drawings]

[0032] [Figure 1] Antibody specificity. Reactivity was tested with the standard peptide (QETCIQG) (SEQ ID NO: 1), the extended peptide (QETCIQGA) (SEQ ID NO: 2), the nonsense peptide (GLRPGSEYTV) (SEQ ID NO: 7), and the nonsense peptide (GLRPGSEYTV-K-Biotin) (SEQ ID NO: 8). [Figure 2] Serum PRO-C28 levels in healthy controls (HC) and patients with HFpEF [Example]

[0033] Embodiments of the present disclosure are described and illustrated in the following examples. These examples are presented to aid in the understanding of the present disclosure and should not be construed in any way to limit the scope of the present disclosure, which is defined in the claims that follow. The examples set forth below are presented 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 disclosure, nor are they intended to imply that the experiments described below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. The examples described below used the following materials and methods.

[0034] material and method All reagents used in the experiments were of high quality and were obtained from companies such as Sigma-Aldrich (St. Louis, MO, USA) and Merck (Whitehouse Station, NJ, USA). Synthetic peptides used for immunoassay and assay development were purchased from GenScript (New Jersey, USA). Human serum from healthy donors was purchased from a commercial vendor (Lee Biosolutions, MO 63043, USA). Human serum from HFpEF patients in the TRAINING-HF cohort was obtained through a collaboration with the INCLIVA Health Research Institute in Valencia, Spain.

[0035] Production of monoclonal antibodies targeting PRO-C28 Monoclonal antibodies targeting the C-terminus of type XXVIII collagen were generated by raising antibodies against the seven amino acid sequence QETCIQG (SEQ ID NO: 1) ("PRO-C28") found at the C-terminus of type XXVIII collagen. This seven amino acid sequence was chosen rather than a longer sequence, such as the 10 amino acid C-terminal sequence KECQETCIQG (SEQ ID NO: 5), to reduce the number of cysteine ​​residues and thereby avoid the formation of Cys-Cys bridges within the immunogenic peptide used to generate the antibodies. The protocol used to generate monoclonal antibodies targeting PRO-C28 is as follows:

[0036] Immunization of 6-7 week-old female Balb / C mice (14-18 g body weight) was initiated by subcutaneous injection of 200 μL of an emulsified antigen solution containing 100 μg of immunogenic peptide (KLH-CGG-QETCIQG (SEQ ID NO: 6)) in Stimune Immunogenic Adjuvant (SPECOL) (Cat #7925000, Invitrogen). Here, 'KHL' stands for keyhole limpet hemokine, and CGG is the conjugation linker. Immunizations were repeated every 2 weeks until a stable serum titer level was reached. Mice showing the highest serum titer and best inhibition were selected for fusion. After the final immunization, mice were rested for at least 3 weeks. Subsequently, 3 days before isolating spleens for cell fusion, mice were boosted intravenously with 100 μL of 0.9% sodium chloride solution containing 100 μg of immunogenic peptide. To generate hybridoma cells, mouse spleen cells were fused with SP2 / 0 myeloma cells as described by Gefter et al. Hybridoma cells were cloned in culture dishes using the semisolid medium method. For further growth, clones were seeded into 96-well microtiter plates and limiting dilution was used to promote monoclonal growth. Supernatant reactivity was screened using indirect ELISA performed on streptavidin-coated plates. Biotin-QETCIQG was used as the screening peptide, and the standard peptide (QETCIQG (SEQ ID NO: 1)), extended peptide (QETCIQGA (SEQ ID NO: 2)), nonsense peptide (GLRPGSEYTV) (SEQ ID NO: 7), and nonsense peptide (GLRPGSEYTV-K-Biotin) (SEQ ID NO: 8) were used to further test the specificity of the clones. Supernatants were collected from hybridoma cells and purified using HiTrap affinity columns (GE Healthcare Life Sciences, Little Chalfront, Buckinghamshire, UK) according to the manufacturer's instructions. All animals were handled in accordance with animal welfare guidelines.

[0037] Clone selection and characterization The best antibody-producing hybridomas were screened for reactivity to the standard peptide (QETCIQG (SEQ ID NO: 1)) by competitive ELISA described below, and the clones showing the highest reactivity were selected to produce monoclonal antibodies targeting PRO-C28. Antibody specificity was tested using the standard peptide (QETCIQG (SEQ ID NO: 1)), the extended peptide (QETCIQGA (SEQ ID NO: 2)), the nonsense peptide (GLRPGSEYTV) (SEQ ID NO: 7), and the nonsense peptide (GLRPGSEYTV-K-Biotin) (SEQ ID NO: 8). The isotype of the monoclonal antibodies was determined using the Clonotyping System-HRP Kit, cat. 5300-05 (Southern Biotech, Birmingham, AL, USA).

[0038] PRO-C28 ELISA A 96-well Roche streptavidin-coated ELISA plate, cat. 11940279, was coated with 100 μL / well of the biotinylated peptide, Biotin-QETCIQG (SEQ ID NO: 4), dissolved in assay buffer (25 mM TBS-BTE + 2 g / L NaCl, pH 8), incubated for 30 minutes with shaking at 20°C in the dark, and then washed five times with wash buffer (20 mM Tris, 50 mM NaCl, pH 7.2). 20 μL of calibration peptide or sample was then added to the appropriate wells, followed by 100 μL of purified antibody solution (monoclonal antibody specific for PRO-C28 dissolved in assay buffer). The plate was incubated for 1 hour with shaking at 20°C, and then washed five times with wash buffer. Next, 100 μl of the second antibody solution (horseradish peroxidase (HRP)-labeled anti-mouse antibody dissolved in the same assay buffer as used for the PRO-C28-specific monoclonal antibody) was added to each well and incubated with shaking at 20°C for 1 h, followed by washing five times with wash buffer. Finally, 100 μl of tetramethylbenzidine (TMB) (Kem-En-Tec cat.: 438OH) was added to each well, and the plate was incubated in the dark at 20°C for 15 min. 100 μl of stop solution (1% H2SO4) was added to stop the reaction, and the plate was analyzed at 450 nm with a reference wavelength of 650 nm using an ELISA reader (Molecular Devices, SpectraMax M, CA, USA). A calibration curve was plotted using a four-parameter mathematical fitting model.

[0039] Technical evaluation of PRO-C28 ELISA Linearity was assessed using two-fold dilutions of human serum samples, human urine and EDTA samples, and heparinized or citrated human plasma samples (four samples of each type). Linearity was calculated as the percentage recovery of the undiluted samples. Intra- and inter-assay variations were determined in duplicate by performing 10 independent five-point quality controls (QCs) and two-point kit controls. The precision of the assay was determined on healthy human serum samples spiked with standard proteins and calculated as the percentage of serum recovery in buffer. The lower limit of the measuring range (LLMR) and upper limit of the measuring range (ULMR) were calculated based on 10 individual standard curves obtained from intra- and inter-assay variations.

[0040] Validation of the biological validity of PRO-C28 as a biomarker for HFpEF PRO-C28 was measured in serum samples from a population of patients with HFpEF (heart failure with preserved ejection fraction) and healthy controls using the PRO-C28 ELISA protocol described above. Patient demographics are shown in Table 1.

[0041] [Table 1]

[0042] ★ There was a significant difference in age between the two groups, but there was no correlation between age and PRO-C28 in either group.

[0043] result Clone selection and characterization The best antibody-producing hybridomas were screened for reactivity and selectivity to the standard peptide, and clone NBH218#65 8C11-2F10-1H7 was selected based on its reactivity. This clone was then used to produce a monoclonal antibody targeting PRO-C28 for technical and biological evaluation of the PRO-C28 ELISA. The monoclonal antibody belonged to the IgG2b, k isotype. No reactivity was observed to extended peptides, nonsense peptides, or nonsense coaters (Figure 1).

[0044] Technical evaluation of PRO-C28 ELISA A series of technical validation studies were performed to evaluate the PRO-C28 ELISA assay. A summary of the validation data is shown in Table 2.

[0045] [Table 2]

[0046] Biological evaluation of PRO-C28 as a biomarker for HFpEF PRO-C28 levels were measured in serum samples from a population of patients with heart failure with preserved ejection fraction (HFpEF) and healthy controls (HCs) using the PRO-C28 ELISA. The biomarker levels in serum samples from the two populations were compared using the Mann-Whitney test (nonparametric data). The results are shown in Figure 2 (in this figure, the results are presented as Tukey's box plots). As can be seen, PRO-C28 was significantly elevated in serum from HFpEF patients compared to healthy controls (p<0.0001). Additionally, NT-proBNP levels were measured in serum samples from HFpEF patients at three different time points throughout the study, and the measured NT-proBNP concentrations were compared with PRO-C28 concentrations (measured as described above) in the same samples using Spearman correlation. As shown in Table 3 below, PRO-C28 levels in the HFpEF population were found to be significantly correlated with NT-proBNP, a standard clinically assessed biomarker for diagnosing and monitoring HF.

[0047] [Table 3]

[0048] In this specification, unless otherwise specified, the word "or" is used to mean an operator which returns a true value if one or both of the stated conditions are met, as opposed to the "exclusive or" operator which requires that only one of several conditions 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 common general knowledge in Australia or elsewhere at the date of this specification.

[0049] [References] 1. Lamb CS, Donal E, Crager-Crainer E, Basan RS. Epidemiology and clinical course of heart failure with preserved ejection fraction. Eur J Heart Fail 2011;13:18-28 2. Lloyd-Jones DM, Hong Y, Lavalse D, et al. Defining and setting national goals for improving cardiovascular health and reducing disease: American Heart Association Strategic Impact Goals for 2020 and beyond. Circulation 2010;121::586-613 3. Lamb CS, Donal E, Crager-Crainer E, Basan RS. Epidemiology and clinical course of heart failure with preserved ejection fraction. Eur J Heart Fail 2011;13:18-28 4. Kairinos JA. Deep phenotyping of systemic arterial hemodynamics in HFpEF (part 2): clinical and therapeutic considerations. J Cardiovasc Transl Res 2017;10:261-274 5. Romel KP, von Roedel M, Laczynski K et al. Extracellular volume fraction for characterization of patients with heart failure and preserved ejection fraction. J Am coll Cardiol 2016;67:1815-25

[0050] 6. Mohamud SF, Hussain S, Mirzoev SA, Edwards WD, Maleszewski JJ, Redfield MM. Coronary microvascular rarefaction and myocardial fibrosis in heart failure with preserved ejection fraction. Circulation 2015;131:550-9 7. Kairinos JA, Aker SR, Traille L et al. Heart failure, left ventricular remodeling, and circulating nitric oxide metabolites. J Am Heart Assoc 2016;5 8. Su MY, Lin LY, Tseng YH, et al. CMR-verified diffuse myocardial fibrosis is associated with diastolic dysfunction in HFpEF. JACC Cardiovasc Imaging 2014;7:991-7 9. Mohamud SF, Majleh DT, Redfield MM. Access to the microvasculature in heart failure with preserved ejection fraction. Circ Heart Fail 2016;9 10. Richards AM. Circulating biomarkers of cardiac fibrosis: what are they and what are their uses? Circ Heart Fail 2017;10

[0051] 11. Lin LY, Wu CK, Yuan JM, et al. Myocardial regional interstitial fibrosis is associated with left intraventricular dyssynchrony in patients with heart failure: a cardiovascular magnetic resonance study. Sci Rep 2016;6:20711 12. Duca F, Kamerlander AA, Zotter-Tufaro C et al. Interstitial fibrosis, functional status, and outcome in heart failure with preserved ejection fraction: insights from a prospective cardiac magnetic resonance imaging study. Circ Cardiovasc Imaging 2016;9 13. Roy C, Slimani A, Da Meester C, et al. Association and prognostic significance of diffuse myocardial fibrosis by cardiovascular magnetic resonance in heart failure with preserved ejection fraction. J Cardiovasc Magn Reson 2018;20:55 14. Shelbert EB, Fridman Y, Wong TC et al. Temporal relationship between myocardial fibrosis and heart failure with preserved ejection fraction: association with baseline disease severity and subsequent outcome. JAMA Cardiol 2017 15. Nielsen MJ, Kalsdal MA, Kazankov K et al. Fibrosis is not just fibrosis—basement membrane architecture and collagen metabolism differ between hepatitis B and hepatitis C-induced injury. Aliment Pharmacol Ther 2016;44:1242-1252

[0052] 16. Hajkowski MJ, Kouba EJ, Brubaker PH, Niklas BJ, Eggebeen J, Kitzman DW. Skeletal muscle composition and its relationship to exercise intolerance in elderly patients with heart failure and preserved ejection fraction. Am J Cardiol 2014;113:1211-6 17. Gebauer, J.M., Kobbe, B., Paulson, M. & Wagner, R. Structure, evolution, and expression of collagen XXVIII: lessons from the zebrafish. Matrix Biol, 49, 106-119 (2016) 18. Bate, G. et al. Collagen XXVIII, a novel von Willebrand factor A domain-containing protein with multiple defects in the collagen domain. J. Biol. Chem. 281, 3494-3504 (2006) 19. Annis, DS, Mosher, DF & Roberts, DD NIH Public Access. 27, 339-351 (2009) 20. Schiller, H.B. et al. Time- and compartment-resolved proteomic profiling of the extracellular niche in lung injury and repair. Mol. Syst. Biol. 11, 819-819 (2015) 21. Lai KKY, Xiang S, Roya N, Booth GC, Masse DJ, Faust N et al. (2011) Extracellular matrix dynamics in hepatocellular carcinoma formation: a comparative proteomic study of PDGFC-transduced and Pten null mouse models. PLos Genet 7(6): e1002147 https: / / doi.org / 10.1371 / journal.pgen.1002147

Claims

1. 1. An immunoassay method for detecting and / or monitoring heart failure with preserved ejection fraction (HFpEF) in a patient, the method comprising: (i) contacting a biological fluid sample from a patient with a monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) of the C-terminal epitope of type XXVIII collagen; (ii) detecting and determining the amount of binding between the monoclonal antibody and the peptide in the sample; and (iii) correlating the amount of binding of the monoclonal antibody determined in step (ii) with a value associated with a normal healthy individual and / or a value associated with a known severity of the disease and / or a value obtained from the patient at a previous time point and / or a predetermined cut-off value.

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

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

4. 4. The method of claim 1, wherein the monoclonal antibody is raised against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1).

5. 5. The method of claim 1, wherein the biological fluid is blood, serum, plasma, urine, or a supernatant obtained from a cell or tissue culture.

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

7. 7. The method of claim 1, wherein the immunoassay is a radioimmunoassay or an enzyme-linked immunosorbent assay.

8. A monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) of the C-terminal epitope of type XXVIII collagen, and at least one of the following: - streptavidin-coated well plates; - biotinylated peptide biotin-L-QETCIQG (SEQ ID NO: 4), where L is an optional linking group; - secondary antibodies used in sandwich immunoassays; a calibration peptide comprising the sequence QETCIQG (SEQ ID NO: 1); - antibody biotinylation kit; - Antibody HRP labeling kit; - antibody radiolabeling kit; and - Assay Visualization Kit 1. An immunoassay kit comprising:

9. 9. The immunoassay kit of claim 8, wherein the monoclonal antibody is raised against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1).

10. 10. The assay kit of claim 8 or 9, wherein the monoclonal antibody does not recognize or specifically bind to an extension of the C-terminal amino acid sequence that is QETCIQGA (SEQ ID NO: 2).

11. 11. The assay kit of any one of claims 8 to 10, wherein the monoclonal antibody does not recognize or specifically bind to truncations of the C-terminal amino acid sequence of QETCIQ (SEQ ID NO: 3).

12. A monoclonal antibody that specifically binds to the C-terminal amino acid sequence QETCIQG (SEQ ID NO: 1) of the C-terminal epitope of type XXVIII collagen.

13. 13. The monoclonal antibody of claim 12, which is raised against a synthetic peptide having the amino acid sequence QETCIQG (SEQ ID NO: 1).

14. 14. The monoclonal antibody of claim 12 or 13, which does not recognize or specifically bind to an extension of the C-terminal amino acid sequence QETCIQGA (SEQ ID NO: 2).

15. A monoclonal antibody according to any one of claims 12 to 14, which does not recognise or specifically bind to truncations of the C-terminal amino acid sequence QETCIQ (SEQ ID NO: 3).

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