Method for detecting circulating BMP10 (bone morphogenetic protein 10)

Abstract

The present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising determining the amount of BMP10 in a sample from the subject and comparing the amount of BMP10 with a reference amount, thereby assessing atrial fibrillation. Furthermore, the present invention relates to a method for diagnosing heart failure based on the determination of BMP10 in a sample from the subject. Furthermore, the present invention relates to a method for predicting a subject's risk of hospitalization due to heart failure based on the determination of a BMP10-type peptide in a sample from the subject. The present invention further relates to antibodies that bind to one or more BMP10-type peptides, such as NT-proBMP10.

Description

[Technical Field]

[0001] The present invention relates to a method for assessing atrial fibrillation in a subject, the method comprising the steps of determining the amount of one or more BMP10-type peptides in a sample from the subject and comparing the amount of the one or more BMP10-type peptides with a reference amount, thereby assessing atrial fibrillation. Further, the present invention relates to a method for diagnosing heart failure based on the determination of one or more BMP10-type peptides in a sample from the subject. Furthermore, the present invention relates to a method for predicting the risk of a subject being hospitalized due to heart failure based on the determination of one or more BMP10-type peptides in a sample from the subject. The present invention further relates to an antibody that binds to one or more BMP10-type peptides, such as NT-proBMP10. [Background technology]

[0002] background part Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and one of the most prevalent conditions among older adults. AF is characterized by an irregular heartbeat that often begins with brief, abnormal beats and increases over time, sometimes becoming a permanent symptom. An estimated 2.7 to 6.1 million people in the United States have AF, and approximately 33 million people worldwide have AF (Chugh, S. et al., Circulation 2014;129:837-47).

[0003] Diagnosing cardiac arrhythmias, such as atrial fibrillation, typically involves identifying the cause of the arrhythmia and classifying the arrhythmia. Guidelines for classifying atrial fibrillation by the American College of Cardiology (ACC), American Heart Association (AHA), and European Society of Cardiology (ESC) are primarily based on simplicity and clinical relevance. The first category is called "first detected AF." Individuals in this category are initially diagnosed with AF, and it is unknown whether they have previously experienced undetected episodes. If the first detected episode spontaneously terminates within one week but is followed by another episode, the category changes to "paroxysmal AF." Patients in this category have episodes lasting up to seven days, but in most cases of paroxysmal AF, the episodes terminate within 24 hours. If episodes last for more than one week, they are classified as "persistent AF." If such episodes cannot be terminated by electrical or pharmacological cardioversion and persist for more than one year, the classification changes to "persistent AF." Early diagnosis of atrial fibrillation is highly desirable because it is an important risk factor for stroke and systemic embolism (Hart et al., Ann Intern Med 2007,146(12):857-67; Go AS et al JAMA 2001;285(18):2370-5). Stroke is second only to ischemic heart disease as a cause of disability-adjusted life years lost in high-income countries and as a cause of death worldwide. To reduce the risk of stroke, anticoagulation appears to be the most appropriate treatment.

[0004] Biomarkers that allow assessment of atrial fibrillation are highly desirable.

[0005] Latini R. et al. (J Intern Med. 2011 Feb;269(2):160-71) measured various circulating biomarkers (hsTnT, NT-proBNP, MR-proANP, MR-proADM, copeptin, and CT-proendothelin-1) in patients with atrial fibrillation.

[0006] Bone morphogenetic protein 10 (abbreviated as BMP10) is a ligand of the TGF-beta (transforming growth factor-beta) superfamily of proteins. Ligands of this family bind to various TGF-beta receptors, leading to the recruitment and activation of specific transcription factors that regulate gene expression. BMP10 has been shown to bind to activin receptor-like kinase 1 (ALK1) and to be a functional activator of this kinase in endothelial cells (David et al., Blood. 2007, 109(5):1953-61).

[0007] Bone morphogenetic proteins circulate in the blood in various forms (uncleaved, processed, or complexed forms). Kienast et al., J. Biol. Chem. (2016) 293(28) 10963-10974, investigated circulating variants of BMP9 and found that mature BMP9 accounts for only 0.5% of the total BMP9 in human plasma compared to other BMP9 variants.

[0008] Human preproBMP10 contains a short signal peptide (amino acids 1-21) that is enzymatically cleaved to release the inactive precursor protein, proBMP10 (amino acids 22-424 of human preproBMP10). ProBMP10 is cleaved by proteases to yield a 108-amino acid non-glycosylated C-terminal peptide (approximately 14 kDa; BMP10, amino acids 317-424) and an approximately 50 kDa N-terminal prosegment (amino acids 22-424; Susan-Resiga et al., J Biol Chem. 2011 Jul 1;286(26):22785-94). Both mature BMP10 and the N-terminal prosegment of BMP-10 remain structurally close to form homodimers or heterodimers of BMP10 or combine with other BMP family proteins (Yadin et al., CYTOGFR 2016, 27(2016)13-34). Dimerization occurs through the formation of Cys-Cys bridges or strong adhesion in the C-terminal peptides of both binding partners. Thus, a structure consisting of two subunits is formed.

[0009] U.S. Patent No. 8,287,868 discloses isolated monoclonal antibodies that bind to mature humanized or fully human BMP10 peptides and compete with the BMP10 propeptide for binding to mature BMP10. Additional antibodies against BMP10 or its precursor are disclosed in U.S. Patent No. 5,932,216.

[0010] BMP10 has been shown to play a role in cardiovascular development, including regulation of cardiomyocyte proliferation and heart size, closure of the ductus arteriosus, angiogenesis, and ventricular trabeculation.

[0011] Because soluble BMP10 is involved in regulating tissue repair, it has also been identified as a diagnostic and treatment target involved in tissue fibrosis in cardiovascular diseases (see, e.g., U.S. Patent No. 2013209490), and the involvement of BMP10 in vascular and cardiac fibrosis has been described.

[0012] US Patent Application Publication No. 2012 / 0213782 discloses a BMP10 propeptide that can be used to treat cardiac disorders.

[0013] The general role of BMP10 is in the developmental regulation of vascular remodeling (Ricard et al., Blood. 2012 Jun 21;119(25):6162-6171). Furthermore, BMP10 is a cardiogenesis factor (Huang et al., J Clin Invest. 2012;122(10):3678-3691) and induces cardiomyocyte proliferation during myocardial infarction (Sun et al., J Cell Biochem. 2014;115(11):1868-1876). It has also been described to be derived from endothelial cells (Jiang et al., JBC 2016,291(6):2954-2966).

[0014] Transcriptome analysis reveals that in healthy conditions, BMP10 mRNA is strongly expressed in the right atrium and right atrial appendage of the heart, with expression predominantly in the right compared to the left atrial appendage (Kahr et al., Plos ONE, 2010, 6(10):e26389).

[0015] Larissa Fabritz gave a presentation on biomarkers in atrial fibrillation at the ESC (European Society of Cardiology) Congress held in Paris from August 29, 2019 to September 2, 2019. One of the biomarkers mentioned was BMP (FP number: 2365).

[0016] International patent application PCT / EP2019 / 072042 discloses circulating BMP10 (bone morphogenetic protein 10) in the assessment of atrial fibrillation.

[0017] There is a need for reliable methods for assessing atrial fibrillation, including diagnosing atrial fibrillation, risk stratifying patients with atrial fibrillation (such as for the occurrence of stroke), assessing the severity of atrial fibrillation, and assessing treatment options in patients with atrial fibrillation. Additionally, there is a need for reliable methods for assessing heart failure.

[0018] Tillet et al. (J. Biol. Chem. (2018) 293(28) 10963-10974) found that heterodimers of BMP9 and BMP10 are responsible for the majority of the biological BMP activity found in plasma.

[0019] However, Tillet et al. found lower levels of BMP10 in plasma, suggesting the presence of a masking protein in the plasma.

[0020] The technical problem underlying the present invention can be seen as the provision of a method that addresses the above-mentioned needs. This technical problem is solved by the embodiments characterized in the following claims and in this specification.

[0021] Advantageously, it has been discovered in the context of the present invention that determining the amount of one or more BMP10 peptides in a sample from a subject allows for improved assessment of atrial fibrillation and heart failure.Therefore, for example, it can be determined whether a subject suffers from atrial fibrillation or heart failure, is at risk of suffering from atrial fibrillation-related stroke, or is at risk of recurrent Afib after therapeutic intervention.

[0022] Furthermore, the studies described herein have shown that the amount of one or more BMP10-type peptides correlates with the presence of white matter lesions (WMLs) in patients. Because the degree of WMLs can be caused by clinically silent strokes (Wang Y, Liu G, Hong D, Chen F, Ji X, Cao G. White matter injury in ischemic stroke. Prog Neurobiol. 2016;141:45-60), BMP10-type peptides can be used to assess the degree of white matter lesions and to assess whether a subject has previously experienced one or more silent strokes, i.e., clinically silent strokes. Because WMLs are associated with the risk of dementia, one or more BMP10-type peptides can also be used to predict dementia, such as vascular dementia and / or Alzheimer's disease.

[0023] Advantageously, in the studies described herein, it was found that the use of antibodies directed against the amino acid region 22-316 of human preproBMP10 (i.e., directed against the N-terminal prosegment of BMP10) resulted in superior detection of BMP10-type peptides in blood, serum, or plasma samples compared with the use of antibodies directed against the mature BMP10 hormone itself. Thus, the use of a detection agent that binds to the amino acid region 22-316 of human preproBMP10, rather than mature BMP10, allows for improved assessment of atrial fibrillation and heart failure. Furthermore, the inventors identified a subregion within the amino acid region 22-316 of human preproBMP10 that is a particularly suitable target region for the detection agent. In this context, an antibody that allows for improved detection of BMP10-type peptides has been identified. The antibody can bind to any BMP10-type peptide, including NT-proBMP10 and NT-proBMP10 fragments, such as preproBMP10 and proBMP10. Summary of the Invention

[0024] SUMMARY OF THE INVENTION The present invention provides a method for assessing atrial fibrillation in a subject, comprising: a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP3 (Fatty Acid Binding Protein 3); b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one further biomarker with a reference amount of said at least one further biomarker, thereby assessing atrial fibrillation.

[0025] The present invention further relates to a method for assisting in the assessment of atrial fibrillation, the method comprising: a) providing at least one sample from a subject; b) determining in at least one sample provided in step a) the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides) and, optionally, at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 and FABP3 (fatty acid binding protein 3); c) providing a physician with information regarding the determined amount of one or more BMP10-type peptides, and optionally, information regarding the determined amount of at least one further biomarker, thereby assisting in the assessment of atrial fibrillation.

[0026] The present invention further provides a method for assisting in the assessment of atrial fibrillation, comprising: a) providing an assay for one or more BMP10-type peptides and, optionally, at least one further assay for at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP3 (fatty acid binding protein 3); b) providing instructions for the use of the assay results obtained or obtainable by said assay(s) in the assessment of atrial fibrillation.

[0027] The present invention further relates to a method for assessing the extent of white matter pathology in a subject, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) assessing the extent of white matter pathology in the subject based on the amount determined in step a).

[0028] The present invention further relates to a method for assessing whether a subject has experienced one or more silent strokes, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; b) comparing the amount determined in step a) with a standard; and c) assessing whether the subject has experienced one or more silent strokes.

[0029] The present invention further relates to a method for predicting dementia, such as vascular dementia and / or Alzheimer's disease, in a subject, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; b) comparing the amount determined in step a) with a standard; and c) predicting the risk of dementia in the subject.

[0030] The present invention also provides a method for predicting the risk of AFib recurrence after a therapeutic intervention (such as pulmonary vein isolation (PVI) or ablation therapy in atrial fibrillation) in a sample collected prior to the therapeutic intervention, comprising: a) providing an assay for one or more BMP10-type peptides and, optionally, at least one further assay for at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP3 (fatty acid binding protein 3); b) providing instructions for using the assay results obtained or obtainable by said assay(s) in assessing the risk of recurrence of atrial fibrillation.

[0031] Also provided in accordance with the present invention is a computer-implemented method for assessing atrial fibrillation, comprising: a) receiving in a processing unit a value for the amount of one or more BMP 10 type peptides and optionally at least one further value for the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP3 (fatty acid binding protein 3), wherein the amount of the one or more BMP 10 type peptides and optionally the amount of the at least one further biomarker have been determined in a sample derived from a subject, b) comparing, by said processing unit, the value(s) received in step (a) with reference(s); and c) assessing atrial fibrillation based on the comparing step b).

[0032] The present invention further relates to a method for diagnosing heart failure, the method comprising: (a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 and FABP3 (fatty acid binding protein 3); (b) diagnosing heart failure by comparing the amount of one or more BMP10-type peptides to a reference amount for the BMP10-type peptide, and optionally, comparing the amount of at least one additional biomarker to a reference amount for the at least one additional biomarker.

[0033] The present invention further relates to a method for predicting a subject's risk of hospitalization due to heart failure, the method comprising: (a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 and FABP3 (fatty acid binding protein 3); (b) comparing the amount of one or more BMP10-type peptides to a reference amount, and optionally comparing the amount of at least one additional biomarker to a reference amount of said at least one additional biomarker; and (c) predicting the subject's risk of hospitalization due to heart failure.

[0034] In a preferred embodiment of the method of the present invention, determining the amount of one or more BMP10-type peptides comprises contacting the sample with at least one agent that binds to (i.e., is capable of binding to) the N-terminal prosegment of BMP10 (NT-proBMP10, also referred to as the N-terminal prodomain of BMP10). It therefore does not bind to mature BMP10. Thus, the agent binds to (i.e., is capable of binding to) the amino acid region 22-316 of the polypeptide set forth in SEQ ID NO:1. For example, the sample is contacted with at least one agent that binds to the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:1.

[0035] The present invention further relates to a method for determining the amount of one or more BMP10-type peptides, comprising the steps of contacting a sample containing one or more BMP10-type peptides with at least one agent that binds to the N-terminal prosegment of BMP10, and thus within the amino acid region 22 to 316 of the polypeptide set forth in SEQ ID NO: 1, thereby allowing the formation of a complex between the BMP10-type peptide and the at least one agent, and determining the amount of complex formed.

[0036] In some embodiments, the method for determining the amount of one or more BMP10-type peptides is performed by sandwich immunoassay.

[0037] The present invention further relates to a kit comprising at least one agent that specifically binds to one or more BMP10-type peptides, such as an agent that binds within the amino acid region 22 to 316 of the polypeptide set forth in SEQ ID NO: 1, and at least one further agent selected from the group consisting of an agent that specifically binds to a natriuretic peptide, an agent that specifically binds to ESM-1, an agent that specifically binds to Ang2, and an agent that specifically binds to FABP3.

[0038] Furthermore, the present invention relates to an in vitro use, i) one or more BMP10-type peptides and, optionally, at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP3 (fatty acid binding protein 3), and / or ii) Use of at least one agent that specifically binds to one or more BMP10-type peptides, and optionally at least one further agent selected from the group consisting of an agent that specifically binds to a natriuretic peptide, an agent that specifically binds to ESM-1, an agent that specifically binds to Ang2, and an agent that specifically binds to FABP3, for assessing atrial fibrillation, predicting the risk of stroke or diagnosing heart failure, or predicting the risk of a subject being hospitalized due to heart failure.

[0039] In a preferred embodiment of the aforementioned use, the at least one agent that specifically binds to one or more BMP10-type peptides is an agent that binds to the amino acid region 22 to 316 of NT-proBMP10, i.e., the polypeptide set forth in SEQ ID NO: 1. The amino acid sequence of NT-proBMP10 is also set forth in SEQ ID NO: 6.

[0040] The present invention further provides agents, such as antibodies or antigen-binding fragments thereof, that bind (i.e., are capable of binding) within the amino acid region 22 to 316 of the polypeptide set forth in SEQ ID NO: 1. In some embodiments, the antibody is a monoclonal antibody.

[0041] In some embodiments, the agent is an agent that binds to an epitope contained within amino acid region 37-47 of the polypeptide set forth in SEQ ID NO:1 (SLFGDVFSEQD, SEQ ID NO:2).

[0042] In some embodiments, the agent binds to an epitope contained in the amino acid region 171-185 of SEQ ID NO:1 (LESKGDNEGERNMLV, SEQ ID NO:3), for example, an agent that binds to an epitope contained in the amino acid region 173-181 of SEQ ID NO:1 (SKGDNEGER, SEQ ID NO:4).

[0043] In some embodiments, the agent is an agent that binds to an epitope contained within amino acid region 291-299 of SEQ ID NO:1 (SSGPGEEAL, SEQ ID NO:5).

[0044] The present invention also relates to antibodies (eg, monoclonal antibodies) or fragments thereof that bind to one or more BMP10-type peptides.

[0045] In one embodiment, the antibody or fragment thereof comprises a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 (see Table A) and / or a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 (see Table B), in order of increasing preference.

[0046] Additionally or alternatively, the antibody or fragment thereof of the present invention comprises: (a) (a1) a light chain CDR1 shown in Table D (thus, a light chain CDR1 sequence selected from SEQ ID NOs: 34 to 42); (a2) a light chain CDR2 shown in Table D (thus, a light chain CDR2 sequence selected from SEQ ID NOs: 52 to 60), and (a3) Light chain CDR3 shown in Table D (therefore, a light chain CDR3 sequence selected from SEQ ID NOs: 70 to 78) a light chain variable domain comprising and (b) (b1) a heavy chain CDR1 shown in Table C (thus, a heavy chain CDR1 sequence selected from SEQ ID NOs: 25 to 33); (b2) a heavy chain CDR2 shown in Table C (thus, a heavy chain CDR2 sequence selected from SEQ ID NOs: 43 to 51), and (b3) Heavy chain CDR3 shown in Table C (thus, heavy chain CDR3 sequence selected from SEQ ID NOs: 61 to 69) a heavy chain variable domain comprising: DETAILED DESCRIPTION OF THE INVENTION

[0047] Detailed Description of the Invention / Definitions The present invention provides a method for assessing atrial fibrillation in a subject, comprising: a) determining the amount of one or more BMP10 peptides (bone morphogenetic protein type 10 peptides) in at least one sample from the subject; and b) The amount of BMP10-type peptide is compared with a reference amount for BMP10-type peptide, thereby assessing atrial fibrillation.

[0048] The BMP10-type peptide is preferably selected from the group consisting of BMP10, the N-terminal prosegment of BMP10 (N-terminal proBMP10), proBMP10, and preproBMP10. More preferably, the BMP10-type peptide is selected from the group consisting of the N-terminal prosegment of BMP10 (N-terminal proBMP10), proBMP10, and preproBMP10. Even more preferably, the BMP10-type peptide is proBMP10 and / or N-terminal proBMP10. Most preferably, the BMP10-type peptide is NT-proBMP10.

[0049] According to the present invention, the amount of one or more BMP10-type peptides is determined. As described elsewhere herein, the determination is preferably carried out using at least one agent that binds to NT-proBMP10 or a specific subregion thereof. This allows the amount of all (i.e., the combined) BMP10-type peptides containing the NT-proBMP10 peptide sequence, such as proBMP10, preproBMP10, and NT-proBMP10, to be determined. Therefore, the phrase "determining the amount of one or more BMP10-type peptides" preferably means that the total amount (i.e., the sum of their amounts) of polypeptides having an amino acid sequence containing the NT-proBMP10 amino acid sequence is determined, and in particular, the total amount (i.e., the sum of their amounts) of proBMP10, preproBMP10, and NT-proBMP10 is determined. Since preproBMP10 is generally absent in many samples, this phrase can also mean that the total amount of NT-proBMP10 and proBMP10 is determined. Thus, the amount of NT-proBMP10 and / or BMP10 is determined.

[0050] In one embodiment of the method of the invention, the method further comprises determining the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 (angiopoietin 2) and FABP-3 (fatty acid binding protein 3) in a sample from the subject in step a), and comparing the amount of the at least one further biomarker to a reference amount in step b).

[0051] Accordingly, the present invention provides a method for assessing atrial fibrillation in a subject, comprising: a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); b) comparing the amount of one or more BMP10-type peptides with a reference amount of BMP10-type peptides, and optionally comparing the amount of at least one further biomarker with a reference amount of said at least one further biomarker, thereby assessing atrial fibrillation.

[0052] The assessment of atrial fibrillation (AF) shall be based on the results of comparison step b).

[0053] Therefore, the method of the present invention preferably comprises: a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides and, optionally, the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3), b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one further biomarker with a reference amount of said at least one further biomarker; and c) assessing atrial fibrillation based on the results of the comparison step b).

[0054] The method referred to by the present invention includes a method consisting essentially of the above-mentioned steps or a method including additional steps. Furthermore, the method of the present invention is preferably an ex vivo method, more preferably an in vitro method. Furthermore, it may include steps in addition to those explicitly mentioned above. For example, the additional steps may involve determining further markers and / or taking pre-treatment samples or evaluating the results obtained by the method. This method may be performed manually or may be assisted by automation. Preferably, steps (a), (b) and / or (c) may be assisted, in whole or in part, by automation, for example, by suitable robots and sensory devices for the determination in step (a) or the computer-implemented calculation in step (b).

[0055] In accordance with the present invention, atrial fibrillation is assessed. As used herein, the term "assessing atrial fibrillation" preferably refers to diagnosing atrial fibrillation, diagnosing a recent episode of AF, distinguishing between paroxysmal and persistent atrial fibrillation, predicting the risk of adverse events associated with atrial fibrillation (e.g., stroke and / or recurrence of atrial fibrillation, e.g., recurrence of AF after an intervention), identifying subjects to undergo electrocardiography (ECG), or assessing atrial fibrillation therapy.

[0056] As will be understood by those skilled in the art, the assessment of the present invention is not intended to be accurate for 100% of the subjects to be tested. This term preferably requires that a correct assessment (e.g., diagnosis, differentiation, prediction, discrimination, or assessment of a treatment as used herein) can be made for a statistically significant portion of the subjects. Whether a portion is statistically significant can be determined without further ado by those skilled in the art using various well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, or Mann-Whitney test. Details are described in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.4, 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0057] According to the present invention, the expression "assessment of atrial fibrillation" is understood as an aid in the assessment of atrial fibrillation, and therefore as an aid in the diagnosis of atrial fibrillation, an aid in distinguishing between paroxysmal and persistent atrial fibrillation, an aid in predicting the risk of adverse events associated with atrial fibrillation, an aid in identifying subjects to undergo electrocardiography (ECG), or an aid in the assessment of a treatment for atrial fibrillation. The final diagnosis is generally made by a physician.

[0058] In a preferred embodiment of the present invention, the assessment of atrial fibrillation is a diagnosis of atrial fibrillation, thus diagnosing whether the subject suffers from atrial fibrillation.

[0059] Accordingly, the present invention provides a method for diagnosing atrial fibrillation in a subject, comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides to a reference amount, thereby diagnosing atrial fibrillation.

[0060] In one embodiment, the method described above further comprises: (a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2), and FABP-3 (Fatty Acid Binding Protein 3); and (b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker, thereby diagnosing atrial fibrillation.

[0061] Preferably, the subject to be examined in connection with the method for diagnosing atrial fibrillation is a subject suspected of having atrial fibrillation. However, it is also contemplated that the subject may have previously been diagnosed with AF, and that the previous diagnosis may be confirmed by carrying out the method of the present invention.

[0062] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is a distinction between paroxysmal and persistent atrial fibrillation, thus determining whether the subject suffers from paroxysmal or persistent atrial fibrillation.

[0063] Accordingly, the present invention provides a method for distinguishing between paroxysmal and persistent atrial fibrillation in a subject, comprising:

[0064] a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides to a reference amount, thereby distinguishing between paroxysmal and persistent atrial fibrillation.

[0065] In one embodiment, the method described above further comprises: a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker, thereby distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation.

[0066] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is a prediction of the risk of an adverse event (such as stroke) associated with atrial fibrillation, and thus the subject is predicted to be at risk or not of suffering from the adverse event.

[0067] Accordingly, the present invention provides a method for predicting the risk of an adverse event associated with atrial fibrillation in a subject, comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides to a reference amount, thereby predicting the risk of an adverse event associated with atrial fibrillation.

[0068] In one embodiment, the method described above further comprises: a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker, thereby predicting the risk of an adverse event associated with atrial fibrillation.

[0069] It is envisioned that a variety of adverse events can be predicted, with a preferred adverse event being stroke.

[0070] Therefore, the present invention relates in particular to a method for predicting the risk of stroke in a subject, comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides to a reference amount, thereby predicting the risk of stroke.

[0071] The above method may further comprise step c) of predicting stroke based on the comparison result of step b). Accordingly, steps a), b) and c) are preferably as follows: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; b) comparing the amount of one or more BMP10-type peptides with a reference amount; and c) predicting stroke based on the comparison result of step b).

[0072] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is an assessment of a therapy for atrial fibrillation.

[0073] Accordingly, the present invention provides a method for assessing a therapy for atrial fibrillation in a subject, comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides to a reference amount, thereby assessing a treatment for atrial fibrillation.

[0074] In one embodiment, the method described above further comprises: a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); and b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker, thereby assessing a treatment for atrial fibrillation.

[0075] Preferably, the subjects relevant to the above-mentioned distinction, the above-mentioned prediction, and the assessment of a treatment for atrial fibrillation are subjects who are known to suffer from atrial fibrillation, in particular subjects who are known to suffer from atrial fibrillation (and therefore have a medical history of atrial fibrillation). However, it is also envisaged that, with respect to the above-mentioned prediction methods, the subjects do not have a medical history of atrial fibrillation.

[0076] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is the identification of subjects who should be subjected to electrocardiography (ECG), so that subjects who should be subjected to electrocardiography are identified.

[0077] This method is a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); and b) comparing the amount of one or more BMP10 peptides with a reference amount of one or more BMP10 peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker, thereby identifying subjects to undergo electrocardiogram testing.

[0078] Preferably, the subject in relation to the above-described method of identifying a subject who should be subjected to electrocardiography is a subject who has no history of atrial fibrillation, the term "no history of atrial fibrillation" being defined elsewhere herein.

[0079] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is an assessment of the effectiveness of the subject's anticoagulant therapy, thus assessing the effectiveness of said therapy.

[0080] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is predictive of the risk of stroke in the subject, and thus predicting whether the subject is at risk of stroke as referred to herein.

[0081] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is the identification of a subject who is eligible for administration of at least one anticoagulant or who is eligible for an increased dose of at least one anticoagulant, and thus whether the subject is eligible for said administration and / or said increased dose is assessed.

[0082] In another preferred embodiment of the present invention, the assessment of atrial fibrillation is the monitoring of anticoagulation therapy, thus assessing whether the subject will respond to said therapy.

[0083] The term "atrial fibrillation" (abbreviated as AF or AFib) is well known in the art. As used herein, this term preferably refers to supraventricular tachyarrhythmia, characterized by uncoordinated atrial activity, resulting in deterioration of the mechanical function of the atria. In particular, this term refers to an abnormal heart rhythm characterized by rapid and irregular beating. The term involves the two atria of the heart. In normal heart rhythm, excitation generated by the sinoatrial node spreads throughout the heart, causing myocardial contraction and blood ejection. In atrial fibrillation, the regular electrical impulses of the sinoatrial node are replaced by chaotic and rapid electrical impulses, which cause irregular heartbeats. Symptoms of atrial fibrillation include heart palpitations, fainting, shortness of breath, or chest pain. However, most attacks are asymptomatic. On the electrocardiogram, atrial fibrillation is characterized by the replacement of uniform P waves by rapid oscillations or fibrillation waves that vary in amplitude, shape, and timing associated with irregular and frequently rapid ventricular responses when atrioventricular conduction is intact.

[0084] The American College of Cardiology (ACC), the American Heart Association (AHA), and the European Society of Cardiology (ESC) have proposed the following classification systems: initially detected AF, paroxysmal AF, persistent AF, and permanent AF (see Fuster V. et al., Circulation 2006;114(7):e257-354, which is hereby incorporated by reference in its entirety, see e.g., Figure 3 therein): initially detected AF, paroxysmal AF, persistent AF, and permanent AF.

[0085] All people suffering from AF initially fall into a category called first detected AF. However, subjects may or may not have previously experienced undetected episodes. If atrial fibrillation persists for more than one year, the subject suffers from permanent AF, specifically without conversion back to sinus rhythm (or only with medical intervention). If AF persists for more than seven days, the subject suffers from persistent AF. The subject may require either pharmacological or electrical intervention to terminate atrial fibrillation. Preferably, persistent AF occurs in episodes, but the arrhythmia does not spontaneously (i.e., without medical intervention) convert back to sinus rhythm. Paroxysmal atrial fibrillation refers to intermittent episodes of atrial fibrillation that preferably last up to seven days. In most cases of paroxysmal AF, the episodes last less than 24 hours. The atrial fibrillation episodes terminate spontaneously, i.e., without medical intervention. Therefore, the episode(s) of paroxysmal atrial fibrillation preferably terminate spontaneously, whereas persistent atrial fibrillation preferably does not. Preferably, persistent atrial fibrillation requires electrical or pharmacological cardioversion or other procedures, such as ablation procedures, for termination (Fuster V. et al., Circulation 2006;114(7):e257-354). Both persistent and paroxysmal AF can recur, whereby the distinction between paroxysmal and persistent AF is provided by ECG recording. AF is considered recurrent when a patient has had two or more episodes. When the arrhythmia terminates spontaneously, AF, especially recurrent AF, is called paroxysmal. AF is called persistent if it persists for more than seven days.

[0086] In a preferred embodiment of the present invention, the term "paroxysmal atrial fibrillation" is defined as a self-terminating episode of AF, which lasts less than 24 hours. In an alternative embodiment, a self-terminating episode lasts up to 7 days.

[0087] As used herein, a "subject" is preferably a mammal. Mammals include, but are not limited to, domesticated animals (e.g., cows, sheep, cats, dogs, horses, etc.), primates (e.g., humans, non-human primates such as monkeys), rabbits, and rodents (e.g., mice, rats, etc.). Preferably, the subject is a human subject.

[0088] Preferably, the subject being tested is of any age, more preferably the subject being tested is over 50 years old, more preferably over 60 years old, and most preferably over 65 years old. It is further contemplated that the subject being tested is over 70 years old.

[0089] Furthermore, it is envisioned that the subjects being tested will be over the age of 75. The subjects may also be between the ages of 50 and 90.

[0090] In a preferred embodiment of the method of the present invention for assessing atrial fibrillation, the subject to be tested is suffering from atrial fibrillation.Therefore, the subject must have a history of atrial fibrillation.Therefore, the subject must have had an attack of atrial fibrillation before obtaining test sample, and at least one of the previous attacks of atrial fibrillation must be diagnosed, for example, by ECG.For example, when assessing atrial fibrillation, it is considered that the subject is suffering from atrial fibrillation if the assessment is to distinguish between paroxysmal atrial fibrillation and persistent atrial fibrillation, or when the assessment is to predict the risk of adverse events related to atrial fibrillation, or when the assessment is to assess the treatment of atrial fibrillation.

[0091] In another preferred embodiment of the method for assessing atrial fibrillation, for example, where the assessment of atrial fibrillation is a diagnosis of atrial fibrillation or identification of a subject who should undergo electrocardiography (ECG), the subject being tested is suspected of suffering from atrial fibrillation.

[0092] Preferably, the subject suspected of suffering from atrial fibrillation has shown at least one symptom of atrial fibrillation before carrying out the method for assessing atrial fibrillation.The symptom is usually transient, occurring within a few seconds and disappearing just as quickly.Symptoms of atrial fibrillation include dizziness, fainting, shortness of breath, and especially heart palpitations.Preferably, the subject has shown at least one symptom of atrial fibrillation within 6 months before obtaining sample.

[0093] Alternatively or additionally, subjects suspected of having atrial fibrillation are 70 years of age or older.

[0094] Preferably, a subject suspected of having atrial fibrillation has no history of atrial fibrillation.

[0095] According to the present invention, a subject without a history of atrial fibrillation is preferably a subject who has not been previously diagnosed with atrial fibrillation, i.e., before carrying out the method of the present invention (particularly before obtaining a sample from the subject), although the subject may or may not have had a previously undiagnosed episode of atrial fibrillation.

[0096] Preferably, the term "atrial fibrillation" refers to all types of atrial fibrillation, and thus preferably encompasses paroxysmal, persistent, or permanent atrial fibrillation.

[0097] However, in one embodiment of the present invention, the subject being tested does not suffer from permanent atrial fibrillation, and in this embodiment, the term "atrial fibrillation" refers only to paroxysmal and persistent atrial fibrillation.

[0098] However, in another embodiment of the invention, the subject being examined does not suffer from paroxysmal and persistent atrial fibrillation, and in this embodiment, the term "atrial fibrillation" refers only to persistent atrial fibrillation.

[0099] The test subject may or may not be experiencing an episode of atrial fibrillation when the sample is taken. Accordingly, in a preferred embodiment for assessing atrial fibrillation (e.g., diagnosing atrial fibrillation), the subject is not experiencing an episode of atrial fibrillation when the sample is obtained. In this embodiment, the subject is assumed to have normal sinus rhythm (and thus be in sinus rhythm) when the sample is obtained. Therefore, atrial fibrillation can be diagnosed using biomarkers even when atrial fibrillation is (temporarily) absent on the ECG. According to the method of the present invention, the elevation of the biomarkers mentioned herein should be preserved after an episode of atrial fibrillation, thus providing a diagnosis of a subject suffering from atrial fibrillation ("memory effect"). Preferably, AF is diagnosed within about 3 days, about 1 week, about 1 month, about 3 months, or about 6 months after the method of the present invention is performed (or, more precisely, after the sample is obtained). In a preferred embodiment, a diagnosis of atrial fibrillation can be made within about 6 months after the episode. In a preferred embodiment, the diagnosis of atrial fibrillation can be performed within about six months after the attack. Therefore, the assessment of atrial fibrillation as used herein, particularly the diagnosis, risk prediction, or differentiation as used herein in connection with the assessment of atrial fibrillation, is preferably performed about three days, more preferably about one month, even more preferably about three months, and most preferably about six months after the last attack of atrial fibrillation. Accordingly, it is contemplated that the sample to be tested is preferably obtained about three days, more preferably about one month, even more preferably about three months, and most preferably about six months after the last attack of atrial fibrillation. Therefore, the diagnosis of atrial fibrillation preferably also encompasses the diagnosis of an attack of atrial fibrillation that occurred within about three days, more preferably about one week, even more preferably about three months, and most preferably about six months before obtaining the sample. Thus, the present invention allows for the diagnosis of a recent attack of AF, such as an attack that occurred within about three days or more preferably about one week before performing the method of the present invention (or, more precisely, before obtaining the sample to be tested). Additionally, the most recent bout of AF may have occurred approximately two weeks prior to performing the methods of the present invention.

[0100] Thus, the present invention makes it possible to aid in the diagnosis of a recent episode of atrial fibrillation in a subject in sinus rhythm, comprising determining the amount of one or more BMP10 peptides (and optionally at least one further biomarker described elsewhere herein) and comparing the amount(s) thus determined with a reference amount(s). In one embodiment, the subject is suspected of having recently suffered from an AF episode; for example, the subject may have recently shown symptoms of AF (e.g., within 3 days, 1 week, or 2 weeks prior to carrying out the method of the present invention). Symptoms of atrial fibrillation include heart palpitations, fainting, shortness of breath, or chest pain.

[0101] However, it is also contemplated that the subject may be experiencing an episode of atrial fibrillation at the time the sample is obtained (e.g., for stroke prediction).

[0102] The term "sample" refers to a sample of a bodily fluid, a sample of separated cells, or a sample from a tissue or organ. Bodily fluid samples can be obtained by well-known techniques and include samples of blood, plasma, serum, urine, lymph, sputum, ascites, or other bodily secretions or derivatives thereof. Tissue or organ samples may be obtained from any tissue or organ, for example, by biopsy. Separated cells may be obtained from bodily fluids or tissues or organs by separation techniques such as centrifugation or cell sorting. For example, cell, tissue, or organ samples may be taken from cells, tissues, or organs that express or produce biomarkers. Samples can be frozen, fresh, fixed (e.g., formalin-fixed), centrifuged, and / or embedded (e.g., paraffin-embedded), etc. Of course, cell samples can be subjected to a variety of well-known post-collection fractionation and storage techniques (e.g., nucleic acid and / or protein extraction, fixation, storage, freezing, ultrafiltration, concentration, evaporation, centrifugation, etc.) before assessing the amount of biomarker(s) in the sample.

[0103] In a preferred embodiment of the present invention, the sample is a blood (i.e., whole blood), serum, or plasma sample. Serum is the liquid fraction of whole blood obtained after the blood has been allowed to clot. To obtain serum, the clot is removed by centrifugation, and the supernatant is then collected. Plasma is the cell-free fluid portion of blood. To obtain a plasma sample, whole blood is collected in an anticoagulant-treated tube (e.g., a citrate- or EDTA-treated tube). Cells are removed from the sample by centrifugation, and the supernatant (i.e., the plasma sample) is obtained.

[0104] As noted above, the subject may be in sinus rhythm or may be suffering from an episode of AF rhythm at the time the sample is obtained.

[0105] BMP10-type peptides are well known in the art. Preferred BMP10-type peptides are disclosed, for example, in Susan-Resiga et al. (J Biol Chem. 2011 Jul 1; 286(26): 22785-94), the entire contents of which are incorporated herein by reference (see, for example, Figure 3A of Susan-Resiga et al., or U.S. Patent Application Publication No. 2012 / 0213782).

[0106] In one embodiment, the BMP10-type peptide is unprocessed preproBMP10 (see SEQ ID NO: 1 below). In another embodiment, the BMP10-type peptide is the propeptide proBMP10. This marker comprises an N-terminal prosegment and BMP10. In another embodiment, the BMP10-type peptide is the N-terminal prosegment of BMP10 (N-terminal proBMP10 or NT-proBMP10).

[0107] In one embodiment, the BMP10-type peptide is part of a homo- or heterodimeric complex.

[0108] Human preproBMP10 (i.e., unprocessed preproBMP10) has a length of 424 amino acids. The amino acid sequence of human preproBMP10 is shown, for example, in SEQ ID NO: 1 or FIG. 3 of U.S. Patent Application Publication No. 2012 / 0213782, which is incorporated herein by reference in its entirety. The amino acid sequence of preproBMP10 can also be assessed by Uniprot (see the sequence under accession number O95393-1).

[0109] An exemplary amino acid sequence of human preproBMP10 is shown in SEQ ID NO:1.

[0110] MGSLVLTLCALFCLAAYLVSG SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMK DEFLKTLNLSDIPTQDSAKVDPPEYMLELYNKFATDRTSMPSANIIRSFKNEDLFSQPV SFNGLRKYPLLFNVSIPHHEEVIMAELRLYTLVQRDRMIYDGVDRKITIFEVLESKGDN EGERNMLVLVSGEIYGTNSEWETFDVTDAIRRWQKSGSSTHQLEVHIESKHDEAEDASS GRLEIDTSAQNKHNPLLIVFSDDQSSDKERKEELNEMISHEQLPELDNLGLDSFSSGPGE EALLQMRSNIIYDSTARIRR NAKGNYCKRTPLYIDFKEIGWDSWIIAPPGYEAYECRGVC NYPLAEHLTPTKHAIIQALVHLKNSQKASKACCVPTKLEPISILYLDKGVVTYKFKYEGM AVSECGCR

[0111] Human preproBMP10 contains a short signal peptide (amino acids 1-21) that is enzymatically cleaved to release proBMP10 (the signal peptide is italicized in the sequence above). Thus, human proBMP10 contains amino acids 22-424 of human preproBMP10 (i.e., a polypeptide having the sequence shown in SEQ ID NO: 1). Human proBMP10 is further cleaved into an N-terminal prosegment of BMP10 and active (unglycosylated) BMP10. The N-terminal prosegment of BMP10 contains amino acids 22-316 of a polypeptide having the sequence shown in SEQ ID NO: 1 (i.e., of human preproBMP10). In the sequence above, the sequence of NT-proBMP10 is underlined. Thus, NT-proBMP10 has the following sequence (SEQ ID NO: 6): SPIMNLEQSPLEEDMSLFGDVFSEQDGVDFNTLLQSMK DEFLKTLNLSDIPTQDSAKVDPPEYMLELYNKFATDRTSMPSANIIRSFKNEDLFSQPV SFNGLRKYPLLFNVSIPHHEEVIMAELRLYTLVQRDRMIYDGVDRKITIFEVLESKGDN EGERNMLVLVSGEIYGTNSEWETFDVTDAIRRWQKSGSSTHQLEVHIESKHDEAEDASS GRLEIDTSAQNKHNPLLIVFSDDQSSDKERKEELNEMISHEQLPELDNLGLDSFSSGPGE EALLQMRSNIIYDSTARIRR

[0112] BMP10 comprises amino acids 317 to 424 of a polypeptide having the sequence shown in SEQ ID NO: 1 (shown in bold in the sequence above).

[0113] Preferred BMP10-type peptides are proBMP10 and N-terminal proBMP10. After proBMP10 cleavage, BMP10 and N-terminal proBMP10 remain structurally close to form BMP10 homodimers or heterodimers, or remain combined with other BMP family proteins (Yadin et al., CYTOGFR 2016, 27 (2016) 13-34). Dimerization occurs through the formation of Cys-Cys bridges or strong adhesion in the C-terminal peptides of both binding partners. Thus, a structure consisting of two subunits is formed.

[0114] Preferably, the amount of BMP10-type peptide is determined by using at least one agent that specifically binds to the BMP10-type peptide, for example, one or more antibodies (or antigen-binding fragments thereof) that specifically bind to the BMP10-type peptide.

[0115] As described above, the research underlying the present invention showed that the use of antibodies against the amino acid region 22-316 of human preproBMP10 resulted in better detection of BMP10-type peptides compared to antibodies against the mature BMP10 hormone itself.

[0116] Thus, it is specifically contemplated to determine the amount of one or more BMP10-type peptides by using at least one agent that binds to the N-terminal prosegment of BMP10, and thus binds to the amino acid region 22-316 of SEQ ID NO: 1, i.e., within the amino acid region from amino acid 22 to amino acid 316 of the polypeptide having the amino acid sequence set forth in SEQ ID NO: 1. Thus, the agent shall bind to an epitope contained within this region, such as an epitope described elsewhere herein or set forth in the table of Example 12.

[0117] SEQ ID NO: 1 is the sequence of human preproBMP10. This sequence is used herein as a reference sequence. As used herein, an agent is defined as binding to a specific region within a polypeptide having the sequence set forth in SEQ ID NO: 1, e.g., amino acid region 37-299 or region 171-185 of SEQ ID NO: 5. This means that the agent binds to the regions of BMP10 corresponding to these regions. For example, proBMP10 and NT-proBMP10 lack the first 21 amino acids of human preproBMP10. Thus, the agent binds to amino acid region 16-278 and region 150-164 of NT-proBMP10 or proBMP10.

[0118] A subregion within amino acid region 22-316 of SEQ ID NO: 1 that is particularly suitable for detecting BMP10-type peptides was identified.

[0119] In a preferred embodiment, at least one active agent (such as an antibody or fragment thereof) that binds to a BMP10-type peptide binds within the amino acid region 37 to 299 of SEQ ID NO: 1, i.e., the at least one active agent binds to an epitope contained in the region starting at amino acid 37 and ending at amino acid 299 of SEQ ID NO: 1.

[0120] In another preferred embodiment, at least one agent binds within the amino acid region 110-200 of SEQ ID NO: 1, ie, the at least agent binds to an epitope contained in this region.

[0121] In another preferred embodiment, the at least one active substance binds within the amino acid region 37 to 185, such as within the amino acid region 37 to 195 of SEQ ID NO: 1, i.e., the at least one active substance binds to an epitope contained in this region.

[0122] In another preferred embodiment, the at least one active substance binds within the amino acid region 171 to 299, such as within the amino acid region 160 to 299 of SEQ ID NO: 1, i.e., the at least one active substance binds to an epitope contained in this region.

[0123] In another preferred embodiment, the at least one agent binds within the amino acid region 160-195 of SEQ ID NO:1, such as within the amino acid region 171-185.

[0124] In a further preferred embodiment, the at least one agent binds to an epitope (SLFGDVFSEQD, SEQ ID NO: 2) comprised in amino acid region 37-47 of SEQ ID NO: 1. In one embodiment, the agent's epitope consists essentially of SLFGDVFSEQD (SEQ ID NO: 2).

[0125] In a further preferred embodiment, the at least one agent binds to an epitope (LESKGDNEGERNMLV, SEQ ID NO: 3) comprised in the amino acid region 171-185 of SEQ ID NO: 1. For example, the at least one agent binds to an epitope (SKGDNEGER, SEQ ID NO: 4) comprised in the amino acid region 173-181 of SEQ ID NO: 1. In one embodiment, the agent's epitope consists essentially of SKGDNEGER (SEQ ID NO: 4).

[0126] In a further preferred embodiment, the at least one agent binds to an epitope (SSGPGEEAL, SEQ ID NO: 5) comprised in amino acid region 291 to 299 of SEQ ID NO: 1. In one embodiment, the agent's epitope consists essentially of SSGPGEEAL (SEQ ID NO: 5).

[0127] In a preferred embodiment, one or more antibodies or antigen-binding fragments thereof that specifically bind to the N-terminal prosegment of BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the total amount of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10 can be determined in step a) of the method of the present invention. Therefore, the phrase "determining the amount of the N-terminal prosegment of BMP10" also means "determining the total amount of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10." Because preproBMP10 is essentially absent in many samples, this phrase can also mean "determining the total amount of the N-terminal prosegment of BMP10 and preproBMP10."

[0128] For example, one or more antibodies that specifically bind to BMP10 can be used. Since such antibodies (or fragments) also bind to proBMP10 and preproBMP10, the total amount of BMP10, proBMP10, and preproBMP10 is determined in step a) of the method of the present invention. Therefore, the expression "determining the amount of BMP10" also means "determining the total amount of BMP10, proBMP10, and preproBMP10."

[0129] Furthermore, it is envisaged to determine the total amount of all four BMP10-type peptides mentioned above, namely BMP10, the N-terminal prosegment of BMP10, proBMP10 and preproBMP10.

[0130] Thus, according to the invention, the following amounts of BMP10 type peptides can be determined: Amount of BMP10 Amount of N-terminal prosegment of BMP10 Amount of proBMP10 Amount of preproBMP10 The total amount of BMP10, proBMP10, and preproBMP10 the sum of the amounts of the N-terminal prosegment of BMP10, proBMP10 and preproBMP10, or The total amount of BMP10, the N-terminal prosegment of BMP10, proBMP10, and preproBMP10

[0131] In particular, according to the invention, the following amounts of BMP10 type peptides can be determined: Amount of N-terminal prosegment of BMP10 Amount of proBMP10 Amount of preproBMP10 the sum of the amounts of the N-terminal prosegment of BMP10, proBMP10 and preproBMP10, or The sum of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10

[0132] The term "natriuretic peptide" includes atrial natriuretic peptide (ANP)-type and brain natriuretic peptide (BNP)-type peptides. Thus, natriuretic peptides according to the present invention include ANP-type and BNP-type peptides, as well as variants thereof (see, e.g., Bonow RO. et al., Circulation 1996;93:1946-1950).

[0133] ANP-type peptides include pre-proANP, proANP, NT-proANP, and ANP.

[0134] BNP-type peptides include pre-proBNP, proBNP, NT-proBNP, and BNP.

[0135] The prepropeptide (134 amino acids for pre-proBNP) contains a short signal peptide and is enzymatically cleaved to release the propeptide (108 amino acids for proBNP), which is further cleaved into the N-terminal propeptide (NT-propeptide, 76 amino acids for NT-proBNP) and the active hormone (32 amino acids for BNP and 28 amino acids for ANP).

[0136] Preferred natriuretic peptides according to the present invention are NT-proANP, ANP, NT-proBNP, and BNP. ANP and BNP are active hormones and have a shorter half-life than their respective inactive counterparts, NT-proANP and NT-proBNP. BNP is metabolized in the blood, whereas NT-proBNP circulates in the blood as an intact molecule and, as such, is released by the kidney.

[0137] The most preferred natriuretic peptides according to the present invention are NT-proBNP and BNP, particularly NT-proBNP. As briefly discussed above, human NT-proBNP according to the present invention is preferably a polypeptide comprising 76 amino acids in length, corresponding to the N-terminal portion of the human NT-proBNP molecule. The structures of human BNP and NT-proBNP have already been described in detail in the prior art, for example, in WO 02 / 089657 and WO 02 / 083913, and Bonow RO. Et al., New Insights into the cardiac natriuretic peptides. Circulation 1996;93:1946-1950. Preferably, the human NT-proBNP used herein is the human NT-proBNP disclosed in EP 0648228 B1.

[0138] As used herein, the term "FABP-3" refers to fatty acid binding protein 3. FABP-3 is also known as cardiac fatty acid binding protein or heart-type fatty acid binding protein (abbreviated as H-FABP). Preferably, this term also includes variants of FABP-3. FABP-3 as used herein preferably relates to human FABP-3. The DNA sequence of a polypeptide encoding human FABP-3 polypeptide as well as the protein sequence of human FABP-3 are well known in the art and were first described by Peeters et al. (Biochem. J. 276 (Pt 1), 203-207 (1991)). Furthermore, the sequence of human H-FABP can preferably be found in Genbank entries U57623.1 (cDNA sequence) and AAB02555.1 (protein sequence). The main physiological function of FABP is thought to be the transport of free fatty acids (see, for example, Storch et al., Biochem. Biophys. Acta. 1486 (2000), 28-44). Other names for FABP-3 and H-FABP are: FABP-11 (fatty acid binding protein 11), M-FABP (muscle fatty acid binding protein), MDGI (mammary-derived growth inhibitor), and O-FABP.

[0139] The biomarker endothelial cell-specific molecule 1 (ESM-1) is well known in the art. This biomarker is often also referred to as endocan. ESM-1 is a secreted protein expressed primarily in endothelial cells of human lung and kidney tissues. Public domain data suggest that it is expressed in thyroid, lung, kidney, and cardiac tissues. See, for example, the entry for ESM-1 in the Protein Atlas database (Uhlen M. et al., Science 2015;347(6220):1260419). Expression of this gene is regulated by cytokines. ESM-1 is a proteoglycan composed of a 20 kDa mature polypeptide and a 30 kDa O-linked glycan chain (Bechard D et al., J Biol Chem 2001;276(51):48341-48349). In a preferred embodiment of the present invention, the amount of human ESM-1 polypeptide is measured in a sample from a subject. The sequence of the human ESM-1 polypeptide is well known in the art (see, e.g., Lassale P. et al., J. Biol. Chem. 1996;271:20458-20464) and can be accessed, for example, through the Uniprot database. See entry Q9NQ30 (ESM1_HUMAN). Two isoforms of ESM-1 are generated by alternative splicing: isoform 1 (having the Uniprot identifier Q9NQ30-1) and isoform 2 (having the Uniprot identifier Q9NQ30-2). Isoform 1 is 184 amino acids in length. In isoform 2, amino acids 101-150 of isoform 1 are missing. Amino acids 1-19 form a signal peptide (which can be cleaved).

[0140] In a preferred embodiment, the amount of isoform 1 of the ESM-1 polypeptide is determined, ie isoform 1 has the sequence as shown under UniProt accession number Q9NQ30-1.

[0141] In another preferred embodiment, the amount of isoform 2 of the ESM-1 polypeptide is determined, ie isoform 2 has the sequence as shown under UniProt accession number Q9NQ30-2.

[0142] In another preferred embodiment, the amount of isoform-1 and isoform-2 of the ESM-1 polypeptide, ie total ESM-1, is determined.

[0143] For example, the amount of ESM-1 can be determined using a monoclonal antibody (such as a mouse antibody) and / or a goat polyclonal antibody against amino acids 85 to 184 of the ESM-1 polypeptide.

[0144] The biomarker angiopoietin-2 (abbreviated "Ang-2" and often referred to as ANGPT2) is well known in the art. It is a naturally occurring antagonist of both Ang-1 and TIE2 (see, e.g., Maisonpierre et al., Science 277 (1997) 55-60). The protein can induce tyrosine phosphorylation of TEK / TIE2 in the absence of ANG-1. In the absence of angiogenic inducers such as VEGF, ANG2-mediated loosening of cell-matrix contacts can induce endothelial cell apoptosis with consequent vascular regression. In cooperation with VEGF, it can promote endothelial cell migration and proliferation, thus functioning as a permissive angiogenic signal. The sequences of human angiopoietins are well known in the art. Uniprot lists three isoforms of angiopoietin-2: isoform 1 (Uniprot identifier: O15123-1), isoform 2 (identifier: O15123-2), and isoform 3 (O15123-3). In a preferred embodiment, the total amount of angiopoietin-2 is determined. This total amount is preferably the sum of the amounts of conjugated and free angiopoietin-2.

[0145] Determining the amount of biomarkers The term "determining" the amount of a biomarker (such as one or more BMP10-type peptides or natriuretic peptides) referred to herein refers to quantifying the biomarker, e.g., measuring the level of the biomarker in a sample, using a suitable detection method described elsewhere herein. The terms "measuring" and "determining" are used interchangeably herein.

[0146] In one embodiment, determining the amount of a biomarker comprises contacting the sample with an agent that specifically binds to the biomarker (such as an antibody of the invention), thereby forming a complex between the agent and the biomarker, and detecting the amount of complex formed, thereby determining the amount of the biomarker. Determining may further comprise steps such as contacting the sample with a second agent.

[0147] The biomarkers referred to herein (such as one or more BMP10-type peptides) can be detected using methods commonly known in the art. Detection methods generally include methods that quantify the amount of biomarker in a sample (quantitative methods). Those skilled in the art generally know which of the following methods are suitable for qualitative and / or quantitative detection of biomarkers. Samples can be conveniently assayed for proteins using, for example, Western blots and immunoassays such as ELISA, RIA, fluorescence- and luminescence-based immunoassays, as well as commercially available proximity extension methods. Further suitable methods for detecting biomarkers include measuring physical or chemical properties specific to the peptide or polypeptide, such as its precise molecular weight or NMR spectrum. Such methods include, for example, analytical devices such as biosensors, optical devices coupled with immunoassays, biochips, mass spectrometers, NMR analyzers, or chromatography devices. Further methods include microplate ELISA-based methods, fully automated or robotic immunoassays (available on Elecsys™ analyzers), CBA (enzymatic Cobalt Binding Assay, e.g., available on Roche-Hitachi™ analyzers), and latex agglutination assays (e.g., available on Roche-Hitachi™ analyzers).

[0148] For the detection of biomarker proteins as used herein, a wide range of immunoassay techniques are available using such assay formats; see, e.g., U.S. Patent Nos. 4,016,043, 4,424,279, and 4,018,653. These include both traditional competitive binding assays as well as one-site and two-site or "sandwich" assays of the non-competitive type. These assays also include direct binding of labeled antibodies to target biomarkers.

[0149] Methods using electrochemiluminescent labels are well known. These methods utilize the ability of special metal complexes to undergo oxidation to an excited state, which then relaxes to the ground state, emitting electrochemiluminescence. For a review, see Richter, MM, Chem. Rev. 2004;104:3003-3036.

[0150] In one embodiment, the antibody (or antigen-binding fragment thereof) used to measure the amount of a biomarker is ruthenylated or iridinylated. Thus, the antibody (or antigen-binding fragment thereof) comprises a ruthenium label. In one embodiment, the ruthenium label is a bipyridine-ruthenium(II) complex. Alternatively, the antibody (or antigen-binding fragment thereof) comprises an iridium label. In one embodiment, the iridium label is a complex as disclosed in WO 2012 / 107419.

[0151] In one embodiment of a sandwich assay for determining one or more BMP10-type peptides, the assay comprises a biotinylated first monoclonal antibody (or fragment thereof) and a ruthenylated second monoclonal antibody (or fragment thereof) that specifically binds to one or more BMP10-type peptides. The two antibodies form a sandwich immunoassay complex with one or more BMP10-type peptides in a sample.

[0152] Measuring the amount of a polypeptide (e.g., a BMP10-type peptide or a natriuretic peptide) may preferably include the steps of: (a) contacting the polypeptide with an agent that specifically binds to the polypeptide; (b) (optionally) removing unbound agent; and (c) measuring the amount of bound binding agent, i.e., the agent complex formed in step (a). According to preferred embodiments, the contacting, removing, and measuring steps may be performed by an analyzer unit. According to some embodiments, the steps may be performed by a single analyzer unit of the system or by multiple analyzer units in operative communication with each other. For example, according to specific embodiments, the system disclosed herein may include a first analyzer unit for performing the contacting and removing steps, and a second analyzer unit for performing the measuring step, operably connected to the first analyzer unit by a transport unit (e.g., a robotic arm).

[0153] Agents (also referred to herein as "binders") that specifically bind biomarkers may be covalently or non-covalently attached to a label that allows for detection and measurement of the bound agent. Labeling may be achieved by direct or indirect methods. Direct labeling is achieved by directly (covalently or non-covalently) attaching a label to the binder. Indirect labeling is achieved by attaching (covalently or non-covalently) a second binder to the first binder. The second binder shall specifically bind to the first binder. The second binder may be conjugated with an appropriate label and / or may be a target (receptor) for a third binder that binds to the second binder. Suitable secondary and higher order binders may include antibodies, secondary antibodies, and the well-known streptavidin-biotin system (Vector Laboratories, Inc.). The binder or substrate may also be "tagged" with one or more tags known in the art. Such tags can then be targeted by higher-order binding agents. Suitable tags include biotin, digoxygenin, His-tag, glutathione-S-transferase, FLAG, GFP, myc-tag, influenza A virus hemagglutinin (HA), maltose-binding protein, etc. In the case of peptides or polypeptides, the tag is preferably at the N-terminus and / or C-terminus. Suitable labels are any labels that can be detected by an appropriate detection method. Typical labels include gold particles, latex beads, acridan esters, luminol, ruthenium complexes, iridium complexes, enzymatically active labels, radioactive labels, magnetic labels (including "e.g., magnetic beads," paramagnetic and superparamagnetic labels), and fluorescent labels. Enzymatically active labels include, for example, horseradish peroxidase, alkaline phosphatase, β-galactosidase, luciferase, and derivatives thereof.Suitable substrates for detection include diaminobenzidine (DAB), 3,3'-5,5'-tetramethylbenzidine, NBT-BCIP (4-nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate, available as a ready-made stock solution from Roche Diagnostics), CDP-Star™ (Amersham Biosciences), and ECF™ (Amersham Biosciences). Appropriate enzyme-substrate combinations may produce colored reaction products, fluorescence, or chemiluminescence, which can be determined by methods known in the art (e.g., using light-sensitive film or an appropriate camera system). The above criteria apply equally well for measuring enzymatic reactions. Typical fluorescent labels include fluorescent proteins (e.g., GFP and its derivatives), Cy3, Cy5, Texas Red, fluorescein, and Alexa dyes (e.g., Alexa 568). Additional fluorescent labels are available, for example, from Molecular Probes (Oregon). The use of quantum dots as fluorescent labels is also contemplated.Radioactive labels can be detected by any method known and appropriate, such as a photosensitive film or a phosphor imager.

[0154] The amount of a polypeptide may also preferably be determined as follows: (a) contacting a solid support comprising a binding agent for the polypeptide as described elsewhere herein with a sample containing the peptide or polypeptide, and (b) measuring the amount of peptide or polypeptide bound to the support. Materials for manufacturing supports are well known in the art and include, inter alia, commercially available column material, polystyrene beads, latex beads, magnetic beads, colloidal metal particles, glass and / or silicon chips and surfaces, nitrocellulose strips, membranes, sheets, duracyte, wells and walls of reaction trays, plastic tubes, and the like.

[0155] In a further embodiment, the sample is removed from the complex formed between the binding agent and at least one marker prior to measuring the amount of the complex formed. Thus, in one embodiment, the binding agent may be immobilized on a solid support. In a further embodiment, the sample may be removed from the complex formed on the solid support by applying a washing solution.

[0156] "Sandwich assays" are among the most useful and commonly used assays and encompass several variations of the sandwich assay technique. Briefly, in a typical assay, an unlabeled (capture) binding agent is immobilized or can be immobilized on a solid substrate, and the sample to be tested is contacted with the capture binding agent. After a suitable incubation period sufficient to allow for the formation of a binding agent-biomarker complex, a second (detection) binding agent labeled with a reporter molecule capable of producing a detectable signal is added and incubated for a period sufficient to allow the formation of another binding agent-biomarker-labeled binding agent complex. Any unreacted material can be washed away, and the presence of the biomarker is determined by observation of a signal produced by the reporter molecule bound to the detection binding agent. Results may be either qualitative, by simple observation of the visible signal, or quantitated by comparison with a control sample containing known amounts of biomarker.

[0157] The incubation step of a typical sandwich assay can be modified as needed. Such modifications include, for example, simultaneous incubation, in which two or more binding agents and biomarkers are co-incubated. For example, both the sample to be analyzed and the labeled binding agent are simultaneously added to the immobilized capture binding agent. Alternatively, the sample to be analyzed and the labeled binding agent can be first incubated, and then an antibody bound to a solid phase or an antibody capable of binding to a solid phase can be added.

[0158] The complex formed between the specific binding agent and the biomarker shall be proportional to the amount of biomarker present in the sample. It will be understood that the specificity and / or sensitivity of the applied binding agent will determine the extent to which the proportion of at least one marker contained in the sample can be specifically bound. Details of how to perform the measurement are also described elsewhere in this specification. The amount of complex formed shall be converted into the amount of biomarker, which reflects the amount actually present in the sample.

[0159] The terms "binder," "specific binder," "analyte-specific binder," "detection agent," and "agent that specifically binds to a biomarker" are used interchangeably herein. Preferably, the terms relate to an agent comprising a binding moiety that specifically binds the corresponding biomarker. Examples of "binder," "detection agent," and "agent" are nucleic acid probes, nucleic acid primers, DNA molecules, RNA molecules, aptamers, antibodies, antibody fragments, peptides, peptide nucleic acids (PNAs), or chemical compounds. A preferred agent is an antibody that specifically binds to the biomarker to be determined. The term "antibody" is used herein in the broadest sense and encompasses a variety of antibody structures, including, but not limited to, monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments (i.e., antigen-binding fragments thereof) so long as they exhibit the desired antigen-binding activity. Preferably, the antibody is a polyclonal antibody (or an antigen-binding fragment derived therefrom). More preferably, the antibody is a monoclonal antibody (or antigen-binding fragment thereof), and thus, as described elsewhere herein, it is contemplated that two monoclonal antibodies that bind to different positions of one or more BMP10-type peptides (in a sandwich immunoassay) will be used, and thus at least one antibody will be used to determine the amount of BMP10-type peptide.

[0160] In one embodiment, at least one antibody is a mouse monoclonal antibody. In another embodiment, at least one antibody is a rabbit monoclonal antibody. In a further embodiment, the antibody is a goat polyclonal antibody. In yet another embodiment, the antibody is a sheep polyclonal antibody.

[0161] In some embodiments, the at least one antibody or fragment thereof that specifically binds to a BMP10-type peptide is at least one antibody or fragment thereof described in the next section (entitled "Antibodies of the Invention").

[0162] The term "amount," as used herein, encompasses the absolute amount of a biomarker (e.g., one or more BMP10-type peptides or natriuretic peptides) referred to herein, the relative amount or concentration of the biomarker, as well as any value or parameter that correlates thereto or can be derived therefrom. Such values ​​or parameters include intensity signal values ​​derived from any specific physical or chemical property obtained from the peptide by direct measurement, such as intensity values ​​in a mass spectrum or NMR spectrum. Furthermore, all values ​​or parameters obtained by indirect measurement as specified elsewhere herein are encompassed, such as the response amount determined by a biological readout system in response to a peptide or intensity signal obtained from a specifically bound ligand. It should be understood that values ​​correlating to the above-mentioned amounts or parameters can also be obtained by any standard mathematical operation.

[0163] As used herein, the term "comparing" refers to comparing the amount of a biomarker (e.g., one or more BMP10-type peptides and a natriuretic peptide such as NT-proBNP or BNP) in a sample from a subject with a reference amount of the biomarker as specified elsewhere herein. It should be understood that comparing, as used herein, typically refers to a comparison of corresponding parameters or values, e.g., an absolute amount is compared to an absolute reference amount, a concentration is compared to a reference concentration, or an intensity signal obtained from a biomarker in a sample is compared to an intensity signal of the same type obtained from a first sample. Comparisons can be performed manually or using a computer. Thus, comparisons can be performed by a computing device. The determined or detected amount of a biomarker in a sample from a subject and values ​​for the reference amount can be compared, for example, with each other, or the comparison can be performed automatically by a computer program executing a comparison algorithm. The computer program performing the evaluation provides the desired assessment in an appropriate output format. For computer-assisted comparisons, the determined amount value may be compared by the computer program to values ​​corresponding to appropriate standards stored in a database. The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired rating in a suitable output format. For computer-assisted comparisons, the determined quantity values ​​may be compared by the computer program with values ​​corresponding to suitable standards stored in a database. The computer program may further evaluate the results of the comparison, i.e., automatically provide the desired rating in a suitable output format.

[0164] According to the present invention, the amount of one or more BMP10 peptides, and optionally the amount of at least one additional biomarker (such as a natriuretic peptide), shall be compared with a reference. The standard is preferably a reference amount. The term "reference amount" is well understood by those skilled in the art. It should be understood that the reference amount allows the assessment of atrial fibrillation as described herein. For example, in the context of a method for diagnosing atrial fibrillation, the reference amount preferably refers to an amount that allows a subject to be assigned to either (i) a group of subjects suffering from atrial fibrillation, or (ii) a group of subjects not suffering from atrial fibrillation. A suitable reference amount may be determined from a first sample that is analyzed together with the test sample, i.e., simultaneously or subsequently.

[0165] It should be understood that the amount of one or more BMP10 peptides is compared with a reference amount of one or more BMP10 peptides, while the amount of at least one additional biomarker (such as a natriuretic peptide) is compared with a reference amount of said at least one additional biomarker (such as a natriuretic peptide). When the amounts of two or more markers are determined, it is also envisaged to calculate a total score based on the amounts of two or more markers (such as the amount of one or more BMP10 peptides and the amount of a natriuretic peptide). In a next step, this score is compared with a reference score.

[0166] Reference amounts can, in principle, be calculated for a cohort of subjects, such as those specified above, based on the mean or average value of a given biomarker by applying standard methods of statistics. The accuracy of a test, particularly one intended for diagnosing an event, is best described by its receiver operating characteristic (ROC) (see, in particular, Zweig MH. et al., Clin. Chem. 1993;39:561-577). An ROC graph is a plot of all sensitivity versus specificity pairs generated by continuously varying the decision threshold over the entire range of observed data. The clinical performance of a diagnostic method depends on its accuracy, i.e., its ability to correctly assign subjects to a particular prognosis or diagnosis. An ROC plot shows the overlap between two distributions by plotting sensitivity versus 1-specificity for the entire range of thresholds suitable for discrimination. The y-axis is sensitivity, or the true positive rate, defined as the ratio of the number of true-positive test results to the product of the number of true-positive and false-negative test results. The y-axis is calculated only from the affected subgroup. On the x-axis is the false positive rate, or 1-specificity, which is defined as the ratio of the number of false positive results to the product of the number of true negative and false positive results. The x-axis is an index of specificity, calculated only from the unaffected subgroup. Because the true positive rate and false positive rate are calculated completely separately by using test results from two different subgroups, the ROC plot is independent of the prevalence of the event in the cohort. Each point on the ROC plot represents a sensitivity / 1-specificity pair corresponding to a particular decision threshold. A test with perfect discrimination (no overlap between the two distributions of results) would have an ROC plot that passes through the upper left corner, resulting in a true positive rate of 1.0, or 100% (perfect sensitivity), and a false positive rate of 0 (perfect specificity). The theoretical plot for a test with no discrimination (the distributions of results for the two groups are identical) would be a 45° diagonal line from the lower left corner to the upper right corner. Most plots fall between these two extremes. If the ROC plot falls entirely below the 45° diagonal, this is easily corrected by swapping the criteria for "positive rate" from "higher" to "lower" and vice versa.Qualitatively, the closer the plot is to the upper left corner, the higher the overall accuracy of the test. Depending on the desired confidence interval, a threshold value can be derived from the ROC curve, thereby enabling a diagnosis of a given event with an appropriate balance of sensitivity and specificity, respectively. Therefore, the criterion used in the method of the present invention, i.e., the threshold value that allows assessing atrial fibrillation, can preferably be generated by establishing the ROC of the cohort as described above and deriving a threshold amount therefrom. Depending on the desired sensitivity and specificity of the diagnostic method, the ROC plot can derive an appropriate threshold value. It is understood that optimal sensitivity is desirable, for example, to exclude (i.e., rule out) subjects from having atrial fibrillation, while optimal sensitivity is assumed for subjects assessed as having atrial fibrillation. In one embodiment, the method of the present invention allows prediction that a subject is at risk for adverse events related to atrial fibrillation, such as the occurrence or recurrence of atrial fibrillation and / or stroke.

[0167] In a preferred embodiment, the term "reference amount" herein refers to a predetermined value. The predetermined value is intended to enable assessment of atrial fibrillation, and thus diagnosis of atrial fibrillation, distinguishing between paroxysmal and persistent atrial fibrillation, predicting the risk of adverse events associated with atrial fibrillation, identifying subjects who should undergo electrocardiography (ECG), or assessing a treatment for atrial fibrillation. It should be understood that the reference amount may differ based on the type of assessment. For example, the reference amount of one or more BMP10-type peptides for distinguishing AF will usually be higher than the reference amount for diagnosing AF. However, this is considered by those skilled in the art.

[0168] As previously clarified, the term "assessing atrial fibrillation" preferably refers to diagnosing atrial fibrillation, distinguishing between paroxysmal and persistent atrial fibrillation, predicting the risk of adverse events associated with atrial fibrillation, identifying subjects to be subjected to electrocardiography (ECG), or assessing a therapy for atrial fibrillation. These embodiments of the method of the present invention are described in more detail below. The previous definitions apply where appropriate.

[0169] Method for diagnosing atrial fibrillation As used herein, the term "diagnosing" refers to assessing whether a subject according to the method of the present invention is suffering from atrial fibrillation (AF). In a preferred embodiment, the subject is diagnosed as suffering from paroxysmal AF. In an alternative embodiment, the subject is diagnosed as not suffering from AF.

[0170] According to the present invention, all types of AF can be diagnosed. Thus, atrial fibrillation can be paroxysmal, persistent, or permanent AF. Preferably, paroxysmal or persistent atrial fibrillation is diagnosed in subjects who are not particularly affected by permanent atrial fibrillation.

[0171] The actual diagnosis of whether a subject suffers from AF may include further steps, such as confirming the diagnosis (for example, by ECG, such as Holter ECG). Thus, the present invention makes it possible to assess the likelihood that a patient suffers from atrial fibrillation. A subject with an amount of one or more BMP10 peptides that exceeds the reference amount is likely to suffer from atrial fibrillation, while a subject with an amount of one or more BMP10 peptides that is below the reference amount is unlikely to suffer from atrial fibrillation. Therefore, the term "diagnosing" in the context of the present invention also encompasses assisting a doctor to assess whether a subject suffers from atrial fibrillation.

[0172] Preferably, an increased amount of one or more BMP10-type peptides (and optionally an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a test subject compared to a reference amount(s) indicates the subject is suffering from atrial fibrillation, and / or a decreased amount of one or more BMP10-type peptides (and optionally an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a test subject compared to a reference amount(s) indicates the subject is not suffering from atrial fibrillation.

[0173] In a preferred embodiment, the reference amounts, i.e. the reference amounts of one or more BMP10-type peptides and, if determined, the reference amounts of at least one further biomarker, make it possible to distinguish between subjects suffering from atrial fibrillation and subjects not suffering from atrial fibrillation. Preferably, the reference amounts are predetermined values.

[0174] In one embodiment, the present invention allows for the diagnosis of a subject suffering from atrial fibrillation. Preferably, the subject suffers from AF when the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) is above a reference amount. In one embodiment, the subject suffers from AF when the amount of one or more BMP10-type peptides exceeds the upper reference limit (URL) of a particular percentile (e.g., the 99th percentile) of the reference amount.

[0175] In one embodiment of the method for diagnosing atrial fibrillation, the method further comprises recommending and / or initiating a treatment for atrial fibrillation based on the results of the diagnosis. Preferably, if the subject is diagnosed with AF, a treatment is recommended or initiated. Preferred treatments for atrial fibrillation are disclosed elsewhere herein (e.g., anticoagulation therapy).

[0176] Method for distinguishing between paroxysmal and persistent atrial fibrillation As used herein, the term "distinguishing" means distinguishing between paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject. The term used herein preferably includes differentially diagnosing paroxysmal atrial fibrillation and persistent atrial fibrillation in a subject. Thus, the method of the present invention makes it possible to assess whether a subject suffering from atrial fibrillation suffers from paroxysmal atrial fibrillation or persistent atrial fibrillation. The actual distinction may include further steps, such as confirmation of the distinction. Therefore, the term "distinguishing" in the context of the present invention also encompasses assisting a physician in distinguishing between paroxysmal atrial fibrillation and persistent AF.

[0177] Preferably, an increase in the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a subject compared to one or more reference amounts is indicative of the subject suffering from persistent atrial fibrillation, and / or a decrease in the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) compared to one or more reference amounts is indicative of the subject suffering from paroxysmal atrial fibrillation. In both AF types (paroxysmal and persistent), the amount of BMP10-type peptides is increased compared to the reference amounts in non-AF subjects.

[0178] In a preferred embodiment, the reference amount(s) make it possible to distinguish between subjects suffering from paroxysmal atrial fibrillation and subjects suffering from persistent atrial fibrillation. Preferably, the reference amount(s) are predetermined values.

[0179] In one embodiment of the above method for distinguishing between paroxysmal and persistent atrial fibrillation, the subject does not suffer from permanent atrial fibrillation.

[0180] Methods for predicting risk of adverse events associated with atrial fibrillation The methods of the present invention also contemplate methods for predicting the risk of an adverse event.

[0181] In one embodiment, the risk of an adverse event identified herein can be a prediction of any adverse event associated with atrial fibrillation. Preferably, the adverse event is selected from atrial fibrillation recurrence (such as atrial fibrillation recurrence after cardioversion) and stroke. Optionally, the risk of a subject (suffering from atrial fibrillation) suffering from a future adverse event (such as stroke or atrial fibrillation recurrence) is predicted.

[0182] It is further envisioned that the adverse event associated with atrial fibrillation is the occurrence of atrial fibrillation in a subject with no prior history of atrial fibrillation.

[0183] In a particularly preferred embodiment, the risk of stroke is predicted.

[0184] Accordingly, there is provided a method for predicting the risk of stroke in a subject of the present invention, comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides with a reference amount, thereby predicting the risk of stroke.

[0185] In particular, the present invention provides a method for predicting the risk of stroke in a subject, comprising: (a) determining in at least one sample from the subject the amount of one or more BMP10-type peptides (bone morphogenetic protein 10), and optionally the amount of at least one additional biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2), and FABP-3 (Fatty Acid Binding Protein 3); and (b) comparing the amount of one or more BMP10-type peptides with a reference amount of BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount of said at least one additional biomarker, thereby predicting the risk of stroke.

[0186] It is further contemplated that a subject's risk of atrial fibrillation recurrence after electrical cardioversion may be predicted, for example, after pulmonary vein isolation or ablation therapy. Therefore, the biomarkers described herein can be used as predictors of atrial fibrillation recurrence after therapeutic interventions such as pulmonary vein isolation and ablation therapy. The success rate of such procedures depends on the ablation strategy and patient characteristics, and has been reported to be 50% to 60% for persistent AF (J Am Heart Assoc. 2013;2:e004549). In this embodiment, the subject should be analyzed before the therapeutic intervention; that is, the subject being examined is assumed to be suffering from an episode of AF at the time of the examination, i.e., when the sample is obtained. According to this embodiment, the amount of biomarker(s) is determined in a sample obtained before the (planned) intervention, preferably one month before the (planned) intervention, more preferably one week before the (planned) intervention, and most preferably one day before the (planned) intervention. Therefore, biomarker-based prediction of AF recurrence risk is possible before treatment. According to the method of the present invention, the elevation of the biomarkers mentioned herein should help to stratify subjects with high risk from low risk for recurrent AFib after therapeutic intervention. Depending on the detected or predicted risk, some patients with a low probability of success can be treated differently from patients with a high probability of success depending on the blood biomarkers measured before the procedure as predictors of AF recurrence after PVI or ablation.

[0187] Preferably, as used herein, the term "predicting risk" refers to assessing the likelihood that a subject will suffer from the adverse event referred to herein. Usually, it is predicted whether a subject is at risk (and therefore has a high risk) or not at risk (and therefore has a low risk) of suffering from the adverse event. Therefore, the method of the present invention makes it possible to distinguish between subjects at risk and subjects at no risk of suffering from the adverse event. Furthermore, it is assumed that the method of the present invention makes it possible to distinguish between subjects with reduced, average, and elevated risk.

[0188] As previously explained, the risk (and likelihood) of suffering from the adverse event in question within a certain time frame is to be predicted. In a preferred embodiment of the present invention, the prediction window is a period of about 3 months, about 6 months, or particularly about 1 year. Thus, short-term risk is predicted.

[0189] In another preferred embodiment, the prediction window is a period of about 5 years (e.g., for predicting stroke). Further, the prediction window may be a period of about 6 years (e.g., for predicting stroke). Alternatively, the prediction window may be about 10 years. It is also envisioned that the prediction window is a period of 1 to 3 years. Thus, the risk of suffering from a stroke within 1 to 3 years is predicted. It is also envisioned that the prediction window is a period of 1 to 10 years. Thus, the risk of suffering from a stroke within 1 to 10 years is predicted.

[0190] Preferably, the prediction window is calculated from the completion of the method of the present invention. More preferably, the prediction window is calculated from the time when the sample to be tested is collected. As will be understood by those skilled in the art, risk prediction is usually not intended to be correct for 100% of subjects. However, this term requires that the statistically significant portion of subjects can be appropriately and accurately predicted. Whether a portion is statistically significant can be determined by those skilled in the art without further ado using various well-known statistical evaluation tools, such as determining a confidence interval, determining a p-value, Student's t-test, or Mann-Whitney test. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York, 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0191] In a preferred embodiment, the phrase "predicting the risk of suffering from the adverse event" means that the subject analyzed by the method of the present invention is assigned to either a group of subjects at risk of suffering from the adverse event or a group of subjects at no risk of suffering from the adverse event (such as stroke). Thus, it is predicted whether the subject is at risk of suffering from the adverse event. As used herein, a "subject at risk of suffering from the adverse event" preferably has an increased risk of suffering from the adverse event (preferably within a prediction window). Preferably, the risk is increased compared to the average risk in the subject cohort. As used herein, a "subject not at risk of suffering from the adverse event" preferably has a reduced risk of suffering from the adverse event (preferably within a prediction window). Preferably, the risk is reduced compared to the average risk in the subject cohort. A subject at risk of suffering from the adverse event preferably has a risk of suffering from the adverse event, such as the recurrence or occurrence of atrial fibrillation, of preferably at least 20%, or more preferably at least 30%, within a prediction window of about one year. A subject who is not at risk of suffering from the adverse event preferably has less than a 12%, more preferably less than a 10% risk of suffering from the adverse event within a one year predictive window.

[0192] For the prediction of stroke, the subject at risk of suffering from this adverse event preferably has the risk of suffering from this adverse event of at least 10%, or more preferably at least 13%, within a prediction window of about 5 years, or particularly 6 years.The subject at no risk of suffering from this adverse event preferably has the risk of suffering from this adverse event of less than 10%, more preferably less than 8%, or most preferably less than 5%, within a prediction window of about 5 years, particularly about 6 years.If the subject is not receiving anticoagulant therapy, the risk may be higher.This can be considered by those skilled in the art.

[0193] Preferably, an increase in the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from the subject compared to one or more reference amounts is indicative of the subject being at risk of an adverse event associated with atrial fibrillation, and / or a decrease in the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from the subject compared to one or more reference amounts is indicative of the subject not being at risk of an adverse event associated with atrial fibrillation.

[0194] In a preferred embodiment, the reference amount (or amounts) allows for differentiation between subjects at risk of an adverse event as referred to herein and subjects not at risk of said adverse event. Preferably, the reference amount is a predetermined value.

[0195] The predicted adverse event is preferably stroke. The term "stroke" is well known in the art. As used herein, the term preferably refers to ischemic stroke, particularly cerebral ischemic stroke. Stroke predicted by the method of the present invention is caused by a decrease in blood flow to the brain or a portion thereof, resulting in an insufficient supply of oxygen to brain cells. In particular, stroke results in irreversible tissue damage due to brain cell death. Symptoms of stroke are well known in the art. For example, stroke symptoms include sudden numbness or weakness of the face, arms, legs, or especially one side of the body; sudden confusion; difficulty speaking or understanding; sudden loss of vision in one or both eyes; sudden difficulty walking; dizziness; loss of balance or coordination. Ischemic stroke can be caused by atherothrombosis or embolism of a major cerebral artery; by coagulation disorders or non-atheromatous vascular disease; or by cardiac ischemia leading to a decrease in overall blood flow. The ischemic stroke is preferably selected from the group consisting of atherothrombotic stroke, cardioembolic stroke and pit stroke. Preferably, the predicted stroke is an acute ischemic stroke, in particular a cardioembolic stroke. Cardioembolic stroke (often also called embolic or thromboembolic stroke) can be caused by atrial fibrillation.

[0196] Preferably, the stroke is associated with atrial fibrillation. More preferably, the stroke is caused by atrial fibrillation. However, it is also contemplated that the subject may not have a history of atrial fibrillation.

[0197] Preferably, a stroke is associated with atrial fibrillation if there is a temporal correlation between the stroke and an episode of atrial fibrillation. More preferably, a stroke is associated with atrial fibrillation if the stroke is caused by atrial fibrillation. Most preferably, a stroke is associated with atrial fibrillation if the stroke can be caused by atrial fibrillation. For example, cardioembolic stroke (often also referred to as embolic stroke or thromboembolic stroke) can be caused by atrial fibrillation. Preferably, AF-related stroke can be prevented by oral anticoagulation. Also preferably, a stroke is considered to be associated with atrial fibrillation if the subject being examined suffers from and / or has a history of atrial fibrillation. In addition, in one embodiment, a stroke can be considered to be associated with atrial fibrillation if the subject is suspected of suffering from atrial fibrillation.

[0198] The term "stroke" preferably does not include hemorrhagic stroke.

[0199] In a preferred embodiment of the above-mentioned method for predicting adverse events (such as stroke), the subject being examined suffers from atrial fibrillation. More preferably, the subject has a history of atrial fibrillation. According to the method for predicting adverse events, the subject preferably suffers from permanent atrial fibrillation, more preferably from persistent atrial fibrillation, and most preferably from paroxysmal atrial fibrillation.

[0200] In one embodiment of the method for predicting adverse events, the subject suffering from atrial fibrillation is experiencing an episode of atrial fibrillation when the sample is obtained. In another embodiment of the method for predicting adverse events, the subject suffering from atrial fibrillation is not experiencing an episode of atrial fibrillation when the sample is obtained (and therefore has normal sinus rhythm). Furthermore, the subject whose risk is predicted may be undergoing anticoagulation therapy.

[0201] In another embodiment of the method for predicting an adverse event, the subject being tested does not have a history of atrial fibrillation. In particular, it is envisioned that the subject does not suffer from atrial fibrillation.

[0202] The method of the present invention can support personalized medicine. In a preferred embodiment, the method for predicting a subject's risk of stroke further comprises, if the subject is identified as being at risk of suffering from a stroke, i) recommending anticoagulant therapy or ii) recommending intensification of anticoagulant therapy. In a preferred embodiment, the method for predicting a subject's risk of stroke further comprises, if the subject is identified as being at risk of suffering from a stroke, i) initiating anticoagulant therapy or ii) intensifying anticoagulant therapy.

[0203] If the test subject is receiving anticoagulant therapy, and if the subject is identified (by the method of the present invention) as not being at risk for stroke, the dosage of anticoagulant therapy can be reduced. Therefore, reducing the dosage may be recommended. Reducing the dosage may reduce the risk of side effects (such as bleeding).

[0204] As used herein, the term "recommend" refers to establishing a suggested treatment regimen applicable to a subject. However, it is understood that the term does not encompass the actual administration of the treatment regimen, whatever that may be. The recommended treatment regimen depends on the results provided by the methods of the present invention.

[0205] In particular, the following applies: If the subject being tested is not on anticoagulation therapy, initiation of anticoagulation therapy is recommended if the subject is identified as being at risk for stroke, and therefore anticoagulation therapy should be initiated.

[0206] If the subject being tested is already on anticoagulation therapy, intensification of anticoagulation therapy is recommended if the subject is identified as being at risk for stroke, and therefore anticoagulation therapy shall be intensified.

[0207] In one preferred embodiment, anticoagulation therapy is intensified by increasing the dose of anticoagulant, ie, the dose of the coagulant currently being administered.

[0208] In a particularly preferred embodiment, anticoagulant therapy is intensified by increasing the replacement of currently administered anticoagulants with more effective anticoagulants. Anticoagulant replacement is therefore recommended.

[0209] As shown in Hijazi et al., The Lancet 2016 387, 2302-2311 (Figure 4), it has been described that better prophylaxis in high-risk patients is achieved with the oral anticoagulant apixaban compared to the vitamin K antagonist warfarin.

[0210] Therefore, it is assumed that the subject being examined is a subject being treated with a vitamin K antagonist, such as warfarin or dicoumarol. If the subject is identified (by the method of the present invention) as being at risk of suffering from a stroke, it is recommended to replace the vitamin K antagonist with an oral anticoagulant, particularly dabigatran, rivaroxaban, or apixaban. Therefore, treatment with the vitamin K antagonist is discontinued, and treatment with the oral anticoagulant is initiated.

[0211] Method for identifying subjects to be subjected to electrocardiography (ECG) According to this embodiment of the method of the present invention, it is to be assessed whether the subject being tested with the biomarker is to be subjected to an electrocardiogram (ECG), i.e., an electrocardiogram assessment, which is to be performed in the subject for diagnostic purposes, i.e., to detect the presence or absence of AF.

[0212] As used herein, the term "identifying a subject" preferably refers to using information or data generated regarding the amount of one or more BMP10-type peptides (and optionally the amount of at least one additional biomarker) in a sample of a subject undergoing an ECG to identify the subject. The identified subject has an increased likelihood of suffering from AF. The assessment of the ECG is performed as confirmation.

[0213] Electrocardiography (abbreviated as ECG) is the process of recording the electrical activity of the heart by means of a suitable ECG. The ECG device records the electrical signals generated by the heart and spreads throughout the body to the skin. The recording is of the electrical signals achieved by contacting the skin of the subject with electrodes provided by the ECG device. The process of obtaining the recording is non-invasive and risk-free. The ECG is performed for the diagnosis of atrial fibrillation, i.e., for the assessment of the presence or absence of atrial fibrillation in the subject. In an embodiment of the method of the present invention, the ECG device is a single-lead device (such as a single-lead portable ECG device). In another preferred embodiment, the ECG device is a 12-lead ECG device, such as a Holter monitor.

[0214] Preferably, an increased amount of one or more BMP10-type peptides (and optionally, an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a test subject compared to a reference amount(s) indicates a subject undergoing an ECG, and / or a decreased amount of one or more BMP10-type peptides (and optionally, an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a subject compared to a reference amount(s) indicates a subject not undergoing an ECG.

[0215] In a preferred embodiment, the reference amount makes it possible to distinguish between subjects who shall be subjected to an ECG and subjects who shall not be subjected to an ECG. Preferably, the reference amount is a predetermined value.

[0216] In one embodiment of the aforementioned method, the method includes, inter alia, identifying a subject to undergo electrocardiography if the amount of one or more BMP10-type peptides (and optionally the amount of at least one further biomarker, e.g., ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample derived from the test subject is increased compared to a reference amount (or multiple reference amounts), and subjecting the identified subject to electrocardiography.

[0217] Methods for evaluation of treatments for atrial fibrillation As used herein, the term "evaluating a therapy for atrial fibrillation" preferably refers to the evaluation of a therapy aimed at treating atrial fibrillation, and in particular, the evaluation of the effectiveness of the therapy.

[0218] The therapy being evaluated can be any therapy aimed at treating atrial fibrillation. Preferably, the therapy is selected from the group consisting of administration of at least one anticoagulant, rhythm control, rate control, cardioversion, and ablation. Such therapies are well known in the art and are reviewed, for example, in Fuster V et al. Circulation 2011;123:e269-e367, which is incorporated herein by reference in its entirety.

[0219] In one embodiment, the treatment is the administration of at least one anticoagulant, i.e., anticoagulation therapy. Anticoagulation therapy is preferably a treatment aimed at reducing the risk of anticoagulation in the subject. The administration of at least one anticoagulant aims to inhibit or prevent blood clotting and associated stroke. In a preferred embodiment, the at least one anticoagulant is selected from the group consisting of heparin, coumarin derivatives (i.e., vitamin K antagonists), particularly warfarin or dicoumarol, oral anticoagulants, particularly dabigatran, rivaroxaban, or apixaban, tissue factor pathway inhibitor (TFPI), antithrombin III, factor IXa inhibitors, factor Xa inhibitors, inhibitors of factors Va and VIIIa, and thrombin inhibitors (anti-type IIa). Therefore, it is assumed that the subject is taking at least one of the above medications.

[0220] In a preferred embodiment, the anticoagulant is a vitamin K antagonist such as warfarin or dicoumarol. Vitamin K antagonists such as warfarin or dicoumarol are inexpensive, but are inconvenient and cumbersome, and often have unreliable treatment with time-varying therapeutic ranges, making it necessary to improve patient compliance. NOACs (new oral anticoagulants) include direct factor Xa inhibitors (apixaban, rivaroxaban, darexaban, edoxaban), direct thrombin inhibitors (dabigatran), and PAR-1 antagonists (vorapaxar, atopaxar).

[0221] In another preferred embodiment, anticoagulants and oral anticoagulants, in particular apixaban, rivaroxaban, darexaban, edoxaban, dabigatran, vorapaxar, or atopaxar.

[0222] Thus, the subject being tested may be undergoing treatment with an oral anticoagulant or a vitamin K antagonist at the time of testing (ie, when the sample is received).

[0223] In a preferred embodiment, the assessment of a therapy for atrial fibrillation is monitoring of said therapy. In this embodiment, the reference amount is preferably the amount of one or more BMP10-type peptides in a previously obtained sample (i.e., a sample obtained before the test sample in step a).

[0224] Optionally, the amount of at least one further biomarker referred to herein is determined in addition to the amount of one or more BMP10-type peptides.

[0225] Accordingly, the present invention relates to a method for monitoring a therapy for atrial fibrillation in a subject, the subject preferably suffering from atrial fibrillation, the method comprising: (a) determining in a first sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); (b) determining the amount of one or more BMP10-type peptides, and optionally the amount of at least one additional biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 (angiopoietin 2), and FABP-3 (fatty acid binding protein 3), in a second sample from the subject, wherein the second sample is obtained after the first sample; and (c) comparing the amount of one or more BMP10-type peptides in the first sample with the amount of one or more BMP10-type peptides in the second sample, and optionally comparing the amount of the at least one additional biomarker in the first sample with the amount of the at least one additional biomarker in the second sample, thereby monitoring anticoagulant therapy.

[0226] As used herein, the term "monitoring" preferably relates to assessing the effectiveness of a therapy as referred to elsewhere in this specification. Thus, the effectiveness of a therapy (such as anticoagulant therapy) is monitored.

[0227] The above-mentioned method may include a further step of monitoring the treatment based on the results of the comparison step performed in step c).As will be understood by those skilled in the art, risk prediction is usually not intended to be correct for 100% of subjects.However, this term requires that a statistically significant portion of subjects can be appropriately and accurately predicted.Therefore, actual monitoring may include further steps such as confirmation.

[0228] Preferably, by carrying out the method of the present invention, it is possible to assess whether a subject responds to the treatment.If the subject's condition improves between obtaining the first sample and obtaining the second sample, the subject is responding to the treatment.Preferably, if the subject's condition worsens between obtaining the first sample and obtaining the second sample, the subject is not responding to the treatment.

[0229] Preferably, the first sample is obtained before the start of the therapy. More preferably, the sample is obtained within one week, particularly within two weeks, prior to the start of the therapy. However, it is also contemplated that the first sample may be obtained after the start of the therapy (but before the second sample is obtained). In this case, ongoing therapy is monitored.

[0230] Thus, the second sample is taken after the first sample, with the understanding that the second sample is obtained after the start of the therapy.

[0231] Furthermore, it is specifically contemplated that the second sample be obtained a reasonable period of time after obtaining the first sample. It should be understood that the amounts of biomarkers referred to herein do not change instantly (e.g., within one minute or one hour). Thus, "reasonable" in this context refers to an interval between obtaining the first and second samples that allows for adjustment of the biomarker(s). Thus, the second sample is preferably obtained at least one month, at least three months, or particularly at least six months after the first sample.

[0232] Preferably, a decrease, more preferably a significant decrease, most preferably a statistically significant decrease in the amount(s) of biomarker(s), i.e., one or more BMP10-type peptides, and optionally, natriuretic peptides, in the second sample compared to the amount(s) of biomarker(s) in the first sample is indicative of a subject responding to the treatment. Thus, the treatment is efficient. Also, preferably, no change in the concentration of one or more BMP10-type peptides in the second sample, or an increase, more preferably a significant increase, most preferably a statistically significant increase in the amount(s) of biomarker(s) compared to the amount(s) of biomarker(s) in the first sample is indicative of a subject not responding to the treatment. Thus, the treatment is inefficient.

[0233] The terms "significant" and "statistically significant" are known to those skilled in the art. Therefore, whether an increase or decrease is significant or statistically significant can be readily determined by those skilled in the art using various well-known statistical evaluation tools. For example, a significant increase or decrease is an increase or decrease of at least 10%, particularly at least 20%.

[0234] A subject is considered to be responsive to a therapy if the therapy reduces the subject's risk of recurrence of atrial fibrillation. A subject is considered to be non-responsive to a therapy if the therapy does not reduce the subject's risk of recurrence of atrial fibrillation.

[0235] In one embodiment, if the subject is not responding to the therapy, the intensity of the therapy is increased. Furthermore, if the subject is responding to the therapy, the intensity of the therapy is expected to be decreased. For example, the intensity of the therapy can be increased by increasing the dosage of the administered medication. For example, the intensity of the therapy can be decreased by decreasing the dosage of the administered medication. This may avoid undesirable and harmful side effects such as bleeding.

[0236] In another preferred embodiment, the assessment of a therapy for atrial fibrillation is a guidance for guiding a therapy for atrial fibrillation. As used herein, the term "guidance" preferably relates to adjusting the intensity of a therapy, such as increasing or decreasing the dose of an oral anticoagulant, based on the determination of a biomarker, i.e., a BMP10-type peptide, during the therapy.

[0237] In a further preferred embodiment, the assessment of a treatment for atrial fibrillation is a stratification of a treatment for atrial fibrillation. Thus, subjects eligible for a certain treatment for atrial fibrillation are identified. As used herein, the term "stratification" preferably relates to selecting an appropriate treatment based on a given or specific risk, an identified molecular pathway, and / or the expected effectiveness of a specific drug or procedure. Depending on the detected or predicted risk, patients with minimal or no arrhythmia-related symptoms may be eligible for heart rate control, cardioversion, or ablation, while others may only receive antithrombotic therapy. Depending on the detected or predicted risk, some patients with a low probability of success may be treated differently from patients with a high probability of success, depending on blood biomarkers measured before the procedure as predictors of AF recurrence after PVI or ablation.

[0238] The definitions and explanations herein above apply mutatis mutandis to the methods of the invention below (unless otherwise specified). For example, the terms "subject," "sample," and "determining" are defined above. Furthermore, the determining preferably comprises contacting the sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:1.

[0239] Interestingly, the research underlying the present invention has shown that BMP10-type peptides can be used to predict the risk, presence and / or severity of cerebrovascular damage as a cause of dementia and cognitive impairment in patients, such as patients with atrial fibrillation. Specifically, it has been shown that the biomarker correlates with the presence of white matter lesions (WMLs) in patients. The higher the amount of the biomarker, the higher the degree of WMLs (and vice versa). Therefore, it can be used as a marker for assessing the degree of WMLs.

[0240] The present invention further relates to a method for assessing the extent of white matter pathology in a subject, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) assessing the extent of white matter pathology in the subject based on the amount determined in step a).

[0241] The term "white matter lesion" is well known in the art. White matter refers to regions of the central nervous system (CNS) primarily composed of myelinated axons. White matter lesions (also called "white matter disease") are commonly detected as white matter hyperintensities (WMH) or "leukodystrophy" on brain MRI in aging individuals. The presence and extent of WMH have been described as radiological markers of small cerebrovascular disease and important predictors of lifetime risk of stroke, cognitive impairment, and functional disability (Chutinet A, Rost NS. White matter disease as a biomarker for long-term cerebrovascular disease and dementia. Curr Treat Options Cardiovasc Med. 2014;16(3):292. doi:10.1007 / s11936-013-0292-z). Determining RET allows for assessment of the degree of WMH, i.e., WML burden. Thus, the biomarker allows for the quantification of WML in a subject, ie, is a marker of loss of functional brain tissue volume.

[0242] The extent of white matter lesions can be expressed by the Fazekas score (Fazekas, J.B. Chawluk, A. Alavi, H.I. Hurtig, and R.A. Zimmerman, American Journal of Roentgenology 1987, 149:2, 351-356). The Fazekas score ranges from 0 to 3, with 0 indicating no WML, 1 indicating mild WML, 2 indicating moderate WML, and 3 indicating severe WML.

[0243] The degree of WML can be caused by clinically silent stroke. Therefore, the biomarker RET can be further used to assist in assessing whether a subject has previously experienced one or more silent strokes, i.e., before the sample is obtained.

[0244] Silent strokes, i.e., silent cerebral strokes, are known in the art and are described, for example, in Conen et al. (Conen et al., J Am Coll Cardiol 2019;73:989-99), the entire disclosure of which is incorporated herein by reference. Silent strokes are clinically silent strokes in patients without a clinical history of stroke or transient ischemic attack. Therefore, the subject being tested should not have a history of stroke and / or TIA (transient ischemic attack).

[0245] The present invention further relates to a method for assessing whether a subject has experienced one or more silent strokes, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; b) comparing the amount determined in step a) with a standard; and c) assessing whether the subject has experienced one or more silent strokes.

[0246] Preferably, the following diagnostic algorithm is applied: an amount of the biomarker greater than the reference value indicates a subject who has experienced one or more silent strokes, and / or an amount lower than the reference value indicates a subject who has not experienced a silent stroke.

[0247] Accordingly, the present invention relates to a method for predicting dementia, such as vascular dementia and / or Alzheimer's disease, in a subject, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; b) comparing the amount determined in step a) with a standard; and c) predicting the risk of dementia in the subject.

[0248] As used herein, the term "predicting dementia" preferably refers to assessing the probability that a subject will suffer from dementia. Typically, it is predicted whether a subject is at risk (and therefore high risk) or not at risk (and therefore low risk) of suffering from dementia.

[0249] In one embodiment, the prediction window is a period of 1 to 3 years. Thus, the risk of a subject developing dementia within 1 to 3 years is predicted. In a preferred embodiment, the prediction window is a period of 1 to 10 years. Thus, the risk of a subject developing dementia within 1 to 10 years is predicted. Preferably, the prediction window is calculated from the completion of the method of the present invention. More preferably, the prediction window is calculated from the time the sample to be tested is collected.

[0250] As used herein, the term "dementia" preferably refers to a condition that can be characterized as a usually progressive loss of cognitive and intellectual function without impairment of perception or consciousness caused by various disorders, but is most commonly associated with structural brain disease. The most common type of dementia is Alzheimer's disease, which accounts for 50% to 70% of cases. Other common types include vascular dementia (25%), dementia with Lewy bodies, and frontotemporal dementia. The term "dementia" includes, but is not limited to, AIDS dementia, Alzheimer's dementia, presenile dementia, senile dementia, catatonic dementia, dementia with Lewy bodies (diffuse Lewy body disease), multi-infarct dementia (vascular dementia), paralytic dementia, post-traumatic dementia, early-onset dementia, and vascular dementia.

[0251] In one embodiment, the term dementia refers to vascular dementia, Alzheimer's disease, dementia with Lewy bodies, and / or frontotemporal dementia, and thus the risk of suffering from vascular dementia, Alzheimer's disease, dementia with Lewy bodies, and / or frontotemporal dementia is predicted.

[0252] In one embodiment, the risk of developing "Alzheimer's disease" is predicted. The term "Alzheimer's disease" is well known in the art. Alzheimer's disease is a chronic neurodegenerative disease that usually begins slowly and gradually worsens over time. As the disease progresses, symptoms may include language problems, disorientation, mood swings, loss of motivation, failure to manage self-care, and behavioral problems.

[0253] In one embodiment, the risk of suffering from "vascular dementia" is predicted. The term "vascular dementia" preferably refers to the progressive loss of memory and other cognitive functions caused by vascular damage or disease in the brain. Therefore, this term refers to the dementia symptoms caused by problems with blood circulation to the brain. This may occur after a stroke or may accumulate over time.

[0254] In relation to predicting the risk of dementia, the diagnostic algorithm is preferably as follows: preferably, an amount of the biomarker greater than the reference value indicates a subject at risk of dementia, and / or an amount of the biomarker lower than the reference value indicates a subject not at risk of dementia.

[0255] The definitions and explanations herein above apply mutatis mutandis to the methods of the invention below (unless otherwise specified). For example, the terms "subject," "sample," and "determining" are defined above. Furthermore, the determining preferably comprises contacting the sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:1.

[0256] The present invention further provides a method for assisting in the assessment of atrial fibrillation, the method comprising: a) providing at least one sample from a subject; b) determining in at least one sample provided in step a) the amount of one or more BMP10-type peptides and, optionally, the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 and FABP-3 (fatty acid binding protein 3); and c) providing a physician with information regarding the determined amount of one or more BMP10-type peptides, and optionally, information regarding the determined amount of at least one further biomarker, thereby assisting in the assessment of atrial fibrillation.

[0257] The physician is the attending physician, i.e., the physician who requested the determination of the biomarker(s). The above-described method is intended to assist the attending physician in assessing atrial fibrillation. Therefore, this method does not encompass the diagnosis, prediction, monitoring, differentiation, or identification referred to above in connection with the method of assessing atrial fibrillation.

[0258] Step a) of the above method of obtaining a sample does not involve withdrawing the sample from the subject. Preferably, the sample is obtained by receiving the sample from said subject. Thus, the sample may have been delivered.

[0259] In one embodiment, the method is a method for assisting in the prediction of stroke, the method comprising: a) providing at least one sample from a subject as referred to herein in connection with a method for assessing atrial fibrillation, in particular in connection with a method for predicting atrial fibrillation, b) determining the amount of one or more BMP10-type peptides and the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP-3 (fatty acid binding protein 3); and c) providing a physician with information regarding the determined amount of one or more BMP10-type peptides and, optionally, information regarding the determined amount of at least one further biomarker, thereby assisting in the prediction of stroke.

[0260] The present invention further comprises: a) providing an assay for one or more BMP10-type peptides and, optionally, at least one further assay for at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP-3 (fatty acid binding protein 3); b) providing instructions regarding the use of the assay results obtained or obtainable by said assay(s) in assessing atrial fibrillation.

[0261] The purpose of the above described method is preferably to aid in the assessment of atrial fibrillation.

[0262] The instructions may include a protocol for carrying out the methods for assessing atrial fibrillation described herein. The instructions may further include at least one value of a reference amount of one or more BMP10-type peptides, and optionally at least one value of a reference amount of a natriuretic peptide.

[0263] An "assay" is preferably a kit adapted for determining the amount of a biomarker. The term "kit" is explained below. For example, the kit may comprise at least one detection agent for one or more BMP10-type peptides, and optionally at least one further agent selected from the group consisting of an agent that specifically binds to a natriuretic peptide, an agent that specifically binds to ESM-1, an agent that specifically binds to Ang2, and an agent that specifically binds to FABP-3. Thus, there may be one to four detection agents. The detection agents for one to four biomarkers may be provided in one kit or in separate kits.

[0264] The test result obtained or available by a predictive window test is a value for the amount of biomarker(s).

[0265] In one embodiment, step b) comprises providing instructions for using test results obtained or available from the test(s) in predicting stroke (as described elsewhere herein).

[0266] The present invention further provides a computer-implemented method for assessing atrial fibrillation, comprising: a) receiving in a processing unit a value for the amount of one or more BMP 10 type peptides and optionally at least one further value for the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP-3 (fatty acid binding protein 3), wherein the amount of the one or more BMP 10 type peptides and optionally the amount of the at least one further biomarker have been determined in a sample derived from a subject, b) comparing, by said processing unit, the value(s) received in step (a) with reference(s); and c) assessing atrial fibrillation based on the comparing step b).

[0267] The above method is a computer-implemented method. Preferably, all steps of the computer-implemented method are performed by one or more processing units of a computer (or a computer network). Thus, the assessment of step (c) is performed by a processing unit. Preferably, the assessment is based on the result of step (b).

[0268] The value(s) received in step (a) shall be derived from determining the amount of biomarker from the subject, as described elsewhere herein. Preferably, the value is a concentration value of the biomarker. The value is typically received by the processing unit by uploading or transmitting the value to the processing unit. Alternatively, the value may be received by the processing unit by entering the value via a user interface.

[0269] In one embodiment of the foregoing method, the criterion(s) indicated in step (b) are established from memory. Preferably, the values ​​of the criterion(s) are established from memory.

[0270] In one embodiment of the aforementioned computer-implemented method of the present invention, the results of the assessment performed in step c) are provided via a display configured to present the results.

[0271] In one embodiment of the aforementioned computer-implemented method of the present invention, the method may include the further step of transferring information regarding the assessment made in step c) to the subject's electronic medical record.

[0272] Methods for the diagnosis of heart failure Furthermore, it has been shown in the present invention that determining the amount of one or more BMP10-type peptides in a sample from a subject allows for the diagnosis of heart failure. Thus, the present invention also contemplates a method for diagnosing heart failure based on the amount of one or more BMP10-type peptides (and optionally further based on natriuretic peptides, ESM-1, Ang2 and / or FABP3).

[0273] The definitions set forth herein above in relation to the assessment of atrial fibrillation apply mutatis mutandis to the following (except where otherwise stated).

[0274] Accordingly, the present invention further relates to a method for diagnosing heart failure in a subject, the method comprising: a) determining the amount of one or more BMP10-type peptides in a sample from a subject; and b) comparing the amount of one or more BMP10-type peptides with a reference amount, thereby diagnosing heart failure.

[0275] The method for diagnosing heart failure may further comprise determining the amount of at least one additional biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 (angiopoietin 2) and FABP-3 (fatty acid binding protein 3) and comparing with a suitable reference amount.

[0276] Therefore, the method for diagnosing heart failure includes: a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); b) diagnosing heart failure by comparing the amount of one or more BMP10-type peptides with a reference amount for one or more BMP10-type peptides, and optionally comparing the amount of at least one additional biomarker with a reference amount for the at least one additional biomarker.

[0277] As used herein, the term "diagnosing" means assessing whether a subject referred to in accordance with the method of the present invention is suffering from heart failure. The actual diagnosis of whether a subject is suffering from heart failure may include further steps such as confirmation of diagnosis. Therefore, the diagnosis of heart failure is understood as assistance in diagnosing heart failure. Therefore, the term "diagnosing" in the context of the present invention also encompasses assisting a physician to assess whether a subject is suffering from heart failure.

[0278] The term "heart failure" (abbreviated as "HF") is well known to those skilled in the art. As used herein, the term preferably relates to impaired systolic and / or diastolic function of the heart, accompanied by overt signs of heart failure known to those skilled in the art. Preferably, heart failure as referred to herein is also chronic heart failure. Heart failure according to the present invention includes overt heart failure and / or advanced heart failure. In overt heart failure, the subject exhibits symptoms of heart failure known to those skilled in the art.

[0279] In one embodiment of the present invention, the term "heart failure" refers to heart failure with reduced left ventricular ejection fraction (HFrEF). In another embodiment of the present invention, the term "heart failure" refers to heart failure with preserved left ventricular ejection fraction (HFpEF).

[0280] HF can be classified into various degrees of severity. According to the NYHA (New York Heart Association) classification, heart failure patients are classified as belonging to NYHA class I, II, III, and IV. Patients suffering from heart failure have already experienced structural and functional changes in the pericardium, myocardium, coronary circulation, or heart valves. These patients will not be able to fully recover and require treatment. NYHA class I patients have no obvious symptoms of cardiovascular disease but already have objective evidence of functional impairment. NYHA class II patients have slight limitations in physical activity. NYHA class III patients show significant limitations in physical activity. NYHA class IV patients are unable to perform any physical activity without discomfort. These patients exhibit symptoms of heart failure at rest.

[0281] This functional classification is complemented by a more recent classification by the American College of Cardiology and the American Heart Association (J. Am. Coll. Cardiol. 2001;38;2101-2113, updated in 2005, see J. Am. Coll. Cardiol. 2005;46;e1-e82). Four stages, A, B, C, and D, have been defined. Stages A and B are not HF, but are thought to be useful for early identification of patients before they "true" develop HF. Stage A and B patients are best defined as those with risk factors for the development of HF. For example, patients with coronary artery disease, hypertension, or diabetes mellitus who do not yet demonstrate left ventricular (LV) dysfunction, hypertrophy, or geometric ventricular distortion are considered stage A, while asymptomatic patients who demonstrate LV hypertrophy and / or LV dysfunction are considered stage B. Stage C then represents patients with current or past symptoms of HF associated with underlying structural heart disease (the majority of patients with HF), and stage D represents patients with truly refractory HF.

[0282] As used herein, the term "heart failure" preferably includes stages A, B, C, and D of the ACC / AHA classification described above. The term also includes NYHA classes I, II, III, and IV. Thus, a subject may or may not exhibit typical symptoms of heart failure.

[0283] In a preferred embodiment, the term "heart failure" refers to heart failure stage A according to the aforementioned ACC / AHA classification, or particularly heart failure stage B. Identifying these early stages, particularly stage A, is advantageous because treatment can be initiated before irreversible damage occurs.

[0284] The subject examined according to the method for diagnosing heart failure preferably does not suffer from atrial fibrillation. However, it is also assumed that the subject suffers from atrial fibrillation. The term "atrial fibrillation" is defined in relation to the method for assessing heart failure.

[0285] Preferably, the subject to be examined in connection with the method for diagnosing heart failure is suspected of suffering from heart failure.

[0286] The term "reference amount" is defined in relation to the method for assessing atrial fibrillation. The reference amount to be applied in the method for diagnosing heart failure can in principle be determined as described above.

[0287] Preferably, an increased amount of one or more BMP10-type peptides (and optionally, an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a subject compared to a reference amount indicates the subject is suffering from heart failure, and / or a decreased amount of one or more BMP10-type peptides (and optionally, an amount of at least one further biomarker, such as ESM-1, Ang-2, FABP-3 and / or a natriuretic peptide) in a sample from a subject compared to a reference amount indicates the subject is not suffering from heart failure.

[0288] In one embodiment of the method for diagnosing heart failure, the method further comprises recommending and / or initiating treatment for heart failure based on the results of the diagnosis. Preferably, if the subject is diagnosed with heart failure, treatment is recommended or initiated. Preferably, the heart failure treatment comprises administration of at least one medication selected from the group consisting of angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta-blockers, and aldosterone antagonists. Examples of angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor blockers, beta-blockers, and aldosterone antagonists are described in the following sections.

[0289] Method for predicting a subject's risk of hospitalization It is known that some subjects progress to heart failure more rapidly and are therefore at higher risk of hospitalization due to heart failure. It is important to identify these subjects as early as possible, to allow for therapeutic measures to prevent or delay progression to heart failure.

[0290] Advantageously, the study underlying the present invention has found that the amount of one or more BMP10 peptides in subject's sample allows to identify the subject at risk of being hospitalized with heart failure.For example, the subjects in the fourth quartile of BMP10 in the analyzed group (Example 4) have about four times the risk of being hospitalized with heart failure within three years compared with the subjects in the first quartile.

[0291] Accordingly, the present invention further provides a method for predicting a subject's risk of hospitalization due to heart failure, the method comprising: (a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2), and FABP-3 (Fatty Acid Binding Protein 3); (b) comparing the amount of one or more BMP10-type peptides with a reference amount of one or more BMP10-type peptides, and optionally, comparing the amount of at least one additional biomarker with a reference amount of the at least one additional biomarker.

[0292] The definitions and explanations given in relation to the method for assessing atrial fibrillation and the method for diagnosing heart failure preferably apply to the method for predicting a subject's risk of hospitalization due to heart failure.

[0293] The above method is The method may further comprise a step (c) of predicting the risk of the subject being hospitalized due to heart failure. Accordingly, steps (a), (b) and (c) are preferably as follows: (a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein 10 peptides), and optionally the amount of at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2 (Angiopoietin 2) and FABP-3 (Fatty Acid Binding Protein 3); (b) comparing the amount of the BMP10-type peptide with a reference amount, and optionally comparing the amount of at least one additional biomarker with a reference amount of said at least one additional biomarker; and (c) predicting the subject's risk of hospitalization due to heart failure;

[0294] Preferably, the prediction is based on the result of the comparison in step (b).

[0295] The expression "hospitalization" is well understood by those skilled in the art and preferably means that a subject is admitted to a hospital, particularly on an inpatient basis. The hospitalization must be due to heart failure. Thus, heart failure is the cause of the hospitalization. Preferably, the hospitalization is due to acute or chronic heart failure. Thus, heart failure includes both acute and chronic heart failure. More preferably, the hospitalization is due to acute heart failure. This predicts the risk of the subject being hospitalized due to heart failure.

[0296] The term "heart failure" is defined above. This definition applies accordingly. In some embodiments, the hospitalization is due to heart failure classified as stage C or D according to the ACC / AHA classification. The ACC / AHA classification is well known in the art and is described, for example, in Hunt et al. (Journal of the American College of Cardiology, Volume 46, Issue 6, 20 September 2005, Pages e1-e82, ACC / AHA Practice Guidelines), which is incorporated herein by reference in its entirety.

[0297] According to the above method, the risk of a subject being hospitalized due to heart failure is predicted.Therefore, it is possible to identify subjects who are at risk of being hospitalized due to heart failure or who are not at risk of being hospitalized due to heart failure.Therefore, the term "predicting risk" used in accordance with the above method herein preferably refers to assessing the probability of being hospitalized due to heart failure.In some embodiments, the above method of the present invention allows distinguishing between subjects who are at risk of being hospitalized due to heart failure and subjects who are not at risk.

[0298] According to the present invention, the term "predicting the risk" is understood as aiding in the prediction of the risk of hospitalization due to heart failure. The final prediction is, in principle, made by a doctor and may include further diagnostic results.

[0299] As will be understood by those skilled in the art, risk prediction is usually not intended to be correct for 100% of subjects. This term preferably means that a statistically significant portion of subjects can be predicted appropriately and accurately. Whether a portion is statistically significant can be determined by those skilled in the art without further ado by using various well-known statistical evaluation tools, such as determining confidence intervals, determining p-values, Student's t-test, Mann-Whitney test, etc. Details can be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Preferred confidence intervals are at least 90%, at least 95%, at least 97%, at least 98%, or at least 99%. The p-value is preferably 0.1, 0.05, 0.01, 0.005, or 0.0001.

[0300] Preferably, the risk / probability within a specific time window is predicted. In some embodiments, the prediction window is calculated from the completion of the method of the present invention. In particular, the prediction window is calculated from the time the sample to be tested was taken.

[0301] In a preferred embodiment of the present invention, the prediction window is preferably at least 1 year, at least 2 years, at least 3 years, at least 4 years, at least 5 years, or at least 10 years, or any interval time range. In another preferred embodiment of the present invention, the prediction window is preferably a period of up to 5 years, more preferably up to 4 years, or most preferably up to 3 years. Thus, the risk is predicted within a period of up to 3 years, up to 4 years, or up to 5 years. It is also envisioned that the prediction window is a period of 1 to 5 years. Alternatively, the prediction window may be a period of 1 to 3 years.

[0302] In a preferred embodiment, the risk of hospitalization due to heart failure within three years is predicted.

[0303] Preferably, the subjects analyzed by the above method of the present invention are assigned to either a group of subjects at risk of hospitalization due to heart failure or a group of subjects at no risk of hospitalization due to heart failure. At-risk subjects are preferably subjects at high risk of hospitalization due to heart failure (particularly within a predictive window). Preferably, the risk is elevated compared to the risk in the cohort of subjects (i.e., the group of subjects). Not-at-risk subjects are preferably subjects at low risk of hospitalization due to heart failure (particularly within a predictive window). Preferably, the risk is reduced compared to the average risk in the cohort of subjects (i.e., the group of subjects). Thus, the method of the present invention makes it possible to distinguish between elevated risk and reduced risk. At-risk subjects have a risk of hospitalization due to heart failure of preferably 12% or more, more preferably 15% or more, and most preferably 20% or more, preferably within a 3-year predictive window. Not-at-risk subjects have a risk of hospitalization due to heart failure of preferably less than 10%, more preferably less than 8%, or most preferably less than 7%, preferably within a 3-year predictive window.

[0304] The term "reference amount" is defined elsewhere herein. This definition applies where appropriate. The reference amount applied in the above methods should make it possible to predict the risk of hospitalization due to heart failure. In some embodiments, the reference amount should make it possible to distinguish between subjects at risk of hospitalization due to heart failure and subjects not at risk of hospitalization due to heart failure. In some embodiments, the reference amount is a predetermined value.

[0305] Preferably, an increased amount of one or more BMP10 peptides in a sample from a subject compared to a reference amount indicates the subject is at risk of hospitalization due to heart failure. Also preferably, a decreased amount of one or more BMP10 peptides in a sample from a subject compared to a reference amount indicates the subject is not at risk of hospitalization due to heart failure.

[0306] If more than one biomarker is determined, the following applies: Preferably, an increased amount of one or more BMP10 peptides and an amount(s) of the at least one further biomarker in a sample from the subject compared to the respective reference amounts indicates the subject is at risk of hospitalization due to heart failure. Also preferably, a decreased amount of one or more BMP10 peptides and an amount(s) of the at least one further biomarker in a sample from the subject compared to the respective reference amounts indicates the subject is not at risk of hospitalization due to heart failure.

[0307] The term "sample" is defined elsewhere herein. This definition applies where appropriate. In some embodiments, the sample is a blood, serum, or plasma sample.

[0308] The term "subject" is defined elsewhere herein. This definition applies where appropriate. In some embodiments, the subject is a human subject. Preferably, the subject being tested is 50 years of age or older, more preferably 60 years of age or older, and most preferably 65 years of age or older. It is further contemplated that the subject being tested is 70 years of age or older. It is further contemplated that the subject being tested is 75 years of age or older. The subject may also be between 50 and 90 years of age.

[0309] In one embodiment, the subject being tested has a history of heart failure. In another embodiment, the subject being tested does not have a history of heart failure.

[0310] The method of the present invention can support personalized medicine. In a preferred embodiment, the method for predicting the risk of a subject being hospitalized due to heart failure further comprises the step of recommending and / or initiating at least one appropriate treatment if the subject is predicted to be at risk of hospitalization due to heart failure. Therefore, the present invention also relates to a treatment method.

[0311] Preferably, the term "therapy" as used in the context of a method for predicting a subject's risk of hospitalization due to heart failure encompasses lifestyle changes, dietary changes, physical interventions, and drug treatment, i.e., treatment with a medication(s). Preferably, the therapy aims to reduce the risk of hospitalization due to heart failure. In one embodiment, the therapy is the administration of a medication(s). Preferably, the medication is selected from the group consisting of angiotensin-converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs), aldosterone antagonists, and beta-blockers.

[0312] In some embodiments, the pharmaceutical agent is a beta-blocker such as proprenolol, metoprolol, bisoprolol, carvedilol, bucindolol, and nebivolol. In some embodiments, the pharmaceutical agent is an ACE inhibitor such as enalapril, captopril, ramipril, and trandolapril. In some embodiments, the pharmaceutical agent is an angiotensin II receptor blocker such as losartan, valsartan, irbesartan, candesartan, telmisartan, and eprosartan. In some embodiments, the pharmaceutical agent is an aldosterone antagonist such as epleronolactone, spironolactone, canrenone, mexenone, and prorenone.

[0313] Lifestyle changes include smoking cessation, moderate alcohol intake, increased physical activity, weight loss, sodium (salt) restriction, weight management and healthy eating, daily fish oil, and salt restriction.

[0314] Furthermore, the present invention relates to uses (particularly in vitro uses, e.g. in a sample from a subject), comprising: i) one or more BMP10-type peptides and, optionally, at least one further biomarker selected from the group consisting of natriuretic peptides, ESM-1 (endocan), Ang2 and FABP-3 (fatty acid binding protein 3), and / or ii) Use of at least one agent that specifically binds to one or more BMP10-type peptides, and optionally at least one further agent selected from the group consisting of an agent that specifically binds to a natriuretic peptide, an agent that specifically binds to ESM-1, an agent that specifically binds to Ang2, and an agent that specifically binds to FABP-3, for a) assessing atrial fibrillation, b) predicting the risk of stroke in a subject, and / or c) diagnosing heart failure.

[0315] Terms mentioned in connection with the foregoing usage, such as "sample," "subject," "detection agent," "specific binding," "atrial fibrillation," and "assessing atrial fibrillation," are defined in connection with the method for assessing atrial fibrillation. Definitions and explanations apply where appropriate.

[0316] The present invention further relates to the in vitro use of a BMP10-type peptide and / or at least one agent that specifically binds to a BMP10-type peptide for predicting dementia in a subject.

[0317] The present invention further relates to the in vitro use of a BMP10-type peptide and / or at least one agent that specifically binds to a BMP10-type peptide for assessing the extent of white matter pathology in a subject.

[0318] The present invention further relates to the in vitro use of a BMP10-type peptide and / or at least one agent that specifically binds to a BMP10-type peptide to assess whether a subject has experienced one or more silent strokes.

[0319] The present invention further relates to the use of BMP10 type peptides and / or at least one agent that specifically binds to BMP10 type peptides (particularly in vitro use, for example in a sample derived from a subject) for predicting the risk of a subject being hospitalized due to heart failure.

[0320] Preferably, the above-mentioned use is an in vitro use.Furthermore, the detection agent is preferably an antibody, such as a monoclonal antibody (or an antigen-binding fragment thereof).

[0321] The present invention also relates to a kit, in one embodiment, comprising at least one agent that specifically binds to one or more BMP10-type peptides and at least one additional agent selected from the group consisting of an agent that specifically binds to a natriuretic peptide, an agent that specifically binds to ESM-1, an agent that specifically binds to Ang2, and an agent that specifically binds to FABP-3.

[0322] Preferably, the kit is suitable for carrying out a method of the invention, i.e., a method for assessing atrial fibrillation, or a method for diagnosing heart failure, or a method for predicting a subject's risk of hospitalization due to heart failure. Optionally, the kit comprises instructions for carrying out the method.

[0323] In some embodiments, the at least one agent that specifically binds to one or more BMP10-type peptides is at least one antibody or fragment thereof described in the next section (entitled "Antibodies of the Invention").

[0324] As used herein, the term "kit" refers to a collection of the above-mentioned components, preferably provided separately or in a single container. The container also contains instructions for carrying out the method of the present invention. These instructions may be in manual form or may be provided by computer program code that, when implemented in a computer or data processing device, performs the calculations and comparisons referred to in the method of the present invention and establishes an assessment or diagnosis, as appropriate. The computer program code may be provided on a data storage medium or device, such as an optical storage medium (e.g., a compact disc), or directly on the computer or data processing device. Furthermore, the kit may preferably include a standard amount of a BMP10-type peptide for calibration purposes. In a preferred embodiment, the kit further includes a standard amount of at least one additional biomarker referred to herein (such as a natriuretic peptide or ESM-1) for calibration purposes.

[0325] In one embodiment, the kit is used to assess atrial fibrillation in vitro. In an alternative embodiment, the kit is used to diagnose heart failure in vitro. In an alternative embodiment, the kit is used to predict the risk of hospitalization due to heart failure in vitro.

[0326] In the methods and uses of the invention described hereinabove and in the claims, the antibodies (or fragments thereof) used may be those described in the following paragraphs.

[0327] The definitions and explanations given herein above apply mutatis mutandis hereinafter.

[0328] Antibodies of the Invention The present invention also relates to antibodies (e.g., monoclonal antibodies) or fragments thereof that bind to one or more BMP10-type peptides, in particular, that bind to NT-proBMP10.

[0329] The antibody (or fragment thereof) shall be capable of binding to at least one of the amino acid regions of the polypeptide having the sequence set forth in SEQ ID NO:1 disclosed in the previous section.

[0330] Therefore, the antibody (or fragment thereof) of the present invention must be able to bind to NT-proBMP10, i.e., must be able to bind within the amino acid region 22 to 316 of the polypeptide set forth in SEQ ID NO: 1. Since this region is included in preproBMP10 and proBMP10, the antibody (or fragment thereof) must be able to bind to preproBMP10, proBMP10, and NT-proBMP10. However, the antibody (or fragment) must not bind to BMP10, i.e., mature BMP10.

[0331] In a preferred embodiment, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind within the amino acid region 37 to 299 of the polypeptide set forth in SEQ ID NO: 1, i.e., can bind to an epitope contained in the region starting at amino acid 37 and ending at amino acid 299 of the polypeptide set forth in SEQ ID NO: 1.

[0332] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind within the amino acid region 110 to 200 of the polypeptide set forth in SEQ ID NO: 1, i.e., the at least one active substance binds to an epitope contained in this region.

[0333] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides (e.g., NT-proBMP10) can bind within the amino acid region 37 to 195, for example, within the amino acid region 37 to 185, of the polypeptide set forth in SEQ ID NO: 1, i.e., the at least one active agent binds to an epitope contained in this region.

[0334] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind within the amino acid region 160 to 299 of SEQ ID NO: 1, for example, within the amino acid region 171 to 299, i.e., the at least one active substance binds to an epitope contained in this region.

[0335] For example, antibodies or fragments thereof that bind to one or more BMP10-type peptides, such as NT-proBMP10, can bind within the amino acid region 160-195 of SEQ ID NO:1, such as within the amino acid region 171-185.

[0336] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind to an epitope contained in amino acid region 37-47 of SEQ ID NO:1 (SLFGDVFSEQD, SEQ ID NO:2).

[0337] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind to an epitope (LESKGDNEGERNMLV, SEQ ID NO: 3) contained within amino acid region 171-185 of SEQ ID NO: 1. For example, the at least one agent binds to an epitope (SKGDNEGER, SEQ ID NO: 4) contained within amino acid region 173-181 of SEQ ID NO: 1.

[0338] For example, an antibody or fragment thereof that binds to one or more BMP10-type peptides, such as NT-proBMP10, can bind to an epitope contained in the amino acid region 291 to 299 of SEQ ID NO: 1 (SSGPGEEAL, SEQ ID NO: 5).

[0339] In the research underlying the present invention, the inventors generated monoclonal antibodies (in rabbits) against human NT-proBMP10. 280 antibodies were subjected to detailed kinetic screening. This screening identified antibodies that exhibited favorable kinetic properties that make them particularly useful for detecting BMP10-type peptides, such as NT-proBMP10 and / or proBMP10. For example, antibodies were identified that exhibited a fast rate of complex formation for the NT-proBMP10-antibody complex. Furthermore, the antibodies exhibited slow dissociation of the complex, resulting in a long half-life of the complex. Clones 11A5, 11C10, 13G6, 14C8, 2H8, and 9E7 exhibited comparable fast complex formation rates and complex half-lives with t / diss > 15 min. Clone 13G6 exhibited the slowest dissociation (k d <1.0E-04 s -1 ), resulting in a complex half-life t / 2-diss > 115 min.

[0340] The antibodies identified were: 11A2, 11A5, 11C10, 13G6, 14C8, 2H8, 3H8, 8G5, and 9E7. The antibody sequences are shown in Tables A-D below.

[0341] For four of the antibodies mentioned above, linear epitopes could be detected (see the Examples section and Figure 12). Two antibodies bound to the same region.

[0342] The epitope of 3H8 comprises the sequence shown in SEQ ID NO: 2 (SLFGDVFSEQD).

[0343] The epitope of 11A5 comprises the sequence shown in SEQ ID NO: 3 (LESKGDNEGERNMLV).

[0344] The epitope of 13G6 comprises the sequence shown in SEQ ID NO: 4 (SKGDNEGER).

[0345] The epitope of 2H8 comprises the sequence shown in SEQ ID NO: 5 (SSGPGEEAL).

[0346] Therefore, the antibody (or fragment thereof) or agent used to determine the amount of BMP10-type peptide may bind to the above region.

[0347] Table A summarizes the amino acid sequences of the variable heavy (VH) chains of the antibodies of the invention. [Table A]

[0348] Table B summarizes the amino acid sequences of the variable light chains of the antibodies of the invention (see below). [Table B]

[0349] The heavy chain CDR sequences are shown in Table C. The light chain CDR sequences are shown in Table D below. The complementarity determining regions (CDRs) of antibodies can be identified, for example, using the system described in Kabat et al. in Sequences of Proteins of Immunological Interest, 5th Ed., US Dept. of Health and Human Services, PHS, NIH, NIH Publication no. 91-3242, 1991. [Table C] [Table D]

[0350] The antibodies (or fragments thereof) of the present invention are intended to specifically bind to preproBMP, proBMP10, and NT-proBMP10 polypeptides. Thus, the antigen binding proteins are intended to bind to preproBMP, proBMP10, and NT-proBMP10 within the amino acid region 22-316 of SEQ ID NO: 1, as described in more detail elsewhere herein. It is understood that they do not bind to mature BMP10. However, they may be used in conjunction with antigen binding proteins that bind to mature BMP10.

[0351] The present invention is not limited to the identified antibodies, i.e., 11A2, 11A5, 11C10, 13G6, 14C8, 2H8, 3H8, 8G5, and 9E7, but also encompasses variants thereof that bind to one or more BMP10 types, preferably within the same region. For example, a variant of 13G6 would bind to the same or essentially the same epitope as 13G6.

[0352] Preferably, the antibody or fragment thereof of the present invention comprises the light chain variable domain (or a variant thereof) and heavy chain variable domain (or a variant thereof) of antibody 11A2, 11A5, 11C10, 13G6, 14C8, 2H8, 3H8, 8G5, or 9E7. For example, it may comprise the light chain variable domain (or a variant thereof) and heavy chain variable domain (or a variant thereof) of 13G6. For example, it may comprise the light chain variable domain (or a variant thereof) and heavy chain variable domain (or a variant thereof) of antibody 11C10.

[0353] In a preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises a heavy chain variable domain (or variant thereof) selected from VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8 and VH9 shown in Table A and / or a light chain variable domain (or variant thereof) selected from VL1, VL2, VL3, VL4, VL5, VL6, VL7, VL8 and VL9 shown in Table B. Each heavy chain variable domain shown in Table A above can be combined with each light chain variable domain shown in Table B. In a preferred embodiment, a heavy chain variable domain of Table A is combined with a corresponding light chain variable domain of Table B, e.g., VH1 with VL1, VH2 with VL2, or VH3 with VL3.

[0354] In some embodiments, a variant of a polypeptide as referred to in accordance with the present invention will have an amino acid sequence that differs due to at least one amino acid substitution, deletion and / or addition, and the amino acid sequence of the variant will still be preferably at least 80%, 85%, 90%, 95%, 98%, 99% identical to the amino acid sequence of the polypeptide.

[0355] In some embodiments, the antibody (or fragment thereof) variant comprises a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 (see Table A) and / or a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98% or 99% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 (see Table B), in order of increasing preference.

[0356] Alternatively or additionally, variants of antibodies or fragments thereof of the invention comprise the six CDRs of the parent antibody, which six CDRs differ from their respective parent CDRs by the addition, substitution, and / or deletion of a total of no more than three, two, or particularly no more than one amino acid (see Tables C and D).

[0357] Thus, in some embodiments, an antibody or fragment thereof of the present invention may comprise a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 (see Table A) and / or a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 (see Table B), in increasing order of preference. Each heavy chain variable domain set forth in Tables 1 and 2 can be combined with each light chain variable domain, although it is preferred that corresponding sequences be combined (e.g., SEQ ID NO: 7 and SEQ ID NO: 16).

[0358] "Percent (%) amino acid sequence identity" with respect to a reference polypeptide sequence is defined as the percentage of amino acid residues in a candidate sequence that are identical to those in the reference polypeptide sequence after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Standard parameters are preferably applied to determine the degree of sequence identity of two sequences. Preferably, the degree of identity should be determined by comparing two optimally aligned sequences over a comparison window, and the fragment of the amino acid sequence within the comparison window may, for optimal alignment, contain additions or deletions (e.g., gaps or overhangs) compared to the reference sequence (which does not contain additions or deletions). The percentage is calculated by determining the number of positions where identical amino acid residues occur in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to yield the percentage of sequence identity. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman Add. APL. Math. 2:482 (1981), the homology alignment algorithm of Needleman and Wunsch J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), computer implementations of these algorithms (GAP, BESTFIT, BLAST, PASTA, and TFASTA software packages from the Wisconsin Genetics Software Package (Genetics Computer Group (GCG), 575 Science Drive, Madison, Wisconsin), or visual inspection. Given that two sequences have been identified for comparison, it is preferred to use GAP and BESTFIT to determine their optimal alignment, and therefore the degree of identity. Preferably, the default values ​​of 5.00 for gap weight and 0.30 for gap weight length are used.In one embodiment, percent identity between two amino acid sequences is determined using the Needleman and Wunsch algorithm (Needleman 1970, J. Mol. Biol. (48):444-453) as incorporated into the Needle program of the EMBOSS software package (EMBOSS: The European Molecular Biology Open Software Suite, Rice, P., Longden, I., and Bleasby, A., Trends in Genetics 16(6), 276-277, 2000), a BLOSUM62 scoring matrix, and a gap opening penalty of 10 and a gap extension penalty of 0.5. Preferred, non-limiting examples of parameters used to align two amino acid sequences using the Needle program are the default parameters, including an EBLOSUM 62 scoring matrix, a gap opening penalty of 10, and a gap extension penalty of 0.5.

[0359] In one embodiment, the antibody or fragment thereof of the invention comprises a heavy chain variable domain having the sequence shown in SEQ ID NO:7 and a light chain variable domain having the sequence shown in SEQ ID NO:16.

[0360] In one embodiment, the antibody or fragment thereof of the invention comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO:8 and a light chain variable domain having the sequence set forth in SEQ ID NO:17.

[0361] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO:9 and a light chain variable domain having the sequence set forth in SEQ ID NO:18.

[0362] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO:10 and a light chain variable domain having the sequence set forth in SEQ ID NO:19.

[0363] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO:11 and a light chain variable domain having the sequence set forth in SEQ ID NO:20.

[0364] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO: 12 and a light chain variable domain having the sequence set forth in SEQ ID NO:21.

[0365] In one embodiment, the antibody or fragment thereof of the invention comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO: 13 and a light chain variable domain having the sequence set forth in SEQ ID NO: 22.

[0366] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO: 14 and a light chain variable domain having the sequence set forth in SEQ ID NO:23.

[0367] In one embodiment, the antibody of the invention or fragment thereof comprises a heavy chain variable domain having the sequence set forth in SEQ ID NO: 15 and a light chain variable domain having the sequence set forth in SEQ ID NO:24.

[0368] Alternatively or additionally, the antibody or fragment thereof of the present invention comprises: (a) (a1) a light chain CDR1 that differs from the light chain CDR1 shown in Table D by the addition, substitution and / or deletion of no more than a total of three, two, or in particular one amino acid; (a2) a light chain CDR2 that differs from a light chain CDR2 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion. (a3) a light chain CDR3 that differs from a light chain CDR3 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion. a light chain variable domain comprising: (b) (b1) a heavy chain CDR1 that differs from the heavy chain CDR1 shown in Table C by the addition, substitution, and / or deletion of a total of no more than three, two, or, in particular, one amino acid; (b2) a heavy chain CDR2 that differs from the heavy chain CDR2 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid; and (b3) a heavy chain CDR3 that differs from a heavy chain CDR3 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid. The heavy chain variable domain comprises:

[0369] For example, the antibody or fragment thereof of the present invention may (a) (a1) a light chain CDR1 that differs from light chain CDRL1-4 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion; (a2) a light chain CDR2 that differs from light chain CDRL2-4 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion; (a3) a light chain CDR3 that differs from the light chain CDRL3-4 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion a light chain variable domain comprising: (b) (b1) a heavy chain CDR1 that differs from heavy chain CDRH1-1 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid; (b2) a heavy chain CDR2 that differs from heavy chain CDRH2-4 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid; and (b3) a heavy chain CDR3 that differs from the heavy chain CDRH3-4 shown in Table C by the addition, substitution, and / or deletion of no more than three, two, or, in particular, one amino acid in total; The heavy chain variable domain comprises:

[0370] Preferably, the antibody or fragment thereof of the present invention comprises: (c) (a1) a light chain CDR1 shown in Table D (thus, a light chain CDR1 sequence selected from SEQ ID NOs: 34 to 42); (a2) a light chain CDR2 shown in Table D (thus, a light chain CDR2 sequence selected from SEQ ID NOs: 52 to 60), and (a3) Light chain CDR3 shown in Table D (therefore, a light chain CDR3 sequence selected from SEQ ID NOs: 70 to 78) a light chain variable domain comprising: (d) (b1) a heavy chain CDR1 shown in Table C (thus, a heavy chain CDR1 sequence selected from SEQ ID NOs: 25 to 33); (b2) a heavy chain CDR2 shown in Table C (thus, a heavy chain CDR2 sequence selected from SEQ ID NOs: 43 to 51), and (b3) Heavy chain CDR3 shown in Table C (thus, heavy chain CDR3 sequence selected from SEQ ID NOs: 61 to 69) The heavy chain variable domain comprises:

[0371] More preferably, the antibody or antigen-binding fragment thereof of the invention comprises the six CDRs of 11A2, the six CDRs of 11A5, the six CDRs of 11C10, the six CDRs of 13G6, the six CDRs of 14C8, the six CDRs of 2H8, the six CDRs of 3H8, the six CDRs of 8G5 or the six CDRs of 9E7 (or the six CDRs differ by the addition, substitution and / or deletion of a total of no more than three, no more than two, or especially no more than one amino acid from the heavy chain).

[0372] In a particularly preferred embodiment, the antibody or antigen-binding fragment thereof of the present invention comprises the six CDRs of 13G6. Thus, the antibody or fragment thereof comprises a heavy chain CDRH1 (NYAMS) of SEQ ID NO: 28, a heavy chain CDRH2 (YISASGNTYYASWVKG) of SEQ ID NO: 46, and a heavy chain CDRH3 (GYSGWISGTWA) of SEQ ID NO: 64, a light chain CDRL1 (QSSQSVVNNNRLS) of SEQ ID NO: 37, a light chain CDRL2 (RASTLAS) of SEQ ID NO: 55, and a light chain CDRL3 (LGDYVSYSEAA) of SEQ ID NO: 73.

[0373] In another preferred embodiment, the antibody or antigen-binding fragment thereof of the invention comprises the six CDRs of 11C10. Thus, the antibody or fragment thereof comprises a heavy chain CDRH1 (RNLMS) of SEQ ID NO: 27, a heavy chain CDRH2 (SINFRNITWYASWAKG) of SEQ ID NO: 45, a heavy chain CDRH3 (GVYVNSNGYYSL) of SEQ ID NO: 63, a light chain CDRL1 (QASQSVSNLLA) of SEQ ID NO: 36, a light chain CDRL2 (GASKLES) of SEQ ID NO: 54, and a light chain CDRL3 (QTYWGGDGTSYLNP) of SEQ ID NO: 72.

[0374] In some embodiments, antibodies or fragments thereof comprise the six CDRs of 11A2. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 11A5. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 14C8. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 2H8. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 3H8. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 8G5. In some embodiments, antibodies or fragments of the present invention comprise the six CDRs of 9E7.

[0375] It is understood that light chain CDRs are encompassed within a light chain variable domain, and heavy chain CDRs are encompassed within a heavy chain variable domain.

[0376] Furthermore, the antibody or fragment thereof of the present invention (a) a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 (see Table B), in order of increasing preference; (a1) a light chain CDR1 that differs from the light chain CDR1 shown in Table D by the addition, substitution and / or deletion of no more than a total of three, two, or in particular one amino acid; (a2) a light chain CDR2 that differs from the light chain CDR2 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion; (a3) a light chain CDR3 that differs from a light chain CDR3 shown in Table D by a total of no more than three, two, or, in particular, one amino acid addition, substitution, and / or deletion. a light chain variable domain comprising: (b) a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 (see Table A), (b1) a heavy chain CDR1 that differs from the heavy chain CDR1 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid; (b2) a heavy chain CDR2 that differs from the heavy chain CDR2 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid; and (b3) a heavy chain CDR3 that differs from a heavy chain CDR3 shown in Table C by the addition, substitution, and / or deletion of no more than a total of three, two, or, in particular, one amino acid. It is envisaged that the heavy chain variable domain comprises:

[0377] In one embodiment, the antibody or fragment thereof of the invention comprises: (a) a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24 (see Table B), in order of increasing preference; (a1) a light chain CDR1 shown in Table D; (a2) a light chain CDR2 shown in Table D, and (a3) Light chain CDR3 shown in Table D a light chain variable domain comprising: (b) a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15 (see Table A), (b1) a heavy chain CDR1 shown in Table C; (b2) a heavy chain CDR2 shown in Table C, and (b3) a heavy chain CDR3 shown in Table C The heavy chain variable domain comprises:

[0378] The term "antibody" refers to immunoglobulin or immunoglobulin-like molecules, including, but not limited to, IgA, IgD, IgE, IgG, and IgM, combinations thereof, and similar molecules produced during the immune response in any vertebrate, e.g., mammals such as goats, rabbits, and mice, as well as non-mammalian species, e.g., shark immunoglobulins. In some embodiments, the antibody may be a rabbit antibody. The term "antibody" includes intact immunoglobulins and "antibody fragments" or "antigen-binding fragments" that specifically bind to a molecule of interest. In a preferred embodiment, the antibody is an IgG antibody.

[0379] In particular, the term "antibody" refers to a polypeptide ligand comprising at least light and heavy chain immunoglobulin variable regions that specifically recognizes and binds to an epitope of an antigen. Antibodies are composed of heavy and light chains, each of which has a variable region called the variable heavy (VH) region and the variable light (VL) region. Collectively, the VH and VL regions are responsible for binding the antigen recognized by the antibody. Typically, the antibodies of the present invention have a heavy (H) chain and a light (L) chain interconnected by disulfide bonds. As used herein, the term "light chain" includes a full-length light chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length light chain includes a variable region domain, VL, and a constant region domain, CL. The variable region domain of the light chain is at the amino-terminus of the polypeptide. The term "heavy chain" includes a full-length heavy chain and fragments thereof having sufficient variable region sequence to confer binding specificity. A full-length heavy chain includes a variable region domain, VL, and a constant region domain, CL, H , as well as three constant region domains C H 1. C H 2, and C H 3, including V H The domain is located at the amino terminus of the polypeptide and is C H The domain is located at the carboxyl terminus and is C H 3 is closest to the carboxy terminus of the polypeptide.

[0380] There are five major heavy chain classes (or isotypes) that determine the functional activity of antibody molecules: IgM, IgD, IgG, IgA, and IgE. Each heavy and light chain contains a constant region and a variable region (the regions are also known as "domains"). In combination, the heavy and light chain variable regions specifically bind antigens. The light and heavy chain variable regions contain a "framework" region interrupted by three hypervariable regions, also called "complementarity-determining regions" or "CDRs." The CDRs are primarily responsible for binding to an epitope of an antigen. The CDRs of each chain are usually referred to as CDR1, CDR2, and CDR3, numbered consecutively starting from the N-terminus, and are usually identified by the chain in which a particular CDR is located. Thus, a VH CDR3 is located in the variable domain of the heavy chain of the antibody in which it is found, while a VL CDR1 is the CDR1 from the variable domain of the light chain of the antibody in which it is found.

[0381] The antibodies or fragments thereof of the present invention can be single chain antibodies, IgD antibodies, IgE antibodies, IgM antibodies, IgG antibodies, and fragments thereof. For example, the antibodies can be IgG antibodies, such as IgG1 antibodies, IgG2 antibodies, IgG3 antibodies, or IgG4 antibodies. In one embodiment, the antibodies or fragments thereof are recombinantly produced.

[0382] Fragments of the monoclonal antibodies of the present invention are also encompassed by the present invention. The fragments are intended to be immunologically functional fragments, i.e., antigen-binding fragments. Thus, fragments of the monoclonal antibodies of the present invention are intended to be capable of binding to one or more BMP10-type peptides. Thus, the term "immunologically functional fragment" of an antibody, as used herein, refers to a portion of the antibody that lacks at least some of the amino acids present in the full-length chain but is still capable of specifically binding to one or more BMP10-type peptides. Immunologically functional immunoglobulin fragments include Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments. Methods for producing antigen-binding fragments are well known in the art. For example, fragments can be produced by enzymatic cleavage of the antibodies of the present invention. Furthermore, fragments can be produced synthetically or by recombinant techniques. Fab fragments are preferably produced by papain digestion of the antibody, Fab' fragments by pepsin digestion and partial reduction, and F(ab')2 fragments by pepsin digestion. Fv fragments are preferably produced by molecular biology techniques.

[0383] In one embodiment, a fragment of an antibody of the invention is an F(ab')2 fragment. In another embodiment, a fragment of an antibody of the invention is an F(ab')2 fragment. In another embodiment, a fragment of an antibody of the invention is an Fab fragment. In another embodiment, a fragment of an antibody of the invention is an Fv fragment.

[0384] The antibody fragment may be a diabody, which is a small antibody fragment that has two antigen-binding sites. Diabodies preferably contain a heavy chain variable domain connected to a light chain variable domain in the same polypeptide chain.

[0385] The antibodies of the present invention are preferably monoclonal antibodies. As used herein, the term "monoclonal antibody" refers to an antibody produced by a single clone of B lymphocytes or by a cell into which antibody light and heavy chain genes have been transfected.

[0386] In one embodiment, the antibody of the present invention is an isolated antibody. Thus, the antibody is a purified antibody. Purification of antibodies can be achieved by methods well known in the art.

[0387] The monoclonal antibodies or fragments thereof of the present invention (and the agents referred to in the previous section) shall be capable of binding to one or more BMP10-type peptides, such as NT-proBMP10. The terms "bind", "specifically bind", "capable of binding" and "capable of specifically binding" are preferably used interchangeably herein. The terms "specific binding" or "specifically bind to" refer to a binding reaction in which molecules of a binding pair exhibit binding to each other under conditions in which they do not significantly bind to another molecule. The terms "specific binding" or "specifically bind to", when referring to proteins or peptides as biomarkers, preferably refer to a binding reaction in which a binding agent binds to the corresponding biomarker with at least 10 7 M -1 affinity ("association constant" K a The term "specific binding" or "specifically binds" preferably refers to a binding reaction in which the target molecule binds at least 10 8 M -1 , or even more preferably at least 10 9 M -1 The term "specific" or "specifically" refers to the affinity of the target molecule to which the target molecule is specifically bound. The term "specific" or "specifically" is used to indicate that other molecules present in the sample do not significantly bind to the target molecule-specific binding agent. However, as explained herein above, the agent referred to herein is not only capable of binding to NT-proBMP10, but also to preproBMP10 and proBMP10.

[0388] K D is a dissociation constant that can be determined by binding assays such as surface plasmon resonance technology (BIAcore®, GE-Healthcare, Uppsala, Sweden). Preferably, antibodies (or antigen-binding fragments thereof) that bind to one or more BMP10-type peptides have, in some embodiments, a K for one or more BMP10-type peptides (particularly human NT-proBMP10) in the single-digit nanomolar range or sub-nanomolar range at 37°C. D It has a value.

[0389] Another means of describing the kinetic binding properties of an antibody to its antigen is the decomposition of the dissociation rate constant into kinetic rate contributions, the association rate k a constant and dissociation rate constant k d There exists an association rate k a The constant characterizes the rate of antibody / antigen complex formation and is time- and concentration-dependent. In some embodiments, an antibody (or antigen-binding fragment thereof) of the present invention that binds to one or more BMP10-type peptides as referred to herein (particularly human NT-proBMP10) has a k (for that antigen) of greater than 1.0E+05 M-1 s-1 at 37°C. a It has a value.

[0390] Dissociation rate constant (k d ) indicates the dissociation rate of an antibody from an antigen. Thus, the dissociation rate constant indicates the probability that the complex will disintegrate in time. The lower the dissociation rate constant, the stronger the antibody binds to its antigen. In some embodiments, an antibody (or antigen-binding fragment thereof) of the present invention that binds to one or more BMP10-type peptides as referred to herein (particularly human NT-proBMP10) has a k of less than 1.1E-03 s at 37°C. d It has a value.

[0391] In some embodiments, K D value, k d value and k a The values ​​are determined as in the Examples section.

[0392] The term "epitope" refers to a protein determinant capable of specific binding to an antibody. Thus, the term preferably refers to a portion of a polypeptide referred to herein that can be specifically bound by an antibody (or fragment thereof) of the present invention. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids and usually have specific charge characteristics as well as specific three-dimensional structural features. Conformational and nonconformational epitopes are distinguished in that the binding to conformational epitopes, but not nonconformational epitopes, is lost in the presence of denaturing solvents.

[0393] The antibodies or antigen-binding fragments thereof of the present invention are useful in methods related to determining the amount of the BMP10-type peptides referred to herein. For example, the antibodies or fragments thereof allow for determining the amount of BMP10-type peptides in a sample for use in diagnostic methods.

[0394] Therefore, the present invention relates to a method for determining the amount of one or more BMP10-type peptides by using the antibody or fragment of the present invention. Preferred methods for determining the amount of biomarkers, such as sandwich assays, are described in the section entitled "Determining the amount of biomarkers."

[0395] For example, a method for determining the amount of one or more BMP10-type peptides may include contacting a sample containing one or more BMP10-type peptides with at least one active substance that binds within the amino acid region 22-316 of the polypeptide set forth in SEQ ID NO: 1, thereby allowing the formation of a complex between the BMP10-type peptide and the at least one active substance, and determining the amount of the complex formed.

[0396] Furthermore, the antibody or fragment thereof may contain a label, i.e., may be covalently or non-covalently attached to a label that allows for detection and measurement of the bound agent. Preferred labels are disclosed hereinabove. For example, the label may be biotin, a radioactive label, a fluorescent label, a chemiluminescent label, an electrochemiluminescent label, a gold label, or a magnetic label, as described in more detail above.

[0397] In one embodiment, the antibody or fragment thereof (such as a F(ab')2 fragment) is biotinylated. In another embodiment, the antibody or fragment thereof is ruthenylated (as described above).

[0398] The present invention further relates to the in vitro use of an antibody or a fragment thereof according to the invention for determining the amount of one or more BMP10-type peptides in a sample.

[0399] The present invention also relates to a kit comprising at least one monoclonal antibody or fragment thereof of the invention, the term kit being defined above.

[0400] Furthermore, we identified antibody pairs that enable improved detection of BMP10-type peptides in sandwich assays (Example 13). Specifically, biotinylated and ruthenium-conjugated clones 2H8, 3H8, 8G5, 9E7, 11A5, 11C10, 14C8, and 13G6 were tested in multiple sandwich assays to identify sandwich partners. The combination of clones 13G6 and 11C10 was found to exhibit high-grade performance regardless of orientation. Similar observations were made with the combination of 3H8 and 9E7. Other combinations also achieved good ratios, albeit in only one direction.

[0401] Thus, the present invention relates to a kit comprising a first antibody or antigen-binding fragment thereof that binds to one or more BMP10-type peptides and a second antibody or antigen-binding fragment thereof that binds to one or more BMP10-type peptides, preferably wherein the first and second antibodies bind to different epitopes, and the first antibody is selected from 2H8, 3H8, 8G5, 9E7, 11A5, 11C10, 14C8, 13G6 (or a variant thereof), and the second antibody is selected from 2H8, 3H8, 8G5, 9E7, 11A5, 11C10, 14C8, 13G6 (or a variant thereof).

[0402] Both antibodies or fragments thereof can be labeled, for example, the first antibody or fragment thereof can be ruthenylated and the second antibody or fragment thereof can be biotinylated (or vice versa).

[0403] In some embodiments, the first antibody is 2H8. In some embodiments, the first antibody is 3H8. In some embodiments, the first antibody is 8G5. In some embodiments, the first antibody is 9E7. In some embodiments, the first antibody is 11A5. In some embodiments, the first antibody is 11C10. In some embodiments, the first antibody is 14C8. In some embodiments, the first antibody is 13G6.

[0404] Preferred combinations of antibodies are disclosed in Tables 13 and 14 of Example 14.

[0405] For example, the first antibody can be 11C10 and the second antibody can be 13G6 (or vice versa).

[0406] For example, the first antibody can be 3H8 and the second antibody can be 9E7 (or vice versa).

[0407] In one embodiment, the kit comprises a biotinylated first monoclonal antibody or fragment thereof that specifically binds to one or more BMP10-type peptides, wherein the first antibody is 11C10 (or a variant thereof), and a ruthenium-ylated second monoclonal antibody or fragment thereof, wherein the second antibody is 13G6 (or a variant thereof).

[0408] In one embodiment, the kit comprises a biotinylated first monoclonal antibody or fragment thereof that specifically binds to one or more BMP10-type peptides, wherein the first antibody is 13G6 (or a variant thereof), and a ruthenium-ylated second monoclonal antibody or fragment thereof, wherein the second antibody is 11C10 (or a variant thereof).

[0409] The present invention also relates to host cells producing the antibodies or antigen-binding fragments thereof of the present invention. In a preferred embodiment, the host producing the antibodies of the present invention is a hybridoma cell. Furthermore, the host cell can be any type of cell line that can be engineered to produce the antibodies of the present invention. For example, the host cell can be an animal cell, particularly a mammalian cell such as an HEK cell. In some embodiments, HEK293 (human embryonic kidney cell) or CHO (Chinese hamster ovary) cell, such as the HEK293-F cell used in the Examples section, is used as the host cell. In another embodiment, the host cell is a non-human animal or mammalian cell.

[0410] The host cell preferably contains at least one polynucleotide encoding an antibody of the present invention or a fragment thereof. For example, the host cell contains at least one polynucleotide encoding the light chain of an antibody of the present invention and at least one polynucleotide encoding the heavy chain of an antibody of the present invention. The polynucleotide(s) should be operably linked to a suitable promoter. [Brief explanation of the drawings]

[0411] The drawings show: [Figure 1]Measurement of BMP10 ELISA in three patient groups (patients with paroxysmal atrial fibrillation, persistent atrial fibrillation, and sinus rhythm) [Figure 2] ROC curve of BMP10 in paroxysmal Afib; AUC=0.68 [Figure 3] ROC curve of BMP10 in persistent Afib; AUC=0.90 (Exploratory AFib panel: patients with a history of atrial fibrillation, including 14 paroxysmal AFib, 16 persistent AFib, and 30 controls) [Figure 4] BMP10 (unit: ng / ml) in differentiating patients with and without heart failure [Figure 5] BMP10 in the differentiation of heart failure; ROC curve of BMP10; AUC=0.76 [Figure 6] Kaplan-Meier curves showing the risk of HF hospitalization by quartile of BMP-10 in patients with a prior history of heart failure. [Figure 7] Kaplan-Meier curves showing the risk of HF hospitalization by quartile of BMP-10 in patients without a previous history of heart failure. [Figure 8] Kaplan-Meier curve showing the risk of stroke according to the median BMP-10 concentration. [Figure 9] Kaplan-Meier curves showing the risk of AFib recurrence by quartile of BMP-10 in patients undergoing pulmonary vein isolation (BEAT-PVI). [Figure 10-1] Kinetic signatures for propeptide BMP10 binding for a selection of 8 mAbs at 37 °C obtained via SPR. Sensorgram overlays for increasing propeptide BMP10 concentrations ranging from c = 3.7 to 300 nM are shown. A) 2H8 B) 3H8 C) 8G5 D) 9E7 E) 11A5 F) 11C10 G) 14C8 H) 13G6 [Figure 10-2]Kinetic signatures for propeptide BMP10 binding for a selection of 8 mAbs at 37 °C obtained via SPR. Sensorgram overlays for increasing propeptide BMP10 concentrations ranging from c = 3.7 to 300 nM are shown. A) 2H8 B) 3H8 C) 8G5 D) 9E7 E) 11A5 F) 11C10 G) 14C8 H) 13G6 [Figure 11] Overlay of normalized dissociation phases for 8 mAbs binding to the propeptide BMP10 at 37 °C. [Figure 12] Epitopes of the four monoclonal antibodies of the present invention [Figure 13] Detection of NT-proBMP 10 versus mature BMP10 [Figure 14] Measurement of circulating BMP-10 in EDTA plasma samples from the SWISS AF study in Fazekas score <2 (no) vs. Fazekas score ≥2 (yes): detection of WML / prediction of risk of silent stroke: Circulating BMP-10 levels were assessed. [Example]

[0412] The present invention is illustrated solely by the following examples, which should not be construed in any way as limiting the scope of the invention.

[0413] Example 1: Mapping study - Diagnosing patients with atrial fibrillation compared to patients based on their different circulating NT-proBMP10 levels This mapping study involved patients undergoing open-heart surgery. Samples were obtained before anesthesia and surgery. Patients were electrophysiologically characterized using high-density epicardial mapping (high-density mapping) with a multielectrode array. The study included 14 patients with paroxysmal atrial fibrillation, 10 patients with persistent atrial fibrillation, and 28 controls, best matched (for age, sex, and comorbidities) as possible. NT-proBMP10 was determined in serum samples from the MAPPING study. Elevated NT-proBMP10 levels were observed in patients with atrial fibrillation compared with controls. NT-proBMP10 levels were elevated in patients with paroxysmal atrial fibrillation versus matched controls, as well as in patients with persistent atrial fibrillation versus controls.

[0414] Furthermore, the biomarker ESM-1 was determined in samples from the MAPPING cohort. Interestingly, it was shown that the combined determination of NT-proBMP10 and ESM-1 allowed the AUC for the differentiation between persistent AF versus SR (sinus rhythm) to increase to 0.92.

[0415] Furthermore, the biomarker FABP-3 was determined in samples from the MAPPING cohort. Interestingly, it was shown that the combined determination of NT-proBMP10 and FABP-3 allowed for the differentiation between paroxysmal AF and SR (sinus rhythm) to increase the AUC to 0.73.

[0416] Example 2: Heart Failure Panel The heart failure panel included 60 patients with chronic heart failure. Heart failure was diagnosed in patients with typical signs and symptoms and objective evidence of structural or functional cardiac abnormalities at rest, according to the ESC guideline criteria. Patients aged 18 to 80 years with ischemic or dilated cardiomyopathy or significant valvular disease and who were able to sign a consent form were included in the study. Patients with acute myocardial infarction, pulmonary embolism, or stroke in the past 6 months, as well as severe pulmonary hypertension and end-stage renal disease, were excluded. Patients primarily had NYHA stage II to IV heart failure.

[0417] The healthy control cohort included 33 subjects. Health status was verified by assessing the status of ECG and echocardiogram results. Participants with any abnormalities were excluded.

[0418] Elevated NT-proBMP10 levels were observed in serum samples from heart failure patients compared to controls.

[0419] Example 3: Measurement of biomarkers NT-proBMP10 was measured with a research-grade ECLIA assay for bone morphogenetic protein 10 (BMP10) (ECLIA assay from Roche Diagnostics, Germany).

[0420] To detect NT-proBMP10 in human serum and plasma samples, we used an antibody sandwich that specifically binds to the N-terminal prosegment of BMP10. Such antibodies also bind to proBMP10 and preproBMP10. Therefore, we calculated the total amount of the N-terminal prosegment of BMP10, proBMP10, and preproBMP10. Structural predictions based on knowledge from other BMP-type proteins, such as BMP9, indicate that BMP10 remains in a complex with proBMP10. Therefore, detection of the N-terminal prosegment also reflects the amount of prodomain-bound BMP10. Furthermore, we were able to detect homodimeric forms of BMP10 as well as heterodimeric structures, such as those in combination with BMP9 or other BMP-type proteins.

[0421] Example 4: SWISS AF Study - Risk prediction for heart failure hospitalization Data from the SWISS-AF study included 2,387 patients, 617 of whom had a history of heart failure (HF). BMP-10 was measured in these patients to assess its ability to predict the risk of hospitalization due to HF.

[0422] Because HF hospitalizations can occur in patients with and without a history of HF, the ability to predict future HF hospitalizations was assessed separately for these groups. A total of 233 patients had a documented HF hospitalization during follow-up. Of the 233 hospitalizations, 125 were in patients with previously known HF.

[0423] Predicting HF hospitalization in patients with a history of HF Table 1 shows the results of a Cox proportional hazards model including patients with a history of HF. The dependent variable was time to HF hospitalization, and the independent variable was the log-2 transformed NT-proBMP10 values.

[0424] As evidenced by the hazard ratio and low p-value, NT-proBMP10 significantly predicts the risk of HF hospitalization in patients with a history of HF. Because NT-proBMP10 values ​​were log-2 transformed before entering the model, the hazard ratio can be interpreted as a 3.43 increase in patient risk for a doubling of NT-proBMP10 levels. [Table 1]

[0425] Figure 6 shows Kaplan-Meier curves showing the risk of HF hospitalization by quartile of NT-proBMP10. It can be seen that the risk increases continuously with increasing NT-proBMP10 levels, with the greatest risk observed for patients with NT-proBMP10 levels within the highest quartile.

[0426] Predicting HF hospitalization in patients without a history of HF Table 2 shows the results of a Cox proportional hazards model including patients without a history of HF. The dependent variable was time to HF hospitalization, and the independent variable was log-2 transformed NT-proBMP10 levels.

[0427] As evidenced by the hazard ratio and low p-value, NT-proBMP10 significantly predicts the risk of HF hospitalization in patients without a history of HF. Because NT-proBMP10 values ​​were log-2 transformed before entering the model, the hazard ratio can be interpreted as a 3.43 increase in patient risk for a doubling of NT-proBMP10 levels. [Table 2]

[0428] Figure 7 shows Kaplan-Meier curves showing the risk of HF hospitalization by quartile of NT-proBMP10. Risk increases with increasing NT-proBMP10 levels, with the highest risk observed for patients with NT-proBMP10 levels within the two highest quartiles.

[0429] Example 5: SWISS AF Study - Stroke Risk Prediction The ability of circulating NT-proBMP10 to predict the risk of stroke was validated in a prospective multicentric registry of patients with confirmed atrial fibrillation (see Example 3) (Conen D., Swiss Med Wkly 2017 Jul 10;147-w14467).

[0430] NT-proBMP10 results were available for 65 patients with events and 2269 patients without events.

[0431] To quantify the univariate prognostic value of NT-proBMP10, a proportional hazards model was used for outcome stroke.

[0432] The univariate prognostic performance of NT-proBMP10 was assessed by two different incorporations of the prognostic information conferred by NT-proBMP10.

[0433] The first proportional hazards model included NT-proBMP10 dichotomized at the median (2.2 ng / mL), thus comparing the risk of patients with NT-proBMP10 below the median versus those with NT-proBMP10 above the median.

[0434] The second proportional hazards model included the original NT-proBMP10 levels but transformed them to the log2 scale. The log2 transformation was performed to allow for better model calibration.

[0435] A weighted version of the Kaplan-Meier plot was created to obtain estimates for absolute survival in the two groups based on the dichotomized baseline NT-proBMP10 measurement (<=2.2 ng / mL vs. >2.2 ng / mL).

[0436] To assess whether the prognostic value of NT-proBMP10 was independent of known clinical and demographic risk factors, a weighted proportional Cox model was calculated, additionally including the variables age and history of stroke / TIA / thromboembolism, which were the only significant clinical risk predictors in the entire cohort (including all controls).

[0437] To assess the ability of NT-proBMP10 to improve existing risk scores for stroke prognosis, the CHADS2, CHA2DS2-VASc, and ABC scores were extended with NT-proBMP10 (log2 transformed). Extensions were performed by creating partial hazard models that included NT-proBMP10 and each risk score as independent variables.

[0438] The c-indices of the CHADS2 and CHA2DS2-VASc and ABC scores were compared with the c-indices of these extended models.

[0439] result Table 1 shows the results of two univariate weighted proportional hazards models that included dichotomized or log2-transformed NT-proBMP10. The association between the risk of experiencing a stroke and baseline levels of NT-proBMP10 was not significant in the model using log2-transformed NT-proBMP10 as a risk predictor, but it approached the 0.05 significance level.

[0440] For the model using binarized NT-proBMP10, the p-value is slightly higher, but with a larger number of events, it can be argued that the effect is statistically significant.

[0441] The dichotomized hazard ratio for NT-proBMP10 indicates that patients with NT-proBMP10 >2.2 ng / mL have a 1.5-fold increased risk of stroke compared with patients with baseline NT-proBMP10 ≤2.2 ng / mL, as can be seen in Figure 8, which shows the Kaplan-Meier curves for the two groups.

[0442] The results of a proportional hazards model including NT-proBMP10 as a log2-transformed linear risk predictor suggest that the log2-transformed NT-proBMP10 level is proportional to the risk of experiencing a stroke. The hazard ratio of 2.038 can be interpreted as meaning that a two-fold increase in NT-proBMP10 is associated with a 2.038 increase in the risk of stroke. [Table 1]

[0443] Table 2 shows the results of the proportional hazards model including NT-proBMP10 (log2 transformed) combined with clinical and demographic variables. It can be seen that the prognostic value of NT-proBMP10 is somewhat reduced, but this can also be partly explained by the low statistical power of the model. [Table 2]

[0444] Table 3 shows the results of a weighted proportional hazards model combining the CHADS2 score with NT-proBMP10 (log2 transformed). In this model, NT-proBMP10 can add prognostic information to the CHADS2 score, but with a p-value of greater than 0.05, which is acceptable for a small sample size. [Table 3]

[0445] Table 4 shows the results of a weighted proportional hazards model combining the CHA2DS2-VASc score with NT-proBMP10 (log2 transformed). In this model, NT-proBMP10 also adds prognostic information to the CHA2DS2-VASc score, but with a p-value greater than 0.05, which is acceptable for a small sample size. [Table 4]

[0446] Table 5 shows the results of a weighted proportional hazards model combining the ABC score with NT-proBMP10 (log2 transformed). In this model, the estimated hazard ratio was reduced, suggesting that NT-proBMP10 likely does not add any prognostic value. [Table 5]

[0447] Table 6 shows the estimated C-indexes of the weighted proportional hazards model for case cohort selection: NT-proBMP10 alone, CHADS2, CHA2DS2-VASc, ABC score, and the combination of CHADS2, CHA2DS2-VASc, ABC score, and NT-proBMP10 (log2). It can be seen that the addition of NT-proBMP10 improves the C-index of CHADS2 and CHA2DS2-VASc score, but not the ABC score.

[0448] The differences in the indices were 0.019, 0.015, and −0.002 for CHADS2, CHA2DS2-VASc, and ABC scores, respectively. [Table 6]

[0449] Example 6: BEAT-AF-PVI Study - Predicting the risk of recurrent AFib after pulmonary vein isolation and catheter ablation. The ability of NT-proBMP10 to predict the risk of future recurrent atrial fibrillation episodes was assessed in the BEAT-AF-PVI study. The BEAT-AF-PVI study (Knecht S, International Journal of Cardiology, Volume 176, Issue 3, 2014, Pages 645-650) is a prospective cohort study including patients with atrial fibrillation who underwent pulmonary vein isolation. One of the study endpoints collected was the time to first recurrence of atrial fibrillation. Therefore, the ability of circulating NT-proBMP10 to predict the risk of AFib recurrence after PVI and catheter ablation was examined in a prospective multicenter registry of patients with documented atrial fibrillation (Zeljkovic I, Biochem Med. 2019;29:020902).

[0450] Information on NT-proBMP10 measurements and atrial fibrillation recurrence was available for 719 patients. Atrial fibrillation recurrence was observed in 310 of the 719 patients. NT-proBMP10 was measured with a research-grade ECLIA assay for bone morphogenetic protein 10 (NT-proBMP10) from Roche Diagnostics (Germany).

[0451] The ability of NT-proBMP10 to predict the risk of recurrent atrial fibrillation was assessed by Cox (proportional hazards) regression model. The results of the proportional hazards model are shown in Table 1. The results show that the risk of recurrent atrial fibrillation significantly increases as the value of NT-proBMP10 increases. Because NT-proBMP10 was included in the model log2 transformed to improve model calibration, the hazard ratio can be interpreted as a 1.91 increased risk for a 2-fold increase in NT-proBMP10. [Table 7]

[0452] Alternatively, NT-proBMP10 can be used in a dichotomized form (e.g., divided at the median of 1.7 ng / mL) to predict the risk of atrial fibrillation. Table 8 shows that the risk increases by 32% in patients with NT-proBMP10 levels above the median. This risk difference is also statistically significant. [Table 8]

[0453] Example 7: Evaluation of recurrent AF with NT-proBMP10 The GISSI AF study involved patients in sinus rhythm (SR) with a history of atrial fibrillation (AF) but without significant left ventricular dysfunction or heart failure. All patients underwent NT-proBMP10 biochemistry assessment and electrocardiograms three times during a one-year follow-up.

[0454] Circulating NT-proBMP10 levels were determined in samples from n=281 patients who underwent blood sampling and biomarker assays at the 6-month SR visit, and in samples from n=33 patients who underwent blood sampling and biomarker assays at the 6-month visit and had progressing AF. An antibody against the propeptide BMP10 was used. [Table 9]

[0455] As shown in Table 9, NT-proBMP10 was observed in patients with ongoing AF at the time of sampling compared to patients with SR at the time of blood draw at the 6-month visit in the GISSI AF study.

[0456] It is clear that for NT-proBMP10, a small but highly significant delta change in marker elevation in patients with AF versus those in SR can be detected: in 33 patients with ongoing AF at the time of sampling, a median NT-proBMP10 of 2.31 [2.04-2.67] vs. 1.97 [1.75-2.33] ng / mL was observed for 281 patients in SR at the time of blood draw.

[0457] A sample of n = 105 patients was in SR at the 6-month visit but experienced multiple recurrences of AF between randomization and the 6-month visit. All 105 patients spontaneously converted to SR. - 0-7 days n=11 patients - 8-30 days n=17 patients - >30 days n=77 patients [Table 10]

[0458] As shown in Table 10, NT-proBMP10 titers were observed to increase up to 7 days after AF in patients who spontaneously converted to SR. In 11 patients with preceding AF up to 7 days before sampling, a median value of 2.30 [1.65-2.45] vs. 1.90 [1.75-2.25] ng / mL was observed for 77 patients with preceding AF more than 30 days before blood sampling. Notably, very similar median values ​​for NT-proBMP10 were observed up to 7 days after AF before sampling as in patients with ongoing AF. In 11 patients with SR after AF up to 7 days before sampling, a median NT-proBMP10 value of 2.30 [1.65-2.45] ng / mL was observed. As shown in Table 9, a median NT-proBMP-10 value of 2.31 [2.04-2.67] ng / mL was observed in 33 patients with ongoing AF at the time of blood sampling during SR.

[0459] Data evaluation showed that patients with NT-proBMP10 levels (independent of other biomarkers) above the reference value (>2.0 ng / mL in the study) are suspected of having recurrent atrial fibrillation after therapeutic intervention, e.g., after cardioversion. Differentiation between poor and good responders aids decision-making, as patients may not benefit from treatment, avoiding expensive treatment and associated burdens, but with poor outcomes for the patient.

[0460] It has even been shown that elevated NT-proBMP10 levels can be detected in patients presenting in sinus rhythm up to 7 days after a preceding AF episode.

[0461] In summary, the diagnosis of paroxysmal atrial fibrillation in patients presenting with sinus rhythm within 7 days can be achieved by detecting enhanced levels of NT-proBMP10 alone or in combination with markers of cardiac damage (e.g., cTNThs) and / or markers of heart failure (NT-ProBNP).

[0462] Example 8: Detection of NT-proBMP 10 versus mature BMP10 To compare the detection of mature BMP10 and NT-proBMP10, the Elecsys prototype detection method described in Example 14 was measured in a head-to-head analysis using a BMP-10 ELISA (R&Dsystems DuoSet DY 2926-05) that detects mature BMP-10 homodimer (aa 317-424). The measured samples were derived from the mapping cohort described in Example 1. Compared with the commercially available immunoassay for detecting mature BMP10 from R&Dsystems, the novel method for detecting NT-proBMP10 was applied to compare patients diagnosed with atrial fibrillation based on their different circulating BMP10 levels. Serum samples from the patients were electrophysiologically characterized using high-density epicardial mapping with a multielectrode array (MAPPING study) (Figure 13, or the table below). [Table 10A]

[0463] Mature BMP10 levels were detectable in only 6 of the 52 samples, reflecting 4 patients in sinus rhythm, 1 in paroxysmal AF, and 1 in persistent AF. However, as described in Example 1, NT-proBMP10 levels were detectable in all samples.

[0464] This finding, that only 11.5% of samples had detectable levels of mature BMP10, suggests that physiologically the mature form is underrepresented in the circulation due to internalization upon receptor binding. Therefore, NT-proBMP10 circulates in a more stable form and at higher detectable concentrations, enabling clinical decision-making based on circulating NT-proBMP10 levels.

[0465] Example 9: Immunization of rabbits for the generation of antibodies against BMP-10 Here, we describe the development of antibodies capable of binding to bone morphogenetic protein-10 (NT-proBMP10). To generate such antibodies, we immunized 12- to 16-week-old NZW rabbits with rec. NT-proBMP10, a polypeptide containing the first 312 amino acids of preproBMP10. All rabbits were subjected to repeated immunizations. During the first month, animals were immunized weekly. From the second month onward, animals were immunized once a month. For the first immunization, we dissolved 500 μg of immunogen in 1 mL of 140 mM NaCl and emulsified the solution with 1 mL of CFA. For all subsequent immunizations, CFA was replaced with IFA.

[0466] Example 10: Development of antibodies that bind to NT-proBMP10 To develop antibodies that bind to BMP-10, we used B cell cloning as described in Seeber et al. (2014), PLoS One. 2014 Feb 4;9(2). First, a PBMC pool was prepared from the whole blood of immunized animals by Ficoll gradient centrifugation. To enrich for antigen-reactive B cells from the PBMC pool, random biotinylated NT-proBMP10 was immobilized on streptavidin-coated magnetic beads (Miltenyi). For bead coating, the protein was used at a concentration of 1 μg / ml. Therefore, we incubated the prepared PBMC pool from immunized animals with NT-proBMP10-coated beads for 1 hour. For enrichment of antigen-reactive B cells, we used a MACS column (Miltenyi). B cell sorting and incubation were performed as described in Seeber et al. (2014), PLoS One. 2014 Feb 4;9(2). To identify NT-proBMP10-reactive clones by ELISA, we immobilized NT-proBMP10 on the surface of a 96-well plate at a concentration of 250 ng / ml. After washing, the plate was blocked with 5% BSA to reduce background signals. The plate was washed again, and 30 μl of primary rabbit B cell supernatant was transferred to the 96-well plate and incubated at room temperature for 1 hour. For detection of antibodies bound to the screening peptide, HRP-labeled F(ab')2 goat anti-rabbit Fcγ (Dianova) and ABTS (Roche) were added as a substrate. Clones that bound to plate-bound NT-proBMP10 were selected for subsequent molecular cloning as described in Seeber et al. (2014), PLoS One. 2014 Feb 4;9(2).

[0467] Example 11: Kinetic screening Informative antigen: Propeptide BMP10 (R&D-Systems) and in-house construct "312" (prepropeptide BMP10) both represent the prodomain of the N-terminal leader peptide of BMP10 ± 19 aa. Recombinant human BMP-10, R&D-Systems, Catalog No. 2926-BP / CF, Lot: Qual 0518031, disulfide-linked homodimer, MW 24.40 kDa.

[0468] Kinetic screening Kinetic screening was performed on a GE Healthcare Biacore 4000 instrument at 37 °C. Biacore CM5 Series S sensors were installed in the instrument, hydrodynamically addressed, and preconditioned according to the manufacturer's instructions. The system buffer was HBS-EP (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20). System buffer supplemented with 1 mg / mL CMD (carboxymethyl dextran, Fluka) was used as the sample buffer.

[0469] A rabbit antibody capture system was immobilized on the sensor surface: the polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (code number 111-005-046; Jackson Immuno Research) was amine-coupled using EDC / NHS chemistry according to the manufacturer's instructions.

[0470] GARbFcγ (30 μg / mL) in 10 mM sodium acetate buffer (pH 4.5) was preconcentrated onto spots 1, 2, 4, and 5 of flow cells 1, 2, 3, and 4 and covalently bound to the CMD surface at a density of approximately 10,000 RU. Subsequently, the free activated carboxyl groups were saturated with 1 M ethanolamine, pH 8.5.

[0471] Spots 1 and 5 were used for interaction measurements, and spots 2 and 4 were used as references. Each rabbit antibody supernatant suspension was diluted 1:5 with sample buffer and injected at a flow rate of 10 μL / min for 2 min. The rabbit antibody capture level (CL) in resonance units (RU) was monitored.

[0472] Construct "312" was injected onto each surface at a single concentration of c = 150 nM, and anti-propeptide BMP10 rabbit mAb was presented at 30 μL / min. The association and dissociation phases were monitored for 5 min each. After each cycle of kinetic determination, the rabbit clone was thoroughly washed from the sensor surface by injecting 10 mM glycine pH 1.5 at 20 μL / min for 30 s. The reporting points, Binding Late (BL) just before the end of the propeptide BMP10 injection and Stability Late (SL) just before the end of dissociation, were extracted from the resulting sensorgrams and used to characterize the antibody / antigen binding stability. Furthermore, the dissociation rate constant k was calculated using the Langmuir 1:1 model. d [s -1 The half-life of the antigen / antibody complex stability (minutes) was calculated using the formula ln(2) / 60*k d The molar ratio representing the binding stoichiometry was calculated using the following formula: MW(antibody) / MW(antigen)*BL(antigen) / CL(antibody)

[0473] Using this approach, 280 rabbit antibodies were tested, and 18 Abs with suitable kinetic properties that met the criteria of the Elecsys platform were identified.

[0474] Dynamic characterization Detailed kinetic studies were performed using a GE Healthcare BIAcore 8K instrument. Rabbit mAb<propeptide BMP 10> clones 2H8, 3H8, 8G5, 9F7, 11A5, 11C10, 14C8, and 13G6, identified by kinetic screening, were extensively kinetically characterized for binding to propeptide BMP 10 at 37°C.

[0475] A Biacore CM 5 Series S sensor (Lot No. 10281824 / 10276998) was installed in the instrument.

[0476] Amine coupling of capture molecules A rabbit antibody capture system was immobilized on the sensor surface. The polyclonal goat anti-rabbit IgG Fc capture antibody GARbFcγ (code number 111-005-046, lot number 131053; Jackson Immuno Research) was amine-coupled using EDC / NHS chemistry according to the manufacturer's instructions: running buffer: HBS-N buffer (10 mM HEPES, 150 mM NaCl, pH 7.4). After activation with a mixture of EDC / NHS, the capture-Ab was diluted in coupling buffer NaAc, pH 5.0, c = 30 μg / mL; the last remaining activated carboxyl groups were blocked by injection of 1 M ethanolamine, pH 8.5; the Ab density reached 11,200–12,700 RU.

[0477] Kinetic characterization of propeptide BMP10 binding to a selection of mAbs at 37°C The system and sample buffer was HBS-EP (10 mM HEPES, 150 mM NaCl, 1 mM EDTA, 0.05% (w / v) P20, pH 7.4).

[0478] Flow cell 2 of channels 1, 2, 3, 4, 5, 6, 7, and 8 was used for interaction measurements, and flow cell 1 of each channel was used as a reference. Each rabbit antibody was diluted to 3 nM in sample buffer and injected for 2 min at a flow rate of 5 μL / min. The rabbit antibody capture level (CL) in resonance units (RU) was monitored.

[0479] A series of increasing concentrations of the propeptide BMP10 (BMP10(312)) from 3.7 nM to 300 nM was injected at 60 μL / min onto each surface-displayed anti-propeptide BMP10 rabbit mAb, with duplicates for a concentration of 33.3 nM. The association phase was monitored for 3 min, and the dissociation phase was monitored for 10 min. After each cycle of kinetic determination, the rabbit clones were eluted from the capture system with a 1 min injection of 10 mM glycine pH 2.0, followed by two consecutive 1 min injections of 10 mM glycine pH 2.25 at 20 μL / min.

[0480] dissociation rate constant k dwas evaluated using the Langmuir 1:1 fitting model according to GE Healthcare's BIAcore™ evaluation software Insight SW V 2.0. The antigen / antibody complex stability half-life time (min) was calculated using the formula ln(2) / 60*k d .Calculated according to.

[0481] The molar ratio representing the binding stoichiometry was calculated using the following formula: MW(antibody) / MW(antigen)*BL(antigen) / CL(antibody)

[0482] Since the propeptide BMP10 is a dimeric molecule, the affinities obtained are avidin-loaded and therefore represent unambiguous data.

[0483] result Kinetic screening 280 rabbit antibodies were tested using a kinetic screening approach. Eighteen Abs with suitable kinetic properties were identified and further characterized.

[0484] Detailed dynamic characterization From 280 kinetically screened rabbit monoclonal antibodies, 18 antibodies were selected.

[0485] Detailed concentration-dependent kinetic studies showed that the propeptide-BMP10 interaction did not behave according to a Langmuir 1:1 interaction.

[0486] The propeptide BMP10 is a dimeric molecule, and the interaction is likely avidity-weighted. Kinetic data represent apparent data, but can be characterized by visual inspection of the sensorgram and quantification of the antibody linear dissociation phase.

[0487] Complex half-lives vary from t / 2diss = 6 to over 115 minutes.

[0488] The binding stoichiometry is 1.2-1.4, indicating a 2:1 binding stoichiometry.

[0489] All Abs except 14C8 and 8G5 show appropriate kinetic profiles: comparable fast complex formation rates and complex half-lives (t / 2diss) of greater than 15 minutes. All Ab molar ratios show a 2:1 binding stoichiometry. Clones 8G5 and 14C8 show slightly slower association and less complex stability than the other clones. Both encompass the same epitope region. Clones 2H8 and 3H8 each encompass a unique epitope region.

[0490] Clone 13G6 exhibited the slowest dissociation (k d <1.0E-04 s -1 ) resulting in a complex half-life t / 2-diss > 115 min. Clone 11A5 displays the appropriate kinetic signature with a fast complexation rate and a complex half-life of 30 min. The molar ratio indicates a fully functional Ab with a 2:1 binding stoichiometry.

[0491] The results are summarized in Table 11 and the sensorgram overlay is shown in Figure 10. The normalized antibody dissociation phases of the eight clones are shown in Figure 11. [Table 11]

[0492] Example 12: Epitope mapping using peptide microarrays Epitope mapping of antibody clones was performed using a library of overlapping immobilized peptide fragments (length: 15 amino acids, 14 amino acid overlap) corresponding to the sequence of human bone morphogenetic protein 10. Peptides were synthesized using an automated synthesizer (Intavis MultiPep RS) on modified cellulose discs that were dissolved after synthesis. Solutions of individual peptides were then spotted onto coated microscope slides. Synthesis was carried out stepwise on amino-modified cellulose discs in a 384-well synthesis plate using 9-fluorenylmethoxycarbonyl (Fmoc) chemistry. In each coupling cycle, the corresponding amino acid was activated with a solution of DIC / HOBt in DMF. Between coupling steps, unreacted amino groups were capped with a mixture of acetic anhydride, diisopropylethylamine, and 1-hydroxybenzotriazole. Upon completion of synthesis, the cellulose discs were transferred to a 96-well plate and treated with a mixture of trifluoroacetic acid (TFA), dichloromethane, triisopropylsilane (TIS), and water for side-chain deprotection. After removal of the cleavage solution, the cellulose-bound peptides were dissolved with a mixture of TFA, TFMSA, TIS, and water, precipitated with diisopropyl ether, and resuspended in DMSO. These peptide solutions were then spotted onto Intavis CelluSpot™ slides using an Intavis slide spotting robot.

[0493] For epitope analysis, prepared slides were washed with ethanol and then with Tris-buffered saline (TBS; 50 mM Tris, 137 mM NaCl, 2.7 mM KCl, pH 8), followed by a blocking step using 5 mL of 10x Western Blocking Reagent (Roche Applied Science), 2.5 g sucrose in TBS, and 0.1% Tween 20 at 37°C for 1 h. After washing (TBS + 0.1% Tween 20), the slides were incubated with a solution of the antibody clone (1 μg / mL) in TBS + 0.1% Tween 20 at 37°C for 1 h. After washing, the slides were incubated with anti-rabbit secondary HRP antibody (1:20,000 in TBS-T) for detection, followed by incubation with DAB substrate. Positive spots were assigned to the corresponding peptide sequences. [Table 12]

[0494] Example 13: Selection of antibodies for sandwich assays Biotinylated and ruthenylated clones 2H8, 3H8, 8G5, 9E7, 11A5, 11C10, 14C8, and 13G6 were tested in multiple sandwiches to identify sandwich partners for further Elecsys immunoassay development. The relationship between recognition of recombinant NT-proBMP10 (22-312) at 0.01 ng / ml and blank values ​​across sandwich combinations (Table 13) reflects signal-to-noise ratios of 1.16 and 1.22 for the combination of clones 11A2 and 11C2 on either the biotinylated or ruthenylated side. The combinations 13G6 and 11C10 reflect ratios of 1.22 and 1.23, respectively, indicating a high performance grade regardless of orientation. A similar observation is made for the combination of 3H8 and 9E7, with ratios of 1.22 and 1.23, respectively, but with higher blank values ​​compared to the combination of 11C10 and 13G6. Other combinations described achieve good ratios, but only for one sandwich orientation. [Table 13]

[0495] The identification of 20 natural samples from healthy donors (Table 14) shows the detection range of NT-proBMP10 in natural serum samples, allowing the determination of baseline values ​​for clinical sample measurements. These are in comparable ranges for both the Ru- and Bi-orientations of 11C10 and 13G6. Similar ranges are detected for 11C10 and 11A5 in both orientations. [Table 14]

[0496] Example 14: Biomarker Measurement (An example of a method for the detection of NT-proBMP10) Serum and plasma concentrations of the biomarker NT-proBMP10 were measured using the commercially available Elecsys® reagent from Roche Diagnostics (Mannheim, Germany). The biomarker NT-proBMP10 was measured using the prototype Elecsys® reagent from Roche Diagnostics (Mannheim, Germany).

[0497] Biotinylated rabbit MAB <nt-probmp10>F(ab')2-Bi and ruthenium-conjugated MAB <nt-probmp10>NT-proBMP10 assay with -Ru: An electrochemiluminescence immunoassay (ECLIA) for the specific measurement of NT-proBMP10 in human serum or plasma samples was developed using the Elecsys® cobas analyzer e601. The Elecsys NT-proBMP10 immunoassay is an electrochemiluminescence immunoassay (ECLIA) that works via the sandwich principle. The assay contains two antibodies: a biotinylated monoclonal antibody F(ab')2 fragment MAB <bmp10>(MAB<BMP10_22-312> Bi; capture antibody such as 11C10) and ruthenium-conjugated monoclonal anti-BMP10 antibody MAB <bmp10>(MAB<BMP10_22-312> The assay includes a detection antibody (e.g., 13G6) and an antibody (e.g., 13G6) that forms a sandwich immunoassay complex with NT-proBMP10 in the sample. The complex is then bound to solid-phase streptavidin-coated microparticles. These are magnetically captured on an electrode surface, and application of a voltage to the electrode results in chemiluminescence, which is measured by a photomultiplier tube. Results are determined via an instrument-specific calibration curve established using a series of six calibrators with different concentrations of NT-proBMP10 across the measurement range. Samples are assayed using assay protocol 2 with a pipetting volume of 20 μl of sample, 75 μl of Reagent 1 (R1), 75 μl of Reagent 2 (R2), and 30 μl of magnetic beads. R1 is a MAB in phosphate reaction buffer. <bmp10>Reagent 2 (R2) contains MAB in the same reaction buffer. <bmp10>-Contains Ru.

[0498] Example 15: Prediction of silent cerebral infarction (LNCCI and SNCI) based on circulating BMP-10 levels BMP-10 in the assessment of silent cerebral infarction 1. Method for predicting the risk of silent cerebral infarction in patients with atrial fibrillation based on circulating BMP-10 levels in serum / plasma (SWISSAF study, Tables 15 and 16) 2. To provide a method to improve the prediction of clinical accuracy of clinical stroke risk scores for silent cerebral infarction based on circulating BMP-10 levels in serum / plasma (e.g., CHA2DS2-VASc, CHADS2 score) (SWISS AF study, Table 17).

[0499] The ability of circulating BMP-10 to predict the risk of silent infarction was assessed in the SWISS AF study (Conen D., Forum Med Suisse 2012;12:860-862; Conen et al., Swiss Med Wkly. 2017;147). Patients in the SWISS AF cohort had a median age of 74 years, a prevalence of previous clinical stroke or TIA of 20%, a prevalence of vascular disease of 34%, and a history of diabetes in 17%.

[0500] BMP-10 was measured in the complete SWISS AF study using a pre-commercial assay, Bone Morphogenetic Protein 10 (BMP-10) (High-Throughput Elecsys® Immunoassay; Roche Diagnostics, Mannheim, Germany). For the detection of BMP-10, a sandwich immunoassay was developed for the cobas Elecsys® ECLIA platform.

[0501] Because estimates from a naive proportional hazards model for the case-control cohort are biased (due to the changing ratio of cases to controls), a weighted proportional hazards model was used. Weights are based on the inverse probability of each patient being selected into the case-control cohort. To obtain estimates of absolute survival in the two groups based on dichotomized baseline BMP-10 measurements (≤median vs >median), a weighted version of the Kaplan-Meier plot was created.

[0502] To assess the ability of BMP-10 to improve existing risk scores for stroke prognosis, the CHADS2, CHA2DS2-VASc, and ABC scores were extended with BMP-10 (log2 transformed). Extensions were performed by creating partial hazard models that included BMP-10 and each risk score as independent variables.

[0503] The c-indices of the CHADS2, CHA2DS2-VASc, and ABC scores were compared with the c-indices of these extended models. For calculation of the c-indices in the case-cohort setting, a weighted version of the c-index was used as proposed in Ganna (2011).

[0504] result [Table 15]

[0505] Patients with LNCCI or SNCI on bMRI were older (75.0 vs. 68.1 years, p<0.0001), more frequently had persistent AF (28.4 vs. 17.8%, p=0.0002), higher systolic BP levels (136.7 vs. 131.3 mmHg, p<0.0001), and higher CHA2DS2-VASc scores (3.2 vs. 2.1 points, p<0.0001), but showed no difference in the rate of oral anticoagulation therapy (90.3 vs. 88.5%, p=0.32). As shown in Table 15, BMP-10 levels were significantly higher in patients with brain lesions.

[0506] As shown in Table 15, the risk of silent cerebral infarction in patients with atrial fibrillation can be assessed based on circulating BMP-10 levels in serum / plasma. [Table 16]

[0507] Model 1 was adjusted for age and sex.

[0508] Model 2 was further adjusted for systolic blood pressure, previous major bleeding, diabetes, peripheral vascular disease, BMI, smoking status, and use of oral anticoagulants and antiplatelet medications.

[0509] Biomarkers were logarithmized.

[0510] As shown in Table 16, BMP-10 was significantly associated with LNCCI after multivariate adjustment for age and sex (Model 1) or age, sex, systolic blood pressure, previous major bleeding, diabetes, peripheral vascular disease, BMI, smoking status, oral anticoagulant use, and antiplatelet drug use.

[0511] Therefore, the risk of silent cerebral infarction in patients with atrial fibrillation can be assessed based on circulating BMP-10 levels in serum / plasma.

[0512] [Table 17]

[0513] When we added individual biomarkers to the CHA2DS2-VASc score, the AUC (95% CI) was improved by BMP-10 0.699 (0.673-0.724), as shown in Table 17.

[0514] The combination of BMP-10 and the CHA2DS2-VASc score clinical parameters predicted clinically silent cerebral infarction well and outperformed the CHA2DS2-VASc score.Early clinical identification of patients at risk of cognitive decline may allow for better diagnosis and preventive measures.

[0515] Example 16: Prediction of white matter lesions based on circulating BMP-10 levels Data in the SWISS-AF data show that BMP-10 correlates with the presence of large non-cortical and cortical infarcts (LNCCI) in patients.

[0516] The extent of parenchymal involvement can be expressed by the Fazekas score (Fazekas, JB Chawluk, A Alavi, HI Hurtig, and RA Zimmerman, American Journal of Roentgenology 1987 149:2, 351-356). The Fazekas score ranges from 0 to 3, with 0 indicating no WML, 1 indicating mild WML, 2 indicating moderate WML, and 3 indicating severe WML.

[0517] To compare the association between BMP-10 and large non-cortical and cortical restriction (LNCCI), patients were categorized into two groups: Fazekas score < 2 (absent) vs. Fazekas score ≥ 2 (present). Figure 14 shows that BMP-10 is increased in patients with moderate or severe WML compared with patients with mild or no WML.

[0518] The extent of WML can be caused by clinically silent stroke (Wang Y, Liu G, Hong D, Chen F, Ji X, Cao G. White matter injury in ischemic stroke. Prog Neurobiol. 2016;141:45-60. doi:10.1016 / j.pneurobio.2016.04.005), further supporting the utility of BMP-10 for predicting the risk of clinical stroke.

[0519] The ability of circulating BMP-10 to distinguish patients with a Fazekas score <2 (absent) versus a Fazekas score ≥2 (present) is demonstrated by an AUC of 0.62. White matter changes in the brain of patients with dementia. Age and changes in WML score have been described to be associated with the severity of dementia in patients with Alzheimer's disease (Kao et al., 2019).

[0520] Age is also an important predictor of clinical stroke. Therefore, significantly elevated circulating BMP-10 levels may indicate not only moderate or severe large non-cortical and cortical infarction (LNCCI), but also age-related brain diseases such as vascular dementia.

Claims

1. 1. A method for assessing atrial fibrillation in a subject, comprising: a) determining the amount of one or more BMP10 peptides (bone morphogenetic protein type 10 peptides) and the amount of at least one further biomarker in at least one sample from said subject; and b) comparing the amount of the BMP10 peptide with a reference amount for the BMP10 peptide and comparing the amount of the at least one additional biomarker with a reference amount for the at least one additional biomarker, thereby providing an index for assessing atrial fibrillation. Including, A method wherein step a) comprises contacting said sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:

1.

2. The method of claim 1 , wherein the agent is a monoclonal antibody or an antigen-binding fragment thereof.

3. the monoclonal antibody or antigen-binding fragment thereof a) an epitope contained in the amino acid region 171-185 of SEQ ID NO: 1 (LESKGDNEGERNMLV, SEQ ID NO: 3); b) an epitope (SLFGDVFSEQD, SEQ ID NO: 2) contained in the amino acid region 37-47 of the polypeptide set forth in SEQ ID NO: 1; or c) an epitope contained in the amino acid region 291-299 of SEQ ID NO: 1 (SSGPGEEAL, SEQ ID NO: 5) The method of claim 2, wherein the

4. The method according to claim 3, wherein the epitope (LESKGDNEGERNMLV, SEQ ID NO: 3) contained in the amino acid region 171 to 185 of SEQ ID NO: 1 is the epitope (SKGDNEGER, SEQ ID NO: 4) contained in the amino acid region 173 to 181 of SEQ ID NO:

1.

5. The monoclonal antibody or antigen-binding fragment thereof: a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO:7 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO:16; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO:8 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO:17; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO:9 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO:18; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 10 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 19; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 11 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 20; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 12 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 21; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 13 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 22; a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 14 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 23; or a heavy chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 15 and a light chain variable domain that is at least 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 24; wherein the monoclonal antibody or antigen-binding fragment thereof is: （１） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 34; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 52, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 70 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 25; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 43, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 61 a heavy chain variable domain comprising: （２） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 35; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 53, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 71 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 26; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 44, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 62 a heavy chain variable domain comprising: （３） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 36; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 54, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 72 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 27; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 45, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 63 a heavy chain variable domain comprising: （４） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 37; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 55, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 73 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 28; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 46, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 64 a heavy chain variable domain comprising: （５） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 38; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 56, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 74 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 29; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 47, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 65 a heavy chain variable domain comprising: （６） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 39; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 57, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 75 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 30; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 48, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 66 a heavy chain variable domain comprising: （７） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 40; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 58, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 76 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 31; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 49, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 67 a heavy chain variable domain comprising: （８） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 41; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 59, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 77 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 32; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 50, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 68 a heavy chain variable domain comprising: （９） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 42; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 60, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 78 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 33; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 51; and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 69 a heavy chain variable domain comprising: Including, The method of claim 2.

6. The method of any one of claims 1 to 5, wherein the assessment of atrial fibrillation is a diagnosis of atrial fibrillation.

7. The method according to any one of claims 1 to 5, wherein the assessment of atrial fibrillation is a prediction of the risk of an adverse event associated with atrial fibrillation.

8. 8. The method of claim 7, wherein the adverse event associated with atrial fibrillation is atrial fibrillation recurrence and / or stroke.

9. 9. The method of claim 8, wherein the recurrence of atrial fibrillation is recurrence of atrial fibrillation after pulmonary vein isolation (PVI) or ablation therapy.

10. The method of any one of claims 7 to 9, wherein the amount of the BMP10 peptide indicates a subject at risk of suffering from an adverse event associated with atrial fibrillation, and / or the amount of the BMP10 peptide below the reference amount indicates a subject not at risk of suffering from an adverse event associated with atrial fibrillation.

11. 1. A method for diagnosing heart failure, comprising: (a) determining in at least one sample from the subject the amount of one or more BMP10 peptides (bone morphogenetic protein type 10 peptides), and optionally the amount of at least one additional biomarker selected from the group consisting of natriuretic peptides, ESM-1 (Endocan), Ang2, and FABP3 (fatty acid binding protein 3); (b) comparing the amount of the BMP10 peptide with a reference amount for the BMP10 peptide, and optionally comparing the amount of the at least one additional biomarker with a reference amount for the at least one additional biomarker, thereby providing an indication for diagnosing heart failure. Including, 6. A method, wherein step a) comprises contacting the sample with at least one agent capable of binding within the amino acid region 37 to 299 of the polypeptide set forth in SEQ ID NO: 1 according to any one of claims 2 to 5.

12. The method of claim 11 , wherein the agent is a monoclonal antibody, or a fragment thereof.

13. To assess atrial fibrillation, to diagnose heart failure, or to predict a subject's risk of hospitalization due to heart failure; (a) In vitro use of at least one agent that specifically binds to one or more BMP10-type peptides, wherein the agent is capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:

1.

14. The use according to claim 13, wherein the agent is a monoclonal antibody or an antigen-binding fragment thereof according to any one of claims 2 to 5.

15. A monoclonal antibody or antigen-binding fragment thereof that binds to one or more BMP10-type peptides, a) an epitope contained in the amino acid region 171-185 of SEQ ID NO: 1 (LESKGDNEGERNMLV, SEQ ID NO: 3); b) an epitope (SLFGDVFSEQD, SEQ ID NO: 2) contained in the amino acid region 37-47 of the polypeptide set forth in SEQ ID NO: 1; c) an epitope contained in the amino acid region 291-299 of SEQ ID NO: 1 (SSGPGEEAL, SEQ ID NO: 5) A monoclonal antibody or antigen-binding fragment thereof capable of binding to

16. 16. The monoclonal antibody or antigen-binding fragment thereof according to claim 15, wherein the epitope (LESKGDNEGERNMLV, SEQ ID NO: 3) contained in the amino acid region 171 to 185 of SEQ ID NO: 1 is the epitope (SKGDNEGER, SEQ ID NO: 4) contained in the amino acid region 173 to 181 of SEQ ID NO:

1.

17. 1. A monoclonal antibody or antigen-binding fragment thereof that binds to one or more BMP10-type peptides, comprising a heavy chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a heavy chain variable domain comprising the sequence set forth in SEQ ID NO: 7, 8, 9, 10, 11, 12, 13, 14 or 15, and / or a light chain variable domain that is at least 80%, 85%, 90%, 95%, 98%, 99% or 100% identical to a light chain variable domain comprising the sequence set forth in SEQ ID NO: 16, 17, 18, 19, 20, 21, 22, 23 or 24, The monoclonal antibody or antigen-binding fragment thereof: （１） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 34; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 52, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 70 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 25; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 43, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 61 a heavy chain variable domain comprising: （２） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 35; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 53, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 71 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 26; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 44, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 62 a heavy chain variable domain comprising: （３） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 36; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 54, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 72 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 27; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 45, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 63 a heavy chain variable domain comprising: （４） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 37; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 55, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 73 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 28; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 46, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 64 a heavy chain variable domain comprising: （５） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 38; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 56, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 74 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 29; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 47, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 65 a heavy chain variable domain comprising: （６） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 39; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 57, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 75 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 30; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 48, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 66 a heavy chain variable domain comprising: （７） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 40; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 58, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 76 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 31; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 49, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 67 a heavy chain variable domain comprising: （８） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 41; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 59, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 77 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 32; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 50, and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 68 a heavy chain variable domain comprising: （９） (a) (a1) the light chain CDR1 sequence shown in SEQ ID NO: 42; (a2) a light chain CDR2 sequence set forth in SEQ ID NO: 60, and (a3) the light chain CDR3 sequence shown in SEQ ID NO: 78 a light chain variable domain comprising and (b) (b1) the heavy chain CDR1 sequence shown in SEQ ID NO: 33; (b2) the heavy chain CDR2 sequence set forth in SEQ ID NO: 51; and (b3) the heavy chain CDR3 sequence shown in SEQ ID NO: 69 a heavy chain variable domain comprising: Including, A monoclonal antibody or an antigen-binding fragment thereof.

18. A kit comprising at least one monoclonal antibody or fragment thereof according to any one of claims 2 to 5 or 15 to 17.

19. The method according to any one of claims 1 to 12 or the use according to claim 13 or 14, wherein the BMP10-type peptide is NT-proBMP10.

20. The monoclonal antibody or fragment thereof according to any one of claims 15 to 17, or the kit according to claim 18, wherein the BMP10-type peptide is NT-proBMP10.

21. 1. A method for assessing the extent of white matter pathology in a subject, comprising: a) determining the amount of one or more BMP10 type peptides in a sample from said subject, and providing an index for assessing the degree of white matter lesions in the subject based on said determined amount. Including, A method wherein step a) comprises contacting said sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:

1.

22. 1. A method for predicting dementia, vascular dementia, Alzheimer's disease, dementia with Lewy bodies, and / or frontotemporal dementia in a subject, comprising: a) determining the amount of one or more BMP10 type peptides in a sample from said subject; and b) comparing the amount determined in step a) with a standard, thereby providing an index for predicting the risk of dementia in the subject. Including, A method wherein step a) comprises contacting said sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:

1.

23. 1. A method for assessing whether a subject has experienced one or more cryptogenic strokes, comprising: a) determining the amount of one or more BMP10 type peptides in a sample from said subject; and b) comparing the amount determined in step a) with a reference, thereby providing an index for assessing whether the subject has experienced one or more cryptogenic strokes. Including, A method wherein step a) comprises contacting said sample with at least one agent capable of binding within the amino acid region 37-299 of the polypeptide set forth in SEQ ID NO:1.

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