HK1237281B - Soluble human st-2 antibodies and assays - Google Patents

Description

Soluble human ST-2 antibody and assay

This application is a divisional application of the Chinese invention patent application "Soluble human ST-2 antibody and analysis method" with application number 201180028646.5 and international filing date on April 8, 2011.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 61/322,578, filed April 9, 2010, and U.S. Provisional Application No. 61/345,837, filed May 18, 2010, the contents of each of which are incorporated herein by reference in their entirety.

Technical Field

Described herein are antibodies and antigen-binding fragments of antibodies that bind to human soluble growth stimulatory gene expression 2 (ST2) protein, kits containing the antibodies and antibody fragments, and analytical assays using the antibodies and antibody fragments.

background

ST2 is a member of the interleukin-1 receptor family with two subtypes: transmembrane (ST2L) and soluble (sST2 or soluble ST2) (Iwahana et al., Eur. J. Biochem. 264:397-406, 1999). Recent publications have expanded our understanding of the relationship between ST2 and inflammatory diseases (Arend et al., Immunol. Rev. 223:20-38, 2008; Kakkar et al., Nat. Rev. Drug Discov. 7:827-840, 2008; Hayakawa et al., J. Biol. Chem. 282:26369-26380, 2007; Trajkovic et al., Cytokine Growth Factor Rev. 15:87-95, 2004). Circulating concentrations of human soluble ST2 are elevated in patients with a variety of diseases associated with abnormal type 2 helper T cell (Th2) responses, including systemic lupus erythematosus and asthma, as well as in inflammatory conditions that are largely independent of Th2 responses, such as septic shock or trauma (Trajkovic et al., Cytokine Growth Factor Rev. 15:87-95, 2004; Brunner et al., Intensive Care Med. 30:1468-1473, 2004). Furthermore, interleukin-33/ST2L signaling represents a key myocardial protective mechanism during mechanical overload (Seki et al., Circulation Heart Fail. 2:684-691, 2009; Kakkar et al., Nat. Rev. Drug Discov. 7:827-40, 2008; Sanada et al., J. Clin. Invest. 117:1538-1549, 2007). Elevated human soluble ST2 also predicts worse prognosis in patients with heart failure (HF) and myocardial infarction (Kakkar et al., Nat. Rev. Drug Discov. 7:827-40, 2008; Weinberg et al., Circulation 107:721-726, 2003; Shimpo et al., Circulation 109:2186-2190, 2004; Januzzi et al., J. Am. Coll. Cardiol. 50:607-613, 2007; Mueller et al., Clin. Chem. 54:752-756, 2008; Rehman et al., J. Am. Coll. Cardiol. 52:1458-65, 2008; Sabatine et al., Circulation 117:1936-1944, 2008). People's soluble ST2 level raises and also indicates that individuality occurs death within 1 year (referring to for example WO 07/127749).In a word, people's soluble ST2 is considered to be relevant with some inflammatory diseases and myocardial protection paracrine system, and is the predictor of heart failure patient prognosis and experimenter's death within 1 year.

Overview

The present invention is at least partially based on the exploitation of new antibodies of human soluble ST2 protein specificity.These antibodies and Fab thereof can be used for example to carry out quantitative, the risk of prediction experimenter's death within 1 year to the human soluble ST2 protein in biological sample (for example clinical sample), determine whether to allow experimenter to be discharged from hospital or start or continue treatment (for example, hospitalization), and select the experimenter of participation clinical research.These antibodies and Fab thereof, the test kit that contains these antibodies and antibody fragment, and the various method using these antibodies and antibody fragment are provided in the literary composition.

Provided herein are isolated antibodies and antigen-binding fragments thereof, which are antibodies produced by hybridomas or compete for binding with antibodies produced by hybridomas deposited with the American Type Culture Collection (ATCC) on October 20, 2009, and designated as Patent Deposit Designations PTA-10431 and PTA 10432. The microbial taxonomic designations for PTA-10431 and PTA 10432 are monoclonal mouse hybridomas: ST2-BA-1.1-07E04-02B07-02G06 and ST2-BA-1.1-09F08-02D04-01F05, respectively. The address of the American Type Culture Collection (ATCC) is Manassas, Virginia, USA. In some embodiments, the antibody or fragment thereof does not compete for binding with one or both of the D066-3 or D067-3 antibodies (MBL International) (described in U.S. Patent 7,087,396), or binds to human soluble ST2 with a _KD equal to or less than 1.51 x ^10-9 M. In some embodiments, the antibody or fragment thereof binds to human soluble ST2 with a _KD equal to or less than 8.59 x ^10-10 M. In some embodiments, the antibody or fragment thereof is chimeric or humanized. In some embodiments, the fragment is selected from the group consisting of a Fab fragment, a F(ab') ₂ fragment, and a scFv fragment. In some embodiments, the antibody or fragment thereof is glycosylated. In some embodiments, the antibody or fragment thereof contains one or more complementarity determining regions of a light chain or heavy chain of an antibody produced by a hybridoma, wherein the hybridoma is deposited with the ATCC and designated Patent Deposit Designation PTA-10431 or PTA-10432. In some embodiments, the antibody is an antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10431, or an antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10432, or an antigen-binding fragment thereof.

Also provided are antibodies and Fab thereof that are specifically bound to the separation of soluble ST2, the production process of which comprises using the recombinant human soluble ST2 immune non-human mammals (for example mouse, rat, rabbit, goat, cattle, pig, monkey or horse) separated from human cells (for example human fibroblasts, neural cells, epithelial cells or endothelial cells, embryos or adult cells, particularly human embryonic kidney cells). In some embodiments, the recombinant human soluble ST2 separated is completely glycosylated, i.e. has substantially the same glycosylation state as the natural endogenous human soluble ST2 present in human serum. In some embodiments of all antibodies described herein and fragments, antibody or its fragment carry a mark.

Also provided are cells of the hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10431, and cells of the hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10432.

Also provide the method for the people's soluble ST2 level of quantitatively from the sample of experimenter.Said method comprises that sample is contacted with at least one antibody described herein or its fragment, and the binding situation of detection antibody or its fragment and people's soluble ST2.In some embodiments, method comprises and uses at least two different antibodies described herein or its fragment.

Also provided is a method for predicting the risk of death within one year of a subject. Said method comprises obtaining a subject sample and utilizing at least one antibody as described herein or its fragment to determine the level of human soluble ST2 in the sample, wherein compared with a human soluble ST2 reference level, an increase in the level of human soluble ST2 in the sample indicates that the risk of death within one year of the subject increases (e.g., relative to those compared with the same control, the risk of death within one year of the subject (e.g., the subject suffering from the same disease) decreases in the level of human soluble ST2), and a decrease or a substantially identical level of human soluble ST2 indicates that the risk of death within one year of the subject decreases (e.g., relative to those compared with the same control, the risk of death within one year of the subject (e.g., the subject suffering from the same disease) decreases in the level of human soluble ST2).

Also provide and determine whether to allow inpatient to be discharged from hospital or to start or continue the method for hospitalization to experimenter, described method comprises obtaining experimenter sample and utilizes at least one antibody described herein or its fragment to determine the people's soluble ST2 level in sample, wherein compared with people's soluble ST2 reference level, the people's soluble ST2 level of rising shows and should start or continue hospitalization, decline or equal people's soluble ST2 level shows and can consider to allow experimenter to be discharged from hospital.In some embodiments, experimenter has at least one or more in following symptom: chest pain or discomfort, shortness of breath, nausea, vomiting, belching, sweating, palpitation, dizziness, fatigue and fainting.

Also provide the method for the experimenter of selecting to participate in clinical research, described method comprises obtaining experimenter sample, utilizes at least one antibody described herein or its fragment to determine the people's soluble ST2 level in the sample, and if compared with people's soluble ST2 reference level, experimenter's people's soluble ST2 level shows that he should be selected to participate in clinical research and then selects this experimenter to participate in clinical trial.In some embodiments, there is the people's soluble ST2 level that rises to show that experimenter should be selected to participate in clinical research.

Also provide the method for selecting treatment regimen to experimenter, described method comprises utilizing at least one antibody described herein or its fragment to determine the people's soluble ST2 level in the biological sample from experimenter, wherein the people's soluble ST2 level of experimenter of relative people's soluble ST2 reference level is used to select treatment regimen to experimenter.In some embodiments, the people's soluble ST2 level that has rising is used to select treatment regimen to experimenter.

In some embodiments of any of the methods described herein, the subject is undiagnosed or does not have two or more (e.g., at least three, four, or five) symptoms of a disease state; the subject has been diagnosed with a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, stroke, or any of the diseases described herein); or the subject has one or more of the following: hypertriglyceridemia, hypercholesterolemia, hypertension, and a body mass index ≥ 30. In some embodiments of any of the methods described herein, the determination utilizes at least two antibodies or fragments thereof described herein.

In some embodiments of any one method described herein, people's solubility ST2 reference level is the threshold level of people's solubility ST2.In some embodiments, average people's solubility ST2 level in healthy patient colony (for example healthy male patient colony or healthy female patient colony) on the threshold level.In some embodiments of any one method described herein, reference level is the people's solubility ST2 level existing in such subject's sample, and described experimenter does not present two or more disease symptoms, is not diagnosed with certain disease or is not accredited as the risk of developing disease.

In any of the methods described herein, the subject has not been diagnosed with a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, stroke, or any of the diseases described herein). In any of the methods described herein, the sample comprises blood, serum, or plasma. Any of the antibodies and fragments thereof described herein can be used in any of the methods described herein.

Also provided are methods for diagnosing a disease in a subject, the method comprising obtaining a sample from the subject, determining the level of human soluble ST2 and the level of at least one additional marker in the sample using at least one antibody or fragment thereof described herein, wherein the level of human soluble ST2 in the subject, if compared to a reference level of human soluble ST2; wherein an elevated level of human soluble ST2 in the sample compared to the reference level of human soluble ST2, and an altered level of the at least one additional marker relative to the reference level of the at least one additional marker, indicates that the subject has a disease (e.g., cardiovascular disease, pulmonary disease, sepsis, Kawasaki disease, or a Th2-related disease, or any of the other diseases described herein).

Also provide and determine whether experimenter has normal people soluble ST2 level (therefore may not suffer from serious disease (for example cardiovascular disease) and for example the risk of death or being admitted to hospital in 1 year is normal) method, described method comprises obtaining experimenter sample, utilize at least one antibody described herein or its fragment to determine the people soluble ST2 level in sample, if wherein people soluble ST2 level drops on specific scope (for example, at the about 25.3ng/mL of approximately 14.5-, perhaps between the about 19.9ng/mL of approximately 18.1-), then determine that experimenter has normal people soluble ST2 level.In some embodiments, if the people soluble ST2 level of male subject is between any scope that table 9 is enumerated, then experimenter is defined as having normal people soluble ST2 level.In some embodiments, if the people soluble ST2 level of female subject is between any scope that table 9 is enumerated, then experimenter is defined as having normal people soluble ST2 level.

Also provided is a test kit, which contains at least one (e.g., two, three, four, or five) antibody or its Fab as described herein. Some embodiments of these test kits contain two antibodies or their Fab as described herein. In some embodiments of these test kits, at least one antibody or its fragment has a K _D equal to or less than 8.59x 10 ^-10 M for the combination of human soluble ST2 (e.g., recombinant human soluble ST2). In some embodiments, the test kit is provided as an enzyme-linked immunosorbent assay (ELISA). In some embodiments, the test kit also contains the recombinant human soluble ST2 separated from human cells (e.g., human embryonic kidney cells). In some embodiments, the recombinant human soluble ST2 is fully glycosylated.

The term "soluble ST2" means a soluble protein comprising a sequence that is at least 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBI Accession No. NP_003847.2 (SEQ ID NO: 1); or a nucleic acid comprising a sequence that is at least 90% identical (e.g., at least 95%, 96%, 97%, 98%, 99%, or 100% identical) to NCBI Accession No. NM_003856.2 (SEQ ID NO: 2).

The term "elevated" or "elevated" means that the level of soluble ST2 determined or measured (e.g., the level of soluble ST2 determined or measured) is different, for example, statistically significant (e.g., increasing). In some embodiments, the level of soluble ST2 determined or measured is a threshold level, and any level that exceeds the threshold level is considered to be "elevated." Other reference levels of soluble ST2 are described herein.

The term "healthcare facility" means a place where a subject can receive medical care from a health care professional (e.g., a nurse, physician, or physician's assistant). Non-limiting examples of health care facilities include hospitals, clinics, and assisted care facilities (e.g., nursing homes).

The term "inpatient" means a subject who is admitted to a medical institution (eg, a hospital or assisted living facility).

The term "hospitalization" means monitoring and/or medical treatment of a subject who is admitted to a medical facility (e.g., a hospital or assisted living facility). For example, a subject who is hospitalized may be given one or more therapeutic agents or undergo a medical procedure (e.g., surgery (e.g., organ transplant, heart bypass surgery), angioplasty, imaging (e.g., magnetic resonance imaging, ultrasound imaging, and computerized tomography)) by medical personnel. In other embodiments, one or more markers of disease or condition severity may be periodically measured by medical personnel to assess the severity or progression of the disease or condition of the subject.

The term "reference level" means the threshold level in control subjects or control patient colonies. Reference level depends on the test carried out and can be determined by those skilled in the art. Reference level can be the level measured at an earlier or later time point in basal level or the same patient. Some non-limiting examples of human soluble ST2 reference levels include the human soluble ST2 level in such a patient, the patient: not diagnosed with disease; two or more symptoms of disease are not presented; there is no high-risk CVD; there is no renal failure; there is no hypertriglyceridemia, hypercholesterolemia, hypertension and/or body mass index < 30 (for example, BMI is less than 25); there is no risk of developing disease; and/or not suffering from the disease (for example, cardiovascular disease, lung disease, sepsis, Kawasaki disease or Th2 related disease, or any other disease described herein) associated with ST2 levels. Other control patient colonies are also described herein. Other examples of human soluble ST2 reference levels are human soluble ST2 threshold levels. Methods known in the art can be utilized to determine human soluble ST2 reference levels; some exemplary levels are described herein.

In some embodiments, the ratio of two people's soluble ST2 levels in the experimenter has been calculated.The parameter ratio of the people's soluble ST2 level measured in reference ratio and experimenter (for example any control subject described in the literary composition or same experimenter) can be compared, for example reference ratio can be disease (for example, heart disease (for example, heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome and angina pectoris), renal insufficiency or apoplexy, or as described herein with the people's soluble ST2 of raising the ratio of human soluble ST2 levels before and after the onset of symptoms of a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, and angina pectoris), renal insufficiency, or stroke, or any other disease as described herein associated with increased human soluble ST2 levels); the ratio of human soluble ST2 levels before and after diagnosis of a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, and angina pectoris), renal insufficiency, or stroke, or any other disease as described herein associated with increased human soluble ST2 levels); the ratio of human soluble ST2 levels before and after treatment of a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, and angina pectoris), renal insufficiency, or stroke, or any other disease as described herein associated with increased human soluble ST2 levels; the ratio of human soluble ST2 levels at two different time points during treatment (e.g., inpatient or outpatient treatment) of a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, and angina pectoris), renal insufficiency, or stroke, or any other disease as described herein associated with increased human soluble ST2 levels; or the ratio of human soluble ST2 levels before and after a cardiac event (e.g., myocardial infarction).

In some embodiments, the ratio of the human soluble ST2 level measured in the subject can be compared with a threshold reference ratio. The reference ratio of human soluble ST2 can be determined using methods known in the art; the reference ratio of human soluble ST2 can be calculated using data described herein. For example, the reference ratio of human soluble ST2 can be between about 0.7 and about 1.1, or about 1.

The term "therapeutic treatment" or "treatment" means administering one or more agents to a subject or performing a medical procedure on the subject's body (e.g., surgery such as an organ transplant or heart surgery). Non-limiting examples of agents that can be administered to a subject include nitrates, calcium channel blockers, diuretics, thrombolytics, digitalis, renin angiotensin aldosterone system (RAAS) modulators (e.g., beta-adrenergic blockers, angiotensin-converting enzyme inhibitors, aldosterone antagonists, renin inhibitors, and angiotensin II receptor blockers), and cholesterol-lowering agents (e.g., dyes). The term therapeutic treatment also includes adjusting (e.g., increasing or decreasing) the dose or frequency of one or more agents used by a subject, administering one or more new agents to a subject, or removing one or more agents from a subject's treatment regimen.

As used herein, a "subject" is a mammal, such as a human.

As used herein, a "biological sample" includes one or more of blood, serum, plasma, urine, and body tissue. Generally, a biological sample is a sample containing serum, blood, or plasma.

As used herein, the term "antibody" refers to a protein that typically contains heavy chain polypeptides and light chain polypeptides. Antigen recognition and binding occur in the variable regions of the heavy and light chains. Single-domain antibodies with one heavy chain and one light chain, and heavy chain antibodies without light chains are also known. A given antibody contains one of five different types of heavy chains, known as α, δ, ε, γ, and μ, which are classified based on the amino acid sequence of the heavy chain constant region. These different types of heavy chains form five classes of antibodies, namely IgA (including IgA1 and IgA2), IgD, IgE, IgG (IgG1, IgG2, IgG3, and IgG4), and IgM. A given antibody also contains one of two types of light chains, known as κ and λ, which are classified based on the amino acid sequence of the light chain constant domain. IgG, IgD, and IgE antibodies typically contain two identical heavy chains, two identical light chains, and two antigen-binding domains, each consisting of a heavy chain variable region (VH) and a light chain variable region (VL). Typically, IgA antibodies are composed of two monomers, each consisting of two heavy chains and two light chains (for IgG, IgD, and IgE antibodies). Thus, IgA molecules have four antigen-binding domains, each also consisting of VH and VL. Some IgA antibodies are monomeric in nature, as they are composed of two heavy chains and two light chains. Secreted IgM antibodies are generally composed of five monomers, each consisting of two heavy chains and two light chains (for IgG and IgE antibodies). Thus, secreted IgM molecules have ten antigen-binding domains, each also consisting of VH and VL. There is also a cell-surface form of IgM, which, similar to IgG, IgD, and IgE antibodies, has a two heavy chain/two light chain structure.

As used herein, the term "chimeric antibody" refers to an antibody that has been modified to include at least one human constant region. For example, one or all (e.g., one, two, or three) light chain variable regions and/or one or all (e.g., one, two, or three) heavy chain variable regions of a mouse antibody (e.g., a mouse monoclonal antibody) can each be linked to a human constant region, such as, but not limited to, an IgG1 human constant region. Relative to non-chimeric antibodies, chimeric antibodies are generally less immunogenic to humans and therefore confer therapeutic benefits in certain cases. Those skilled in the art are aware of chimeric antibodies and understand suitable techniques for preparing them. See, for example, U.S. Patents 4,816,567, 4,978,775, 4,975,369, and U.S. Patent 4,816,397.

As used herein, the term "fully human antibody" refers to an antibody or antigen-binding fragment of an antibody that contains only amino acid sequences derived from humans. For example, fully human antibodies can be produced by human B cells or human hybridoma cells. In other embodiments, antibodies can be produced by transgenic animals that contain human heavy chain immunoglobulin and human light chain immunoglobulin loci, or contain nucleic acids encoding the heavy and light chains of specific human antibodies.

The term "complementarity determining region" or "CDR" as used herein refers to short polypeptide sequences within the variable regions of heavy and light chain polypeptides that are primarily responsible for mediating specific antigen recognition. CDRs are described in Kabat, et al., J. Biol. Chem. 252, 6609-6616, 1977; Chothia et al., J. Mol. Biol. 196: 901-917, 1987; and MacCallum et al., J. Mol. Biol. 262: 732-745, 1996. There are three CDRs (designated CDR1, CDR2, and CDR3) in each VL and each VH.

The term "fragment" or "antibody fragment" as used herein refers to a polypeptide derived from an antibody polypeptide molecule (e.g., an antibody heavy chain and/or light chain polypeptide), which does not comprise a full-length antibody polypeptide but still contains at least a portion of a full-length antibody polypeptide capable of binding to an antigen. An antibody fragment may comprise a portion of a cleavage of a full-length antibody polypeptide, although this term is not limited to such cleavage fragments. Antibody fragments may include, for example, Fab fragments, F(ab')2 fragments, scFv (single-chain Fv) fragments, linear antibodies, monospecific or multispecific antibody fragments, such as bispecific, trispecific and multispecific antibodies (e.g., dimers, trimers, tetrabodies), miniantibodies, chelating recombinant antibodies, trimers or dimers, intrabodies, nanoantibodies, small modular immunopharmaceuticals (SMIPs), binding domain immunoglobulin fusion proteins, camelized antibodies, and antibodies containing VHHs. Other examples of antigen-binding antibody fragments are known in the art.

The term "framework region" as used herein refers to amino acid sequences within the variable regions of heavy and light chain polypeptides that are not CDR sequences and are primarily responsible for maintaining the correct positioning of the CDR sequences for antigen binding. Although the framework regions themselves are generally not directly involved in antigen binding, as is known in the art, certain residues within the framework regions of certain antibodies can directly participate in antigen binding or can affect the ability of one or more amino acids in the CDRs to interact with the antigen.

The term "humanized antibody" as used herein refers to an antibody that has been modified to contain one or more human framework regions together with non-human (e.g., mouse, rat, or hamster) heavy and/or light chain complementary determining regions (CDRs) within the variable region. In some embodiments, a humanized antibody comprises sequences that are entirely human except for the CDR regions. Relative to non-humanized antibodies, humanized antibodies are generally less immunogenic to humans and therefore provide therapeutic advantages in certain cases. Humanized antibodies are known in the art, and suitable techniques for preparing humanized antibodies are also known. See, for example, Hwang et al., Methods 36:35, 2005; Queen et al., Proc. Natl. Acad. Sci. U.S.A. 86:10029-10033, 1989; Jones et al., Nature 321:522-25, 1986; Riechmann et al., Nature 332:323-27, 1988; Verhoeyen et al., Science 239:1534-36, 1988; Orlandi ... al., Proc. Natl. Acad. Sci. U.S.A. 86:3833-3837, 1989; U.S. Patents 5,225,539, 5,530,101, 5,585,089, 5,693,761, 5,693,762, and 6,180,370; and WO 90/07861.

As used herein, the term "Th2-related disease" refers to a disease associated with an aberrant type 2 helper T cell (Th2) response.

As used herein, the term "cardiovascular disease" refers to disorders of the heart and blood vessels, including disorders of the arteries, veins, arterioles, venules, and capillaries.

As used herein, the term "pulmonary disease" refers to disorders of the lungs.

The term "other marker" means a protein, nucleic acid, lipid or carbohydrate or a combination thereof (e.g., two or more) that can diagnose the presence of a particular disease. The methods described herein for diagnosing a subject as having a disease include detecting the level of soluble human ST2 and at least one other marker in a sample from the subject. Several other markers that can be used for disease diagnosis are known in the art (e.g., proANP, NT-proANP, ANP, proBNP, NT-proBNP, BNP, troponin, CRP, creatinine, blood urine nitrogen (BUN), liver function enzymes, albumin, and bacterial endotoxins; and those described in U.S. Patent Applications 2007/0248981, 2011/0053170, 2010/0009356, 2010/0055683, 2009/0264779, all of which are incorporated herein by reference).

The term "hypertriglyceridemia" means a triglyceride level greater than or equal to 180 ng/mL (eg, greater than or equal to 200 ng/mL).

The term "hypercholesterolemia" means that the level of at least one form of cholesterol or total cholesterol in a subject is increased. For example, a subject with hypercholesterolemia may have a high-density lipoprotein (HDL) level of ≥40 mg/dL (e.g., ≥50 mg/dL or ≥60 mg/mL), a low-density lipoprotein (LDL) level of ≥130 mg/dL (e.g., ≥160 mg/dL or ≥200 mg/dL), and/or a total cholesterol level of ≥200 mg/dL (e.g., 240 mg/dL).

The term "hypertension" means elevated systolic and/or diastolic blood pressure levels. For example, a subject with hypertension may have a systolic blood pressure ≥120 mmHg (e.g., ≥140 mmHg or ≥160 mmHg) and/or a diastolic blood pressure ≥80 mmHg (e.g., ≥90 mmHg or ≥100 mmHg).

The term "healthy subject" means that the subject does not suffer from a disease (e.g., cardiovascular disease or pulmonary disease). For example, a healthy subject has not been diagnosed as having a disease and does not exhibit one or more (e.g., two, three, four or five) symptoms of a disease state.

"Risk of death" means the risk of a subject dying from a disease or a complication associated with the disease compared to a reference group (e.g., a healthy control group). The term "risk of death" as used herein does not include intentional or accidental death, such as death caused by being crushed or crushed (e.g., in a car accident).

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The methods and materials used in the present invention are described herein. Other suitable methods and materials known in the art may also be used. The materials, methods, and examples are provided for illustrative purposes only and are not intended to limit the invention. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated herein by reference in their entirety. In the event of conflict, the present specification, including definitions, will take precedence.

Other features and advantages of the invention will be apparent from the following detailed description and drawings, and from the claims.

Description of the drawings

Figure 1 shows an SDS-PAGE gel analysis of fractions purified from recombinant human soluble ST2 protein. Lanes (10 μL per sample lane) are as follows: 1 - MWM (5 μL); 2 - His ladder (3 μL); 3 - untransfected negative control; 4 - supernatant before purification; 5 - column flow-through; 6 - first binding buffer wash; 7 - second 5 mM wash; 8 - third 5 mM wash; 9 - 200 mM elution fraction 2; 10 - 200 mM elution fraction 3; 11 - 200 mM elution fraction 4; 12 - 200 mM elution fraction 5; 13 - 200 mM elution fraction 6; 14 - 200 mM elution fraction 7; and 15 - 0.3 μg soluble ST2.

Figure 2 shows a Western blot of purified fractions used to detect the histidine tag in recombinant human soluble ST2 protein. Lanes (10 μL per sample lane) are as follows: 1 - MWM (5 μL); 2 - His ladder (3 μL); 3 - supernatant before purification; 4 - column flow-through; 5 - first binding buffer wash; 6 - second binding buffer wash; 7 - second 5 mM wash; 8 - third 5 mM wash; 9 - 200 mM elution fraction 2; 10 - 200 mM elution fraction 3; 11 - 200 mM elution fraction 4; 12 - 200 mM elution fraction 5; 13 - 200 mM elution fraction 6; 14 - 200 mM elution fraction 7; and 15 - 0.3 μg soluble ST2.

Figures 3A-3C show a Coomassie gel (Figure 3A) and two Western blots of purified recombinant soluble ST2, compared with the commercial anti-ST2 antibody D066 (MBL International) (Figure 3B) and a hexahistidine antibody (Figure 3C). Lanes (10 μL per sample) are as follows: 1 – MWM; 2 – His ladder; 3 – Serum ST2-His 1000 ng; 4 – Serum ST2-His 500 ng; 5 – Serum ST2-His 200 ng; 6 – Serum ST2-His 100 ng; 7 – Serum ST2-His 50 ng; 8 – Serum-free ST2-His 1000 ng; 9 – Serum-free ST2-His 500 ng; 10 – Serum-free ST2-His 200 ng; 11 – Serum-free ST2-His 100 ng; and 12 – Serum-free ST2-His 50 ng.

FIG4 is a graph showing the results of antigen sensitivity evaluation of the 7E4 and 9F8 antibodies.

Figure 5 is a graph showing the results of antigen sensitivity evaluation of 7E4 and 9F8 antibodies. 7E4 and 9F8 antibodies were loaded into individual wells of a 96-well plate at a concentration of 5 μg/mL-0 and tested against a single concentration of biotin-conjugated recombinant soluble ST2.

Figure 6 is a line graph showing the results of testing their ability to be used together in a monoclonal antibody sandwich enzyme immunoassay (EIA) format, in which either the 7E4 or 9F8 antibody was biotinylated.

Figures 7A-7F are six graphs showing the results of surface plasmon resonance (SPR) analysis of antibody-antigen complex formation. Figure 7A shows an SPR analysis of antibody 9F8 (L1). Figure 7B shows an SPR analysis of antibody 7E4 (L2). Figure 7C shows an SPR analysis of antibody 11A7 (L3). Figure 7D shows an SPR analysis of antibody D066 (L4). Figure 7E shows an SPR analysis of antibody D067 (L5). Figure 7F shows an SPR analysis of an unrelated antibody (L6).

Figures 8A-8F are six graphs showing the results of SPR analysis of antibody-antigen complex formation. Figure 8A shows an SPR analysis of antibody 9F8 (L1). Figure 8B shows an SPR analysis of antibody 7E4 (L2). Figure 8C shows an SPR analysis of antibody 11A7 (L3). Figure 8D shows an SPR analysis of antibody 15D6 (L4). Figure 8E shows an SPR analysis of antibody D066 (L5). Figure 8F shows an SPR analysis of antibody D067 (L6).

FIG9 is a box-and-whisker plot showing the concentration of human soluble ST2 by anticoagulant tube type.

FIG10 is a histogram showing the distribution of human soluble ST2 concentrations in normal healthy donors.

Figure 11 is a box and whisker plot showing human soluble ST2 concentrations in normal healthy donors as a function of sex and age.

Specifically, the present invention relates to the following aspects:

1. An isolated antibody or antigen-binding fragment thereof, wherein the antibody or fragment thereof is produced by a hybridoma deposited with the American Type Culture Collection (ATTC) and assigned patent deposit number PTA-10431 or PTA-10432.

2. The antibody or fragment of claim 1, wherein the antibody or fragment thereof is chimeric.

3. The antibody or fragment of claim 1, wherein the antibody or fragment thereof is humanized.

4. The antibody or fragment of any preceding claim, wherein the fragment is selected from the group consisting of a Fab fragment, a F(ab') ₂ fragment, and a scFv fragment.

5. The antibody or fragment of any preceding item, wherein the antibody or fragment thereof is glycosylated.

6. The antibody or fragment of any of the preceding items, wherein the antibody or fragment thereof comprises one or more complementarity determining regions of the light chain or heavy chain of an antibody produced by a hybridoma deposited with the American Type Culture Collection (ATTC) and assigned patent deposit number PTA-10431 or PTA-10432.

7. The antibody or fragment of any of the foregoing items, wherein the antibody is an antibody produced by a hybridoma deposited with the ATTC and assigned patent accession number PTA-10431, or an antigen-binding fragment of an antibody produced by a hybridoma deposited with the ATTC and assigned patent accession number PTA-10431.

8. The antibody or fragment of any one of items 1 to 6, wherein the antibody is an antibody produced by a hybridoma deposited with the ATTC and assigned patent deposit number PTA-10432, or an antigen-binding fragment of an antibody produced by a hybridoma deposited with the ATTC and assigned patent deposit number PTA-10432.

9. A hybridoma selected from the group consisting of a hybridoma deposited with the American Type Culture Collection (ATCC) and assigned patent accession number PTA-10431 and a hybridoma deposited with the ATCC and assigned patent accession number PTA-10432.

10. A method for quantifying the level of human soluble ST2 in a sample from a subject, the method comprising:

contacting the sample with at least one antibody or fragment thereof according to items 1 to 8; and

The binding of the antibody or fragment thereof to human soluble ST2 is detected.

11. The method of claim 10, wherein the method comprises using at least two different antibodies or fragments thereof of claims 1-8.

12. A method for predicting a subject's risk of death within one year, the method comprising:

obtaining a sample from a subject; and

Determining the level of human soluble ST2 in a sample using at least one antibody or fragment thereof according to items 1-8;

Wherein an elevated level of human soluble ST2 in the sample compared to a reference level of human soluble ST2 indicates that the subject has an increased risk of death within one year, whereas a decreased or identical level of human soluble ST2 in the sample compared to a reference level of human soluble ST2 indicates that the subject has a reduced risk of death within one year.

13. A method of determining whether to discharge a hospitalized subject or to initiate or continue treatment, the method comprising:

obtaining a sample from a subject; and

Determining the level of human soluble ST2 in a sample using at least one antibody or antibody fragment according to items 1-8;

Wherein an elevated level of human soluble ST2 in the sample compared to a reference level of human soluble ST2 indicates that hospitalization of the subject should be initiated or continued, whereas a decreased or equal level of human soluble ST2 in the sample compared to a reference level of human soluble ST2 indicates that the subject should be discharged.

14. A method for selecting subjects to participate in a clinical study, the method comprising:

obtaining a sample from a subject;

Determining the level of human soluble ST2 in a sample using at least one antibody or antibody fragment according to items 1 to 8; and

When the level of human soluble ST2 of the subject relative to a reference level of human soluble ST2 indicates that the subject should be selected for participation in a clinical study, the subject is selected for participation in a clinical study.

15. The method of claim 14, wherein the presence of elevated levels of human soluble ST2 indicates that the subject should be selected for participation in a clinical study.

16. The method of any one of items 10-15, wherein the subject is undiagnosed or does not present with two or more symptoms of the disease.

17. The method of items 10-15, wherein the subject is diagnosed with a disease.

18. The method of claim 17, wherein the disease is selected from the group consisting of heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, and stroke.

19. The method of items 10-15, wherein the subject is identified as being at risk for developing the disease.

20. The method of any one of items 10-19, wherein the subject has one or more of: hypertriglyceridemia, hypercholesterolemia, hypertension, and a body mass index of ≥30.

21. The method of any one of items 12-20, wherein the determining is performed using at least two antibodies or fragments thereof as described in items 1-8.

22. The method of any one of items 12-21, wherein the reference level of human soluble ST2 is a threshold level of human soluble ST2.

23. The method of claim 22, wherein the threshold level of human soluble ST2 is the average level of human soluble ST2 in a healthy patient population.

24. The method of claim 23, wherein the healthy patient population is a healthy male patient population.

25. The method of claim 23, wherein the healthy patient population is a healthy female patient population.

26. The method of any one of items 12-21, wherein the reference level is the level of human soluble ST2 present in a sample from a subject who does not present two or more symptoms of the disease.

27. The method of any one of items 12-21, wherein the reference level is the level of human soluble ST2 present in a sample from a subject not diagnosed as having the disease.

28. The method of claim 27, wherein the disease is selected from the group consisting of heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, and stroke.

29. The method of any one of items 10-28, wherein the sample comprises blood, serum or plasma.

30. The method of claim 12, wherein the subject has at least one or more of the following symptoms: chest pain or discomfort, shortness of breath, nausea, vomiting, belching, sweating, palpitations, dizziness, fatigue, and fainting.

31. A kit comprising at least one antibody or antigen-binding fragment of items 1-8.

32. The kit of claim 31 , wherein the kit comprises two antibodies or antigen-binding fragments thereof.

33. The kit of claim 31 or 32, wherein at least one antibody or fragment has a K _D for binding to human soluble ST2 equal to or less than 8.59 x ^10-10 M.

34. The kit of any one of items 31-33, wherein the kit is provided as an enzyme-linked immunosorbent assay.

35. The kit of any one of items 31-34, further comprising recombinant human soluble ST2 isolated from human cells.

36. The kit of claim 35 , wherein the human cells are human embryonic kidney cells.

Details

Described herein are antibodies and antigen-binding fragments thereof that specifically bind to human soluble ST2, kits containing the antibodies and fragments, and methods of using the antibodies and fragments.

ST2

The ST2 gene is a member of the interleukin-1 receptor family, and its protein product exists in a transmembrane form and a soluble receptor form that can be detected in serum (Kieser et al., FEBS Lett. 372(2-3):189-193, 1995; Kumar et al., J. Biol. Chem. 270(46):27905-27913, 1995; Yanagisawa et al., FEBS Lett. 302(1):51-53, 1992; Kuroiwa et al., Hybridoma 19(2):151-159, 2000). Soluble ST2 has been described to be significantly upregulated in experimental models of heart failure (Weinberg et al., Circulation 106(23):2961-2966, 2002), and data suggest that human soluble ST2 concentrations are also elevated in those with chronic severe heart failure (Weinberg et al., Circulation 107(5):721-726, 2003) and in humans with acute myocardial infarction (Shimpo et al., Circulation 109(18):2186-2190, 2004).

Without wishing to be bound by theory, the transmembrane form of ST2 is thought to play an important role in regulating the response of type 2 helper T cells (Lohning et al., Proc. Natl. Acad. Sci. U.S.A. 95(12):6930-6935, 1998; Schmitz et al., Immunity 23(5):479-490, 2005) and may play a role in the development of tolerance in severe or chronic inflammatory states (Brint et al., Nat. Immunol. 5(4):373-379, 2004), while the soluble form of ST2 is upregulated in growth-stimulated fibroblasts (Yanagisawa et al., 1992, supra). Experimental data showed that the ST2 gene was significantly upregulated under myocardial stretch (Weinberg et al., 2002, supra), and the upregulation pattern was similar to the induction of the BNP gene (Bruneau et al., Cardiovasc. Res. 28(10):1519-1525, 1994).

Tominaga et al. (FEBS Lett. 258:301-304, 1989) isolated a mouse gene that is specifically expressed in BALB/c-3T3 cells upon growth stimulation. Haga et al. (Eur. J. Biochem. 270:163-170, 2003) described the nomenclature of the ST2 gene based on its induction by growth stimulation. The ST2 gene encodes two protein products: a soluble secreted form, ST2 or sST2; and a transmembrane receptor form, ST2L, that is very similar to the interleukin-1 receptor. The HUGO Nomenclature Committee named the human homolog of ST2 (whose clone is described in Tominaga et al., Biochim. Biophys. Acta. 1171:215-218, 1992) interleukin 1 receptor-like 1 (IL1RL1). The two terms are used interchangeably herein.

The mRNA sequence of the shorter soluble hypotype of people ST2 can be found at GenBank accession number NM_003856.2 (SEQ ID NO:2), and peptide sequence is GenBank accession number NP_003847.2 (SEQ ID NO:1). The mRNA sequence of the longer form of people ST2 is GenBank accession number NM_016232.4 (SEQ ID NO:4), and peptide sequence is GenBank accession number NP_057316.3 (SEQ ID NO:3). Other information is provided at public database GeneID:9173, MIM ID#601203 and UniGeneNo.Hs.66. Generally speaking, what is measured in the method described herein is the ST2 polypeptide of people's soluble form.

Antibodies and antigen-binding antibody fragments

Provided herein are isolated antibodies and antigen-binding fragments thereof that bind to human soluble ST2. The provided antibodies and fragments can compete for binding with antibodies produced by hybridomas deposited with the ATCC and designated patent deposit designations PTA-10431 or PTA-10432 (corresponding to the 7E4 and 9F8 antibodies, respectively). In some embodiments, the antibodies or fragments do not compete for binding with the D066-3 and D067-3 antibodies (MBL International) (described in U.S. Patent 7,087,396) and bind to human soluble ST2 with a _KD of 1.51 x ^10-9 M or less. In some embodiments, the antibodies or fragments bind to human soluble ST2 with a _KD of 8.59 x ^10-10 M or less. Methods for determining the affinity ( _KD ) of antibodies or fragments for binding to human soluble ST2 are described herein (e.g., surface plasmon resonance), and other methods are known in the art. Also provided are the 7E4 and 9F8 monoclonal antibodies and antigen-binding fragments thereof produced by the methods described herein.

As used herein, the phrase "competitive binding" refers to a situation in which the binding of one of an antibody or antibody fragment to a given antigen reduces the binding of a second antibody or antibody fragment to the same antigen. In some embodiments, when two antibodies or fragments are substantially bound to the same epitope located on a given antigen (e.g., human soluble human ST2), the antibody or fragment competes with another antibody or fragment for binding. As described in more detail in the examples below, each antibody produced by the hybridomas designated as patent deposits PTA-10431 and PTA-10432 recognizes and identifies different epitopes from the various other antibodies tested (e.g., D066-3 and D067-3 antibodies from MBL International), and therefore will not compete with those test antibodies for binding. In some embodiments, the epitope on the human soluble ST2 that an antibody or fragment is combined with is designated as an antibody produced by the hybridomas designated as patent deposits PTA-10431 or PTA-10432. The method for determining whether two different antibodies or fragments compete for binding is described in the text and is known in the art (e.g., competitive enzyme-linked immunosorbent assay).

In some embodiments, the antibody or fragment binds to or shows improved binding to an epitope that is present in a recombinant human soluble ST2 protein produced by a human cell (e.g., human fibroblasts, epithelial cells, endothelial cells, or neural cells, embryonic or adult cells, or human embryonic kidney cells, such as HEK293), but not in a recombinant human soluble ST2 produced by a non-human cell type. In some embodiments, the antibody or fragment binds to or shows improved binding to an epitope that is present in a fully glycosylated human soluble ST2 protein (e.g., a human soluble ST2 protein isolated from a human cell), but not in a recombinant human soluble ST2 protein that is not glycosylated or misplaced or insufficiently glycosylated (e.g., not fully glycosylated or a pattern of glycosylation (e.g., the number, site, and/or type of sugars) that is not present in the natural human soluble ST2 of a human (e.g., in human serum)). In some embodiments, antibodies and antibody fragments bind better (e.g., have higher affinity) to natural human soluble ST2 than other commercial antibodies.

In some embodiments, the antibody is a monoclonal antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10431 (the 7E4 antibody), or an antigen-binding fragment of an antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10431 (a fragment of the 7E4 antibody). In some embodiments, the antibody is a monoclonal antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10432 (the 9F8 antibody), or an antigen-binding fragment of an antibody produced by a hybridoma deposited with the ATCC and designated as Patent Deposit Designation PTA-10432 (a fragment of the 9F8 antibody). Combinations of two or more antibodies or fragments described herein (e.g., two or more of the 7E4 antibody, a 7E4 antibody fragment, a 9F8 antibody, and a 9F8 antibody fragment) can be used in any of the methods described herein.

The human soluble ST2-binding monoclonal antibodies produced by the hybridomas designated by Patent Deposit Designation PTA-10431 and Patent Deposit Designation PTA-10432 can both be generated by immunizing a non-human mammal with recombinant human soluble ST2 isolated from human embryonic kidney (HEK)-293 cells. Human soluble ST2 undergoes a considerable amount of post-translational modifications. Based on its amino acid sequence, the molecular weight of human soluble ST2 is predicted to be approximately 36 kDa. In contrast, the molecular weight of the native protein is approximately 58 kDa due to post-translational modifications. As is known in the art, such post-translational modifications can affect the ability of an antibody or antibody fragment to bind to a given protein. Therefore, as described in detail in the Examples section below, the human soluble ST2-binding monoclonal antibodies produced by the hybridoma designated by Patent Deposit Designation PTA-10431 have a higher affinity for native human soluble ST2 than other antibodies and are therefore useful as diagnostic and other reagents.

In some embodiments, the antibodies or fragments described herein comprise the heavy and/or light chains (or fragments thereof) of an antibody produced by a hybridoma designated as Patent Deposit Designation PTA-10431 and/or PTA-10432. In some embodiments, the antibodies or fragments described herein comprise the heavy and/or light chain variable regions (or fragments thereof) of an antibody produced by a hybridoma designated as Patent Deposit Designation PTA-10431 and/or PTA-10432.

As is known in the art, the specificity of an antibody for a given antigen is mediated by the heavy and light chain variable regions. Specifically, the specificity of an antibody for a given antigen is primarily determined by short sequences within the heavy and light chain variable regions known as complementarity determining regions or CDRs. In some embodiments, the antibodies or fragments described herein contain one or more (e.g., one, two, three, four, five, or six) CDRs of the heavy and/or light chains of an antibody produced by a hybridoma designated as Patent Deposit No. PTA-10431 and/or PTA-10432. In some embodiments, the antibodies or fragments described herein comprise each CDR of the heavy chain of an antibody produced by a hybridoma designated as Patent Deposit No. PTA-10431 or PTA-10432. In some embodiments, the antibodies or fragments described herein comprise each CDR of the light chain of an antibody produced by a hybridoma designated as Patent Deposit No. PTA-10431 or PTA-10432. In some embodiments, the antibodies or fragments described herein comprise every CDR (all heavy and light chain CDRs) of the antibody produced by the hybridoma designated as Patent Deposit Designation PTA-10431 or PTA-10432.

Also provided are antibodies and antigen-binding antibody fragments of the separation specifically bound to human soluble ST2, the process of producing said antibodies and antigen-binding antibody fragments comprising using recombinant human soluble ST2 immune non-human mammals separated from nephrocytes (e.g., human kidney cells, embryonic kidney cells and human embryonic kidney cells). In some embodiments, recombinant human soluble ST2 is fully glycosylated or contains all post-translational modifications present in native soluble ST2 protein.

In some embodiments, the antibodies or fragments described herein are chimeric in that they comprise at least one human constant region. For example, the constant region of an antibody produced by a hybridoma designated as Patent Deposit Designation PTA-10431 or PTA-10432 can be replaced with a human constant region. Chimeric antibodies are generally less immunogenic in humans than non-chimeric antibodies, and thus can offer therapeutic advantages in certain situations. In some embodiments, the chimeric antibodies described herein comprise an IgG1 constant region. Various human constant regions are known to those skilled in the art. Methods for preparing chimeric antibodies are known in the art.

In some embodiments, the antibodies or fragments described herein are humanized in that they comprise at least one human framework region. For example, one or more (e.g., one, two, three, four, five, or six) framework regions of an antibody produced by a hybridoma designated as Patent Deposit No. PTA-10431 or PTA-10432 can be replaced with one or more (e.g., one, two, three, four, five, or six) human framework regions. Humanized antibodies are generally less immunogenic to humans than non-humanized antibodies and therefore can offer therapeutic advantages in certain situations. Various human framework regions are known to those skilled in the art. Methods for preparing humanized antibodies are known in the art.

For example, humanization can be performed using a method based on CDR homology (see, for example, Hwang et al., Methods 36:35, 2005). These methods generally involve replacing non-human CDRs into human variable domain frameworks based on structurally similar non-human and human CDRs, rather than structurally similar non-human and human framework regions. Determining the similarity of non-human and human CDRs is generally done by identifying human genes of the same type of chain that have the same typical CDR structure combination as non-human (e.g., mouse) binding molecules, thereby maintaining the three-dimensional conformation of the CDR peptide backbone. Secondly, for each candidate variable region gene with a matching typical structure, homology assessment is performed on the residues of the non-human and candidate human CDRs. Finally, in order to produce a humanized binding molecule, the CDR residues that are not identical to the non-human CDRs in the selected candidate CDR are converted to non-human (e.g., mouse) sequences. In some embodiments, mutations in the humanized binding molecule are not introduced into the human framework.

In some embodiments, substitution of non-human CDRs into a human variable domain framework is based on maintaining the correct spatial orientation of the non-human variable domain framework by identifying a human variable domain framework that maintains the same conformation as the non-human variable domain framework from which the CDRs are derived. In some embodiments, this is achieved by obtaining a human variable domain from a human binding molecule whose framework sequence exhibits a high degree of sequence identity to the non-human variable framework domain from which the CDRs are derived. See, for example, Kettleborough et al., Protein Engineering 4:773, 1991; Kolbinger et al., Protein Engineering 6:971, 1993, and WO 92/22653.

In some embodiments, antibody described herein or fragment is monospecific, because it only recognizes a single epitope. Monospecific antibodies are known in the art (see, for example, WO/9639858). In some embodiments, antibody described herein or fragment is bispecific, because it can recognize more than one epitope (e.g., two epitopes). Bispecific antibodies are known in the art (see, for example, U.S. Patent Application Publication 2009/0162360). In some embodiments, the epitope that monospecific or bispecific antibodies described herein or fragment can combine is recognized by such antibodies or antibody fragments, and the antibodies or antibody fragments have the CDRs of the monoclonal antibodies produced by the hybridoma designated as patent deposit nomenclature PTA-10431 or PTA-10432. In some embodiments, bispecific antibodies or fragments bind to human soluble ST2 and different non-ST2 polypeptides. In some embodiments, bispecific antibodies or fragments bind to two different epitopes of human soluble ST2. In some embodiments, antibody described herein or fragment is divalent (see, for example, WO/1999/064460). For a more detailed description of other antibodies and fragments comprising one or more CDRs of the monoclonal antibody produced by the hybridoma designated patent deposit designation PTA-10431 or PTA-10432, see US Patent Application Publication No. 20070105199 and WO/2007/059782.

In some embodiments, the fragment (e.g., antigen binding fragment) is derived from a complete antibody molecule, such as a monoclonal antibody. The antibody can be, for example, cut at the carboxyl end of its hinge region (using trypsin) to produce a F (ab') ₂ fragment, or cut at the amino end of its hinge region (using papain) to produce a Fab fragment. In some embodiments, the antigen binding fragment described herein is a Fab fragment, F (ab') ₂ fragment, scFv fragment, linear antibody, multispecific antibody fragment, such as a bispecific, trispecific or multispecific antibody (e.g., a dibody, tribody or tetrabody), miniantibody, chelated recombinant antibody, intrabody, nanobody, small modular immune drug (SMIP), binding domain immunoglobulin fusion protein, camelized antibody or an antibody containing V _HH . Methods for preparing these fragments are known in the art.

In some embodiments, the human soluble ST2 binding antibodies or antigen-binding antibody fragments described herein comprise polypeptides having one or more amino acid substitutions, deletions, or insertions compared to the heavy and/or light chains of the antibodies produced by the hybridomas designated as Patent Deposit No. PTA-10431 or PTA-10432. By conventional techniques such as site-directed mutagenesis or PCR-mediated mutations, substitutions, deletions, or insertions can be introduced into nucleic acid molecules encoding polypeptides comprising the heavy and/or light chains of the antibodies produced by the hybridomas designated as Patent Deposit No. PTA-10431 or PTA-10432 (e.g., nucleic acids encoding one or more (e.g., one, two, or three) CDR regions of the heavy or light chains). In some embodiments, conservative amino acid substitutions are performed at one or more sites. "Conservative amino acid substitutions" are situations in which an amino acid residue is replaced by an amino acid residue with a similar side chain. Families of amino acids with similar side chains have been defined in the prior art and include basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), non-polar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, an amino acid residue in a polypeptide of an anti-human soluble ST2 antibody or human soluble ST2-binding antibody fragment can be replaced by another amino acid residue from the same side chain family.

In some embodiments, a human soluble ST2-binding antibody or human soluble ST2-binding antibody fragment described herein comprises an amino acid sequence that is at least 90% identical, at least 95%, 96%, 97%, 98%, 99%, or 100% identical to the heavy chain and/or light chain of the antibody produced by the hybridoma designated as Patent Deposit Designation PTA-10431 or PTA-10432 (e.g., or at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% identical to at least one (e.g., one, two, or three) CDR of the heavy chain and/or light chain of the antibody produced by the hybridoma designated as Patent Deposit Designation PTA-10431 or PTA-10432). For example, the human soluble ST2-binding antibodies or human soluble ST2-binding antibody fragments described herein may contain one or more CDRs containing one or more amino acid substitutions, deletions or insertions within the corresponding CDR sequences found in the heavy and/or light chains of the antibody produced by the hybridoma designated as Patent Deposit Designation PTA-10431 or PTA-10432.

In some embodiments, the compositions described herein contain two or more different human soluble ST2 binding antibodies or human soluble ST2 binding antibody fragments described herein. For example, the compositions described herein can contain antibodies produced by one of the hybridomas designated as Patent Deposit No. PTA-10431 and PTA-10432. As described in more detail in the Examples below, this antibody combination shows a higher affinity for the ST2 antigen than any single antibody and other commercial antibodies. The compositions described herein containing antibodies or antigen-binding fragments can be useful in many methods, such as diagnostic methods. In some embodiments, the compositions described herein contain two or more different ST2 binding fragments (e.g., Fab fragments, F(ab) ₂ fragments, or scFv fragments), such as fragments of antibodies produced by hybridomas designated as Patent Deposit No. PTA-10431 or PTA-10432.

In any of the above methods, the antibody or antibody fragment can be glycosylated or labeled. For example, the antibody or antibody fragment can be labeled with a detectable substance, including but not limited to various enzymes, prosthetic groups, fluorescent substances, luminescent substances, bioluminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazineaminofluorescein, dansyl chloride, quantum dots, or phycoerythrin; examples of luminescent substances include luminol; examples of bioluminescent substances include luciferase, luciferin, and aequorin; examples of suitable radioactive substances include ¹²⁵ I, ¹³¹ I, ³⁵ S, or ³ H.

hybridoma

Also provided herein is a hybridoma that can produce an antibody in conjunction with human soluble ST2. As known in the art, term "hybridoma" refers to a cell produced by merging the lymphocyte that produces the antibody with the cancer cell (normally myeloma or lymphoma) that does not produce the antibody. After the fusion, the hybridoma is propagated and produces the specific monoclonal antibody originally produced by the fused lymphocyte. In some embodiments, the hybridoma provided is a hybridoma that is deposited in ATCC and designated as patent deposit named PTA-10431 or PTA-10432. In some embodiments, individual cells, harvested cells and culture are also provided, which contain cells that derive from the hybridoma that is deposited in ATCC and designated as patent deposit named PTA-10431 or PTA-10432.

Methods of using provided antibodies and fragments

In particular, one or more of any of the antibodies or antibody fragments described herein can be used to quantify the level of human soluble ST2 in a sample (e.g., a sample from a subject) for the purpose of predicting the risk of death within one year, determining whether to allow a subject to be discharged from the hospital or to start or continue their inpatient treatment, selecting subjects for participation in clinical trials, diagnosing a subject with a disease, or identifying a subject at risk of developing a disease.

Quantitative method for human soluble ST2 levels

Provided herein is a method for determining the level of human soluble ST2 in a subject's sample, the method comprising contacting sample with at least one antibody or antibody fragment described herein; and detecting the combination of antibody or fragment and human soluble ST2. In some embodiments, at least two (for example, two, three or four) antibodies or antibody fragments described herein have been used to determine the level of human soluble ST2 in a subject's sample. In some embodiments, the subject is not diagnosed or does not present one or more (for example, two, three or four) symptoms of disease. In some embodiments, the subject has been diagnosed as suffering from the disease (for example, any one of heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, apoplexy or other diseases described herein) relevant to the ST2 level increase. In some embodiments, the subject suffers from one or more (for example, two, three or four) of hypertriglyceridemia, hypercholesterolemia, hypertension, renal insufficiency, and body mass index ≥30. In some embodiments, the sample contains blood, serum or plasma.

In some embodiments, sample can be collected from experimenter by medical staff (for example, lancing physician, doctor, nurse, physician's assistant or laboratory technician).Sample can (for example, at≤4 ℃,≤0 ℃ or-80 ℃) preserve a period of time, the combination of detection antibody or fragment before contacting with at least one antibody described herein or fragment.The method that biological sample is contacted and detection antibody or fragment combination is described in literary composition, and additional methods are known in the art.Quantitatively can also comprise the control experiment of the combination of the recombinant human soluble ST2 (for example, separating the recombinant human soluble ST2 of human embryonic kidney cell) that detects at least one antibody described herein or antibody fragment and purification.

In some embodiments, the people's soluble ST2 level in normal or healthy subjects is carried out quantitatively.Described normal or healthy subjects is such experimenter, and it is not involved in ST2 associated condition (for example, ST2 associated condition described herein), is not diagnosed as and suffers from disease (for example, any disease described herein) and two or more (for example, two, three or four) symptoms of disease do not occur.Normal or healthy subjects can confirm by any of multiple technologies known in the art, include but not limited to by biomarker screening or physical examination (for example, by not having the outward manifestation form of two or more symptoms associated with ST2 associated condition or any other disease described herein).For example, can filter out the normal or healthy subjects that do not have occult blood CVD or inflammatory disease by screening one or more markers of low level, wherein said marker includes, but is not limited to brain natriuretic peptide (BNP), procalcitonin (PCT), C-reactive protein (CRP) and interleukin-6 (IL-6).Those skilled in the art know and are used to determine that normal or healthy subjects do not show occult blood CVD or inflammatory disease, or other suitable markers of any other diseases described herein.

In some embodiments, the people's solubility ST2 level of experimenter (for example, normal or healthy subjects), is quantitatively useful in many situations.In some embodiments, the people's solubility ST2 level of experimenter (for example, normal or healthy subjects, the experimenter that the risk of developing disease increases, the experimenter diagnosed with disease or the experimenter presenting two or more symptoms of disease) can be periodically, for example every day, every week, every two weeks, every month, every two months, every year etc., or periodic physical examination.Any one of a kind of multiple technology of those skilled in the art, comprise those describing in the literary composition, can be used for utilizing antibody described herein and the Fab of antibody to carry out quantitatively the people's solubility ST2 in experimenter.

In some embodiments, the human soluble ST2 level in control subjects (such as normal or healthy subjects) is quantitatively obtained reference level, so that for determining that the experimenter does not have the ST2 associated condition, has the risk of developing into disease or has the risk of death within one year.For example, the human soluble ST2 level in the experimenter who does not suffer from disease can be quantitatively obtained human soluble ST2 reference level, described disease is such as, but not limited to cardiovascular disease, heart failure, coronary artery disease, acute coronary syndrome, renal insufficiency, apoplexy, lung disease, sepsis, Kawasaki disease or Th2 associated disease, or any other disease described herein.

In some embodiments, at least one of antibody disclosed herein or Fab can be used for the human soluble ST2 level in quantitative subject (e.g., normal or healthy subjects).For example, human soluble ST2 level can be quantitatively measured in immunoassay using at least one antibody described herein or Fab (e.g., antibody or fragment of the antibody competition binding produced by the hybridoma deposited in ATCC and designated as patent deposit nomenclature PTA-10431 or PTA-104312) or both.

In some embodiments, the human soluble ST2 level in the sample is quantitatively measured to ensure reproducibility, reference range, clinical critical value etc. of routine operation.For example, the human soluble ST2 level in two or more samples (for example, with reference to sample) can be quantitatively measured, and the coefficient of variation (" CV ") of human soluble ST2 level between assessment two or more samples.In addition or alternatively, the human soluble ST2 level in the sample (or experimenter) can be quantitatively measured twice or more (for example, utilizing the reference sample of different batches, or the different samples taken from same experimenter), and determines the CV between the human soluble ST2 level.In some embodiments, the CV between the human soluble ST2 level is less than 20%, for example, is less than 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower.

In some embodiments, provide and determine whether the experimenter has the method for normal people-soluble ST2 level.Determining whether the experimenter has normal people-soluble ST2 level is all useful in many cases.In some embodiments, determine whether the method for experimenter has normal people-soluble ST2 level and comprise the level of the people-soluble ST2 in analyzing and detecting subject sample (for example, any above-described sample, such as but not limited to the sample that contains blood, serum or plasma), if wherein find that the people-soluble ST2 level in sample is similar to known normal or intermediate value people-soluble ST2 level; If perhaps the people-soluble ST2 level in sample drops in certain scope, for example near known normal or intermediate value people-soluble ST2 level (for example, 95% confidence interval or interquartile range, or the scope of listing in any table 9), can determine that experimenter has normal people-soluble ST2 level. For example, if the human soluble ST2 level in the sample is found to be positioned at 95% confidence interval of known normal or intermediate value human soluble ST2 level (intermediate value level in normal or healthy subjects) after analysis from the sample of the experimenter, it can be determined that the experimenter has normal human soluble ST2 level.In addition or alternatively, if the human soluble ST2 level in the sample is found to be positioned at the interquartile range of known normal or intermediate value human soluble ST2 level after analysis from the sample of the experimenter, it can be determined that the experimenter has normal human soluble ST2 level.

In some embodiments, if the people solubility ST2 level in the subject sample is about 18.8ng/mL, can determine that this experimenter has normal people solubility ST2 level.In some embodiments, if the people solubility ST2 level in the sample is about 14.5-25.3ng/mL, can determine that this experimenter has normal people solubility ST2 level.In some embodiments, if the people solubility ST2 level in the sample is about 18.1-19.9ng/mL, can determine that this experimenter has normal people solubility ST2 level.

In some embodiments, if the people's solubility ST2 level from the experimenter's sample is about 16.2ng/mL, can determine that this female experimenter has normal people's solubility ST2 level.In some embodiments, if the people's solubility ST2 level in the sample is positioned within any scope that table 9 is listed, can determine that these women have normal people's solubility ST2 level.

In some embodiments, if the people's solubility ST2 level from the experimenter's sample is about 23.6ng/mL, can determine that this male experimenter has normal people's solubility ST2 level.In some embodiments, if the people's solubility ST2 level in the sample is positioned within any scope that table 9 is listed, can determine that this male has normal people's solubility ST2 level.

In some embodiments, a subject (e.g., a male or female subject) can be determined to have normal soluble ST2 levels if the level of human soluble ST2 in a sample from the subject is below a threshold value (e.g., 25.3 ng/mL, or 19.9 ng/mL (for females) or 30.6 ng/mL (for males)).

The terms "about" or "substantially the same" when used in reference to a value or range of human soluble ST2 levels in a subject (e.g., a range of normal human soluble ST2 levels) refer to a spacing around a reference value or range, e.g., a value or range that one skilled in the art would consider to be equivalent to a reference value or range (e.g., any range listed in Table 9) for the purpose of assessing human soluble ST2 levels (e.g., normal human soluble ST2 levels or human soluble ST2 levels in a patient population having a disease or presenting symptoms of two or more diseases). As used herein, a value or range is "about" a reference value or range when the value or range of human soluble ST2 levels (e.g., normal human soluble ST2 levels) is within +/- 25% of the reference value or range, such as within +/- 20%, +/- 15%, +/- 10%, +/- 9%, +/- 8%, +/- 7%, +/- 6%, +/- 5%, +/- 4%, +/- 3%, +/- 2% or +/- 1% of the value or range.

In some embodiments, at least one or two of any of the antibodies or antigen-binding fragments described herein can be used to determine whether a subject has normal levels of human soluble ST2, has levels of human soluble ST2 associated with a disease, or has levels of human soluble ST2 associated with an increased risk of developing a disease or an increased risk of dying within one year.

Methods for predicting the risk of death within one year

Also provide the method for predicting mortality risk in one year in experimenter, it comprises obtaining sample from experimenter, and uses at least one antibody as described herein or antibody fragment to determine the level of people's soluble ST2 in sample.Compared with the reference level of people's soluble ST2, in sample, the people's soluble ST2 of rising level or substantially the same level shows that described experimenter has the mortality risk of increase in one year (for example, with respect to the experimenter (for example, having or diagnosing the experimenter with same disease) of the people's soluble ST2 that reduces level in sample with same reference level in comparison).Compared with the reference level of people's soluble ST2, in sample, the people's soluble ST2 that reduces level shows that described experimenter has lower mortality risk in one year (for example, with respect to the experimenter (for example, having or diagnosing the experimenter with same disease) of the people's soluble ST2 that reduces level in sample with same reference level in comparison).The mortality risk level in one year determined by method as described herein can depend on morbid state.

In some embodiments, the subject is undiagnosed or does not present one or more (e.g., two, three, four, or five) symptoms of a disease. In some embodiments, the subject is diagnosed with a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, or stroke, or any disease described herein). In some embodiments, the subject has one or more (e.g., one, two, three, or four) of the following: hypertriglyceridemia, hypercholesterolemia, hypertension, and a body mass index of ≥30. In some embodiments, the determination is performed using at least one (e.g., two, three, four, or species) of the antibodies or fragments described herein.

In some embodiments, the reference level of described human soluble ST2 is the threshold level of human soluble ST2 (for example the median level of the human soluble ST2 of healthy patient colony such as healthy male patient whole or healthy female patient colony, or the percentile (for example 75,80,85,90 or 95 percentiles, or the scope or concentration of listing in any table 9) of human soluble ST2 median level). In some embodiments, described reference level can be the human soluble ST2 level in the patient sample of one or more symptoms of the disease relevant to ST2 level increase. In some embodiments, described reference level can be the experimenter with disease (for example heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, apoplexy, or any disease described herein) or be accredited as the level of the human soluble ST2 that exists in the sample of the experimenter who does not have disease (for example any disease described herein) risk. Other reference level can be determined by those skilled in the art.

In some embodiments, described people's soluble ST2 reference level is about 30ng/mL to about 35ng/mL.In some embodiments, when experimenter suffers from heart failure, people's soluble ST2 reference level is about 30ng/mL to about 35ng/mL.In some embodiments, described people's soluble ST2 reference level is about 35ng/mL or about 60ng/mL.

In some embodiments, the sample contains blood, serum, or plasma.Such a sample can be obtained, and determining the level of human soluble ST2 using at least one antibody or fragment described herein can be performed as described herein.

Methods for determining whether to discharge hospitalized subjects or to initiate or continue treatment

Also provided are methods for determining whether to discharge a hospitalized subject or to initiate or continue treatment, comprising obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using at least one (e.g., two) antibodies or antibody fragments described herein, wherein an elevated level of human soluble ST2 in the sample, compared to a reference level of human soluble ST2, indicates that hospitalization (e.g., hospitalization or admission into an assisted-care facility) should be initiated or continued for the subject, while a decreased or equal level of human soluble ST2 in the sample, compared to the reference level of human soluble ST2, indicates that the subject should be discharged. The method can be performed several times (e.g., once a week, twice a week, three times a week, once a month, twice a month, three times a month, and four times a month) on the same subject to determine whether hospitalization should continue.

In some embodiments, the experimenter is undiagnosed, does not present two or more symptoms of morbid state, or is not accredited as having the risk of disease (such as any disease described herein) occurring.In some embodiments, the experimenter is diagnosed as suffering from disease (such as heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, apoplexy, or any disease described herein), presents one or more symptoms of disease (such as any disease described herein), or is accredited as having the risk of disease (such as any disease described herein) occurring.In some embodiments, the experimenter has following one or more (such as a kind of, two, three or four kinds): hypertriglyceridemia, hypercholesterolemia, hypertension, and ≥30 body mass index.In some embodiments, the experimenter is not diagnosed as suffering from heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, or apoplexy, or any disease described herein.In some embodiments, the determination of the level of people's soluble ST2 is carried out using at least two antibodies as described herein or antibody fragment.

In some embodiments, the reference level of people's solubility ST2 can be any reference level described herein.Other people's solubility ST2 reference levels can be determined by those skilled in the art.In some embodiments, described sample contains blood, serum or plasma.Can obtain described sample, and use at least one antibody described herein or fragment to determine that the level of people's solubility ST2 can be carried out as described herein.

Methods for selecting subjects for participation in clinical studies

Also provide the method for the experimenter that is used to select to participate in clinical research.These methods comprise obtaining sample from experimenter, use at least one antibody as described herein or antibody fragment to determine the level of the people's soluble ST2 in the sample, and if the people's soluble ST2 level of this experimenter shows that this experimenter should be selected to participate in clinical research relative to the reference level of people's soluble ST2, select described experimenter to participate in clinical research.In some embodiments, the existence of the rising level of people's soluble ST2 shows that this experimenter should be selected to participate in clinical research.In some embodiments, the existence of the rising level of people's soluble ST2 shows that this experimenter should be excluded from participating in clinical research.

In some embodiments, the subject is undiagnosed, does not present two or more symptoms of a disease state (e.g., any disease described herein), or is not identified as having a risk of developing a disease (e.g., any disease described herein). In some embodiments, the subject is diagnosed with a disease (e.g., heart failure, coronary artery disease, cardiovascular disease, acute coronary syndrome, renal insufficiency, stroke, or any disease described herein), presents one or more symptoms of a disease (e.g., any disease described herein), or is identified as having a risk of developing a disease (e.g., any disease described herein). In some embodiments, the subject has one or more (e.g., one, two, three, or four) of the following: hypertriglyceridemia, hypercholesterolemia, hypertension, and a body mass index of ≥30. The determination is performed using at least two antibodies or antibody fragments described herein.

In some embodiments, the reference level of people's solubility ST2 can be any reference level described herein.Other people's solubility ST2 reference levels can be determined by those skilled in the art.In some embodiments, described sample contains blood, serum or plasma.Can obtain described sample, and use at least one antibody described herein or fragment to determine that the level of people's solubility ST2 can be carried out as described herein.

Choosing a treatment method

Also provided are methods of selecting a treatment for a subject, comprising obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using at least one antibody or antibody fragment described herein, wherein an elevated level of human soluble ST2 in the sample compared to a reference level of human soluble ST2 indicates that the subject should be provided with a particular treatment. For example, the specific treatment can be selected from the group consisting of nitrites, calcium channel blockers, diuretics, thrombolytics, digitalis, renin-angiotensin-aldosterone system (RAAS) modulators (e.g., beta-adrenergic blockers (e.g., alprenolol, bucindolol, carteolol, carvedilol, labetalol, nadolol, penbutolol, pindolol, propanolol (inderal), sotalol, timolol, acebutolol, atenolol, betaxolol, bisoprolol), dapoxetine, ... rolol), celiprolol, esmolol, metoprolol, and nebivolol, angiotensin-converting enzyme inhibitors (e.g., benazepril, captopril, enalapril, fosinopril, lisinopril, moexipril, perindopril, quiapril, ramipril, and trandolapril), aldosterone antagonists (e.g., spironolactone, eplerenone, canrenone), and narcotics. Potassium, prorenone (prorenoate potassium) and mexrenone (mexrenoate potassium)), renin inhibitors (e.g., aliskiren, remikiren and enalkiren), and angiotensin II receptor blockers (e.g., valsartan, telmisartan, losartan, irbesartan and olmesartan), and cholesterol-lowering agents (e.g., statins). Other methods for treatment are also known in the art, for example, Braunwald's Heart Disease: A Textbook of Cardiovascular Medicine, Single Volume, 9th Edition. Specific treatment can also be administering at least one or more therapeutic agents to the subject, changing (e.g., increasing or decreasing) the frequency, dosage, or duration of administration of one or more therapeutic agents to the subject, or removing at least one or more therapeutic agents from the patient's treatment regimen. The treatment can also be inpatient care of the subject (e.g., admitting or re-admitting the subject to a hospital (e.g., intensive care or critical care unit) or an assisted care facility). In some embodiments, the treatment is surgery (e.g., organ or tissue transplantation or angioplasty).

In some embodiments, the reference level of described human soluble ST2 can be any reference level described herein.Other human soluble ST2 reference levels can be determined by those skilled in the art.In some embodiments, described sample contains blood, serum or plasma.Can obtain described sample, and use at least one antibody described herein or fragment to determine that the level of human soluble ST2 can be carried out as mentioned above.

Methods of diagnosing subjects

The methods described herein can be used in a wide variety of clinical settings. For example, such methods can be used for general population screening, including screening by physicians, such as in hospitals and outpatient clinics, and in emergency rooms.

In some embodiments, method described herein can be used for determining the possibility that disease exists in the experimenter.The people's soluble ST2 that increases level is conventionally relevant to the existence of some disease, and described disease is such as but not limited to: cardiovascular disease, pulmonary disease, septicemia, Kawasaki disease, and Th2 associated disease, and any other disease described herein.

Th2 related diseases are diseases related to abnormal type 2 T helper cells (Th2) responses. Th2 related diseases are characterized by several factors, including but not limited to TNF-alpha, IL-4, -5, -6, -10 and -13, but the presence of non-IFN-gamma (Robinson, J.Allergy Clin.Immunol.92:313,1993). CD4+ T cells are classified according to the cytokines they secrete. Th2 cells secrete a large amount of interleukin-4 (IL-4), IL-5 and IL-13, which promote the antibody production of B cells and collagen synthesis of fibroblasts, while Th1 cells secrete a large amount of interferon-γ and related pro-inflammatory cytokines. The cytokines of Th1 type and Th2 type can cross-regulate each other's responses. It is believed that the imbalance of Th1/Th2 causes the morbidity of several infections, allergies and autoimmune diseases. Some exemplary Th2 related diseases include, but are not limited to, systemic lupus erythematosus and asthma, and inflammatory conditions (inflammatory conditions) that are mainly independent of Th2 responses, such as septic shock or trauma (Trajkovic et al., Cytokine Growth Factor Rev.15:87-95,2004; Brunner et al., Intensive Care Med.30:1468-1473,2004). Other exemplary Th2 related diseases include pre-eclampsia and multiple sclerosis. In some embodiments, Th2 related diseases are autoimmune diseases. Autoimmune diseases typically occur when the subject's immune system activates components (cells, tissues, or molecules without cells/tissues) of one or more subjects and attacks the subject's own normal organs, tissues, or cells. Exemplary autoimmune diseases include, but are not limited to, adrenergic drug resistance, alopecia areata, ankylosing spondylitis, antiphospholipid syndrome, autoimmune Addison's disease, autoimmune diseases of the adrenal gland, allergic encephalomyelitis, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inflammatory eye disease, autoimmune neonatal thrombocytopenia, autoimmune neutropenia, autoimmune oophoritis and orchitis. andorchitis, autoimmune thrombocytopenia, autoimmune thyroiditis, Behcet’s disease, bullous pemphigoid, cardiomyopathy, cardiotomy syndrome, celiac sprue-dermatitis, chronic active hepatitis, chronic fatigue immune dysfunction syndrome (CFIDS), chronic inflammatory demyelinating polyneuropathy, Churg-Strauss syndrome, cicatricial pemphigoid, CREST syndrome, cold agglutinin disease, Crohn’s disease disease, dense deposit disease, diseases associated with effects from organ transplantation, discoid lupus, essential mixed cryoglobulinemia, fibromyalgia-fibromyositis, glomerulonephritis (e.g., IgA nephropathy), gluten-sensitive enteropathy, Goodpasture's syndrome, graft vs. host disease (GVHD), Graves' disease (including, for example, Graves' thyroiditis), thyroiditis and Gravesopthalmopathy, Guillain-Barre polyneuritis, hyperthyroidism (Hashimoto’s thyroiditis), idiopathic pulmonary fibrosis, idiopathic Addison’s disease, idiopathic thrombocytopenia purpura (ITP), IgA neuropathy, Insulin Resistance Syndrome, juvenile arthritis, lichen planus, lupus erythematosus, Meniere’s disease, Metabolic Syndrome, mixed connective tissue disease disease, multiple sclerosis, myasthenia gravis, myocarditis, diabetes (e.g., Type 1 or Type 2 diabetes), neuritis, other endocrine gland failure, pemphigus vulgaris, pernicious anemia, polyarteritis nodosa, polychrondritis, polyendocrinopathies, polyglandular syndromes, polymyalgia rheumatica, polymyositis and dermatomyositis, post-myocardial pericarditis infarction, primary agammaglobulinemia, primary biliary cirrhosis, psoriasis, psoriatic arthritis, Raynaud's phenomenon, relapsing polychondritis, Reiter's syndrome, rheumatic heart disease, rheumatoid arthritis, sarcoidosis, scleroderma, Sjogren's syndrome, stiff-man syndrome, systemic lupus erythematosus, Takayasu arteritis, temporal arteritis/giant cell arteritis arteritis, ulcerative colitis, urticaria, uveitis, uveitis opthalmia, vasculitides such as dermatitis herpetiformis vasculitis, vitiligo, and Wegener’s granulomatosis.

Cardiovascular disease is a disorder of the heart and blood vessels and includes disorders of the arteries, veins, arterioles, and capillaries. Cardiovascular disease diagnosed by the methods described herein may include, but is not limited to, congestive heart failure (HF), acute coronary artery disease (CAD), arrhythmia, asymmetric septal hypertrophy (e.g., left ventricular hypertrophy with resultant diastolic dysfunction), cardiomyopathy, valvular dysfunction, pericarditis, atherosclerosis, and acute myocardial infarction (MI).

Pulmonary diseases are conditions of the lungs. Pulmonary diseases diagnosed by the methods described herein may include, but are not limited to, chronic obstructive pulmonary disease (COPD), asthma, pneumonia, pneumothorax, pulmonary embolism, advanced respiratory distress syndrome (ARDS), pleural effusion, metastatic disease, pulmonary edema, gastroesophageal reflux disease with aspiration, interstitial fibrosis, pneumoconiosis, granulomatous disease, collagen vascular disease, and restrictive lung disease.

In some embodiments, the experimenter has the elevated level of human soluble ST2, such as compared with reference level and rises, then the decision of active treatment experimenter can be made, and the experimenter can be accommodated by the hospital for hospitalization, such as in hospital (such as emergency care or critical care unit) or auxiliary nursing institution.Determine whether the experimenter suffers from disease such as cardiovascular disease or lung disease, septicemia, Kawasaki disease, or Th2 related disease, or any disease described herein, is needed in many cases.For example, portable test kit can allow emergency medical personnel to measure the experimenter on the spot, to determine whether it should be transported to the emergency department.And the wounded inspection classification decision (triage decision), such as the wounded inspection classification decision in the emergency department or other clinical situations, also can make based on the information provided by methods described herein.Those patients presenting the human soluble ST2 level of increase should be preferentially disposed relative to those patients with lower levels.

In some embodiments, the level of human soluble ST2 is determined once, for example, when the subject is suspected of having a disease (e.g., when delivered to a medical practitioner or health care facility). In some embodiments, the level of human soluble ST2 is determined 2, 4, 6, 8, 12, 18 and/or 24 hours after the subject is suspected of having a disease (e.g., when delivered to a medical practitioner or health care facility), and/or one or more time points in 1-7 days or longer.

In some embodiments, the determination of the level of people's soluble ST2 is more than once.In some embodiments, when the determination of the level of people's soluble ST2 is more than once, can use the highest level, or can determine and use the variation between the level.The level of people's soluble ST2 also can determine repeatedly to weigh the response of experimenter to treatment.For example, the level of people's soluble ST2 obtained after applying treatment (such as one or more doses or one or more rounds of treatment) can be compared with the level (such as baseline level) of the people's soluble ST2 before initial treatment.The variation between people's soluble sT2 level can show whether treatment is effective, for example, the minimizing of people's soluble ST2 level can show that treatment is effective.

In some embodiments, the level of human soluble ST2 in the assay subject is compared with a human soluble ST2 reference level. Any various techniques known to those skilled in the art can be used to measure the human soluble ST2 level in the assay subject. Exemplary assay methods include, but are not limited to, methods known in the art such as quantitative PCR or Northern blot analysis. In some embodiments, the level of human soluble ST2 in the assay subject is measured using immunoassays such as enzyme-linked immunosorbent assay (ELISA). For example, in some embodiments, an antibody or its antigen-binding fragment is contacted with a sample from the assay subject. The sample can comprise or be derived from a variety of cells or tissues from any assay subject. For example, the sample can contain one or more of blood, serum, or plasma. Then, the combination of the antibody or antibody fragment is detected and optionally quantified, and the protein level is determined based on the level of antibody or antibody fragment binding. In some embodiments, the sample is substantially free of the ST2L form of the ST2 protein, so that the majority of the ST2 in the sample detected according to the methods disclosed herein is human soluble ST2. In some embodiments, the sample is free of detectable ST2L, so that the only detectable ST2 in the sample is human soluble ST2. In some embodiments, the sample that is substantially free of ST2L, or free of detectable ST2L, is a serum or blood sample.In some embodiments, the level of human soluble ST2 in a subject is determined in an immunoassay using at least one antibody or antigen-binding fragment described herein.

As described in more detail in the Examples section below, antibody compositions comprising antibodies produced by the hybridoma deposited with the ATCC and assigned patent deposit number PTA-10431 exhibit increased affinity for human soluble ST2 antigen relative to other commercially available antibodies. Such antibodies can be used in accordance with the methods described herein.

Method described herein can be used to determine that experimenter does not suffer from ST2 related condition of disease.ST2 related condition of disease is the condition of disease relevant to the ST2 of elevated level.Some exemplary ST2 related condition of disease includes but not limited to cardiovascular disease, pulmonary disease, septicemia, Kawasaki disease, and Th2 related disease.ST2 related condition of disease is normally serious, and usually requires active treatment.The experimenter presenting some nonspecific symptoms may suffer from or not suffer from ST2 related condition of disease (for example cardiovascular disease, pulmonary disease, septicemia, Kawasaki disease, or Th2 related disease).Nonspecific symptoms include but not limited to chest pain or discomfort, shortness of breath, nausea, vomiting, belching (eructation), sweating, palpitations, dizziness, fatigue and fainting.Each symptom can have different causes of disease.

In some embodiments, methods described herein can be used for risk stratification (risk stratification), for example, determines that experimenter has the ST2 associated symptom of low severity or low-risk form.For example, some experimenter who suffers from cardiovascular disease (for example myocardial infarction or heart failure) has the risk of adverse consequences (such as death or recurrent cardiac events) low, and presents the people's soluble ST2 of lower concentration, is to observe in the experimenter with the high risk of cardiovascular disease and adverse consequences compared.This type of experimenter can be considered as the ST2 associated cardiovascular symptom with " low severity " or " low-risk " form.However, no matter at high risk or in low-risk colony, all observe with the people's soluble ST2 of higher concentration compared observed in the experimenter not suffering from ST-2 associated symptom. Experimenters suffering from ST2-related conditions of low severity typically have a human soluble ST2 concentration higher than a human soluble ST2 reference level (e.g., the median human soluble ST2 concentration in a healthy or normal individual), while experiments with ST2-related conditions of high severity typically have a human soluble ST2 concentration higher than 80% of a human soluble ST2 reference level (e.g., the human soluble ST2 concentration observed in a healthy or normal individual). Determining that an experimenter presenting certain nonspecific symptoms does not suffer from, or suffers from, a low severity form of the ST2-related condition can lead to improved diagnosis and/or more effective treatment decisions, as such an experimenter may not require active treatment. For example, patients with elevated human soluble ST2 concentrations after acute myocardial infarction exhibit more cardiac remodeling (increased fibrosis) than patients with low human soluble ST2 concentrations, and eplerenone differentially attenuates cardiac remodeling in subjects with high human soluble ST2 concentrations (Weir et al., J. Am. Coll. Cardiol. 55: 243-250, 2010). Therefore, human soluble ST2 concentrations can be used to identify which patients should receive different, possibly nonstandard, hospitalizations, or more aggressive treatments.

chest pain

Chest pain is the primary complaint in approximately 1% to 2% of outpatient visits, and although the cause is usually not cardiac, heart disease remains the leading cause of death in the United States. Therefore, distinguishing between serious and benign causes of chest pain is crucial. The methods described herein can be used to make this determination.

A subject presenting to the emergency department with chest pain may have esophageal pain, ulcers, acute lung problems such as pulmonary embolism (PE) (potentially fatal), ruptured or opened aneurysms (highly fatal), gall bladder attacks, pericarditis (diagnosis of a cyst around the heart), angina (heart pain without damage), or myocardial infarction (potentially fatal). An accurate diagnosis may not be made immediately, but the decision to admit the subject or actively treat the subject should generally be made immediately. If the methods described herein indicate that the subject has elevated soluble ST2 levels, for example, suffering from an ST2-related condition, a decision to actively treat the subject may be made, for example, to avoid potential adverse consequences due to lack of treatment. Other information on the treatment and diagnosis of chest pain can be found in, for example, Cayley (Am. Fam. Phys. 72(10): 2012-2028, 2005).

difficulty breathing

Dyspnea, or shortness of breath (also defined as abnormal or uncomfortable breathing), is a common symptom among subjects presenting to the emergency department. The differential diagnosis of dyspnea includes four major categories: (1) cardiac, (2) pulmonary, (3) mixed cardiac and pulmonary, and (4) neither cardiac nor pulmonary.

Causes of cardiac dyspnea include right-, left-, or biventricular congestive heart failure with resulting systolic dysfunction, coronary artery disease, recent or recent myocardial infarction, cardiomyopathy, valvular dysfunction, left ventricular hypertrophy with resulting systolic dysfunction, asymmetric septal hypertrophy, pericarditis, and arrhythmias.

Pulmonary causes include obstructive (e.g., chronic obstructive pulmonary disease (COPD) and asthma) and restrictive processes (e.g., extrapulmonary causes such as obesity, acute involvement of the spine or chest wall, and intrinsic pulmonary pathology such as interstitial fibrosis, pneumoconiosis, granulomatous disease, or collagen vascular disease). Mixed cardiogenic and pulmonary conditions include COPD with pulmonary hypertension and cor pulmonale, deconditioning, pulmonary embolism, ARDS, and trauma. Conditions that are neither cardiogenic nor pulmonary include metabolic conditions such as anemia, diabetic ketoacidosis, and other less common causes, including metabolic acidosis, pain in the chest wall or elsewhere in the body, and neuromuscular disorders such as multiple sclerosis and muscular dystrophy. Obstructive rhinolaryngeal problems include nasal obstruction due to polyps or a deviated septum, enlarged tonsils, and supraglottic or subglottic airway stricture.

Dyspnea can also present as a physical manifestation of psychiatric illness, such as anxiety disorders, which leads to hyperventilation. Information on the measurement and treatment of dyspnea can be found, for example, in Morgan and Hodge, Am. Fam. Phys. 57(4):711-718, 1998.

Any antibody disclosed herein or Fab can be used to determine that the patient does not suffer from ST2 associated conditions.In some embodiments, measure the level of the people's soluble ST2 in the experimenter (for example, by any of the above methods) and compare with the people's soluble ST2 reference level.If the level of people's soluble ST2 in the experimenter is similar to the people's soluble ST2 reference level, it can be determined that the experimenter has a very low possibility of suffering from the ST2 associated conditions.When the level of people's soluble ST2 in the experimenter and the people's soluble ST2 reference level fully approached on scope, the two were " similar ", and the experimenter is unlikely to suffer from ST2 associated conditions.Usually, when the level of people's soluble ST2 in the experimenter and the people's soluble ST2 reference level differed from each other by about 25%, for example, about 25%, 20%, 15%, 10%, 5% or lower with the time, the two were " similar ".Those skilled in the art can determine suitable people's soluble ST2 reference level for the ST2 associated conditions discussed. Those skilled in the art will also be aware of such normal variations in human soluble ST2 levels and, upon reading this disclosure, will be able to determine whether human soluble ST2 levels are similar to human soluble ST2 reference levels.

In some embodiments, people's soluble ST2 level is determined once, for example, when suspecting that experimenter suffers from ST2 relevant symptom, determine.In some embodiments, people's soluble ST2 level is suspected that experimenter suffers from ST2 relevant symptom after 2,4,6,8,12,18 and/or 24 hours, and/or 1-7 day or more long in one or more time points determine.In some embodiments, the level of people's soluble ST2 is determined to carry out more than once, for example, to confirm or check the level of the people's soluble ST2 that determines in mensuration for the first time.

In some embodiments, the human soluble ST2-binding antibodies and antigen-binding fragments thereof described herein can be used in one or more of the methods described in U.S. Patent Application Publication Nos. US 2007/0248981, US 2009/0264779, US 2009/0305265, and US 2010/0009356, and PCT Application Publication No. WO 2007/131031.

In some embodiments, the method for diagnosing a disease in a subject comprises obtaining a sample from the subject, determining the level of human soluble ST2 in the sample using at least one antibody as described herein or fragment, and determining the level of at least one (e.g., two, three, four or five) other marker, wherein compared with the reference level of human soluble ST2, the elevated level of human soluble ST2 in the sample, and relative to the reference level of at least one (e.g., two, three, four or five) other marker, changing (e.g., increasing or decreasing) the level of at least one (e.g., two, three, four or five) other marker, indicates that the subject suffers from disease (e.g., cardiovascular disease, lung disease, sepsis, Kawasaki disease, or Th2 associated disease, or any other disease described herein). The reference level of at least one other marker can be the level of the marker described in the subject who is not diagnosed as suffering from the disease, the subject who does not present two or more symptoms with the disease, the subject who does not have the risk of the disease, or the level in the same subject at an earlier time point. Other markers are known in the art, and the method for determining the reference level of other markers can be determined by those skilled in the art.

In some embodiments, the reference level of people's soluble ST2 can be any reference level described herein.Other people's soluble reference levels can be determined by those skilled in the art.In some embodiments, described sample contains blood, serum or plasma.Can obtain described sample, and use at least one antibody described herein or fragment to determine that the level of people's soluble ST2 can be carried out as described above.

Treatment

In some embodiments, ST2-binding antibodies or their antigen-binding fragments are administered to subjects to treat any multiple diseases or conditions (e.g., any disease described herein). For example, human soluble ST2 levels increase in subjects suffering from diseases such as, but not limited to, cardiovascular disease, lung disease, septicemia, Kawasaki disease, and/or Th2-related diseases. Any human soluble ST2-binding antibodies or their antigen-binding fragments, as well as antibodies or antigen-binding fragments (e.g., people, chimeric or humanized antibodies or fragments) based on the modification of such antibodies or fragments, can be used to treat such diseases or conditions.

In some embodiments, an antibody or antigen-binding fragment thereof is administered to a subject, wherein the antibody or fragment competitively binds to an antibody produced by a hybridoma deposited with the ATCC and granted accession number PTA-10431 or PTA-104312, or both. In some embodiments, an antibody or antigen-binding fragment described herein is administered to a subject, wherein the antibody or fragment is human, chimeric, or humanized. As is known in the art, such human, chimeric, or humanized antibodies and fragments are generally less immunogenic than non-human, non-chimeric, or non-humanized antibodies. Therefore, such human, chimeric, or humanized antibodies provide therapeutic benefits, such as, but not limited to, a reduced incidence of side effects, tolerance to increased doses, and improved pharmacokinetic and/or pharmacodynamic properties. In some embodiments, the human, chimeric, or humanized antibody or fragment to be administered to a subject is derived from an antibody produced by a hybridoma deposited with the ATCC and granted accession number PTA-10431 or PTA-104312, or both. For example, the heavy chain and/or light chain variable region of such antibodies can be connected to a human constant region or its fragment to construct a chimeric antibody or fragment. Alternatively, one or more CDRs (e.g., each CDR) of such antibodies can be inserted into one or more human framework regions to construct a humanized antibody or fragment.

In some embodiments, an anti-human soluble ST2 antibody or a human soluble ST2 binding antibody fragment is directly administered to a subject. The anti-human soluble ST2 antibody or fragment can be administered in an effective amount at the dosage and duration necessary to achieve the desired result. For example, the therapeutically active amount of the antibody or fragment can vary based on factors such as the subject's disease state, age, sex, and weight, as well as the ability of the antibody or fragment to elicit a desired response in the subject. The dosage regimen can be adjusted to provide the optimal therapeutic response. For example, several separate doses can be administered daily, or the dosage can be proportionally reduced as shown by the urgency of the treatment situation. Those skilled in the art will know the dosage and dosing regimen suitable for administering an anti-human soluble ST2 antibody or fragment to a subject. See, for example, Physicians' Desk Reference , 63rd edition, Thomson Reuters, November 30, 2008.

Anti-human soluble ST2 antibody or human soluble ST2-binding antibody fragment described herein can be formulated to deliver in any possible route, including but not limited to parenteral (e.g., intravenous), intradermal, subcutaneous, oral, nasal, bronchial, ocular, transdermal (topical), transmucosal, rectal and vaginal routes. Antibodies or fragments can comprise a combination of a delivery agent (e.g., cationic polymer, peptide molecule transporter, surfactant, etc.) and a pharmaceutically acceptable carrier. As used herein, term "pharmaceutically acceptable carrier" includes solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, etc. compatible with drug administration. Supplementary active compounds can also be incorporated into pharmaceutical preparations containing antibodies or their antigen-binding fragments described herein.

Reagent test kit

Also provided herein are kits containing reagents comprising at least one (e.g., at least two, three, four, or five) anti-human soluble ST2 antibodies or antigen-binding fragments described herein. Kits typically comprise the following main components: packaging, reagents containing the binding compositions described above, optionally with controls, and instructions. The packaging can be a box-like structure to accommodate a vial (or multiple vials) containing the binding compositions, a vial (or multiple vials) containing controls, and instructions for using the methods described herein. Those skilled in the art can easily modify the packaging to suit various needs.

In some embodiments, the kits provided herein may contain at least one (e.g., at least two, three, four, or five) of any of the antibodies or antigen-binding fragments described herein. For example, the kit may contain at least one (e.g., at least two, three, four, or species) of antibodies or antigen-binding fragments thereof that competitively bind to an antibody produced by a hybridoma deposited with the ATCC and assigned accession number PTA-10431 or PTA-104312, or both.

In some embodiments, provided herein is a test kit containing at least one (for example, at least two, three, four or five) anti-human soluble ST2 antibody or Fab as described herein, and one or more reagents for detecting antibody or Fab and human soluble ST2. For example, the test kit can be designed for chemiluminescent microparticle immunoassay (CMIA), as tested by the ARCHITECT of Abbot Diagnostics (Abbott Park, IL), and therefore can contain the paramagnetic microparticles coated with anti-BNP antibodies, and the paramagnetic microparticles coated with anti-human soluble ST2 antibodies. These microparticles are contacted with the sample, and the human soluble ST2 present in the sample can be bound to the coated microparticles. Optionally, the sample can be divided into at least two aliquots, and the microparticles of each type can be contacted with different aliquots. After washing, the conjugate of anti-human soluble ST2 acridine label can be added to generate a reaction mixture in the second step. After another round of washing, pre-triggering liquid and triggering liquid are added to the reaction mixture. The resulting chemiluminescent reaction is measured, for example, using ARCHITECT iSystem optics (Abbot Diagnostics, Abbott Park, Illinois). There is a direct correlation between the amount of human soluble ST2 in the sample and the chemiluminescence detected.

In some embodiments, the kits provided herein contain at least one (e.g., at least two, three, four, or five) anti-human soluble ST2 antibodies or antigen-binding fragments described herein, and one or more solid phase immunoassay components for detecting human soluble ST2 by solid phase analysis. Solid phase immunoassays use a solid support to which one member of a ligand-receptor pair, such as an antibody or antigen-binding fragment thereof, is bound. Non-limiting examples of solid supports include plates, test tubes, polystyrene beads, and various porous materials such as nylon, nitrocellulose, cellulose acetate, and glass fiber. See, for example, U.S. Patent Nos. 4,703,017; 4,743,560; and 5,073,484. In some embodiments, the kits contain components for a solid phase immunoassay, wherein the solid phase-bound antibody or antigen-binding fragment thereof (e.g., an anti-human soluble ST2 antibody or antigen-binding fragment thereof) is contacted with a sample containing an analyte of interest (e.g., human soluble ST2), and the solid phase is subsequently washed to remove unbound material.

In some embodiments, the kit contains components for flow-through solid phase immunoassays. Flow-through solid phase immunoassays eliminate the need for incubation and washing steps involved in other types of solid phase immunoassays. A variety of flow-through solid phase immunoassays are known in the art. For example, U.S. Patent No. 4,632,901 discloses a flow-through immunoassay, in which an antibody (specific for the target antigen analyte) is bound to a porous membrane or filter paper, and a liquid sample is added thereto. When the liquid flows through the membrane, the target analyte is bound to the antibody. After adding the sample, a labeled antibody is added. Visual detection of the labeled antibody provides an indication of whether the target antigen analyte is present in the sample. And U.S. Patent No. 5,229,073 describes a semi-quantitative competitive immunoassay lateral flow method that uses multiple capture zones or lines containing immobilized antibodies for measuring plasma lipoprotein levels. Other examples of lateral flow tests for detecting analytes are disclosed in U.S. Patent Nos. 4,168,146; 4,366,241; 4,703,017; 4,855,240; 4,861,711; and 5,120,643; European Patent No. 0296724; WO 97/06439; and WO 98/36278. Those skilled in the art will be aware of other suitable solid phase immunoassay methods or devices and will be able to use one or more of the anti-human soluble ST2 antibodies and antigen-binding fragments described herein in such methods and devices.

In some embodiments, other detection methods may be used, for example, colorimetric assays, radioimmunoassays, or chemiluminescent assays. Sandwich assays may also be used, for example, using two monoclonal antibodies, one labeled with iodine 125 and the other adsorbed to beads, such as those used in the IRMA-BNP2 kit from CISBIO International (France), and the ShionoRIA BNP or ANP kit (SHIONOGI USA Inc.).

The kits provided herein can be used according to any of the methods described above (e.g., diagnostic methods). For example, a kit containing at least one (e.g., at least two, three, four, or five) anti-human soluble ST2 antibodies or antigen-binding fragments thereof described herein can be used to determine the level of human soluble ST2 in a sample. Furthermore, a kit containing at least one (e.g., at least two, three, four, or five) anti-human soluble ST2 antibodies or antigen-binding fragments thereof can be used to determine a human soluble ST2 reference level. Those skilled in the art will recognize suitable uses for other kits provided herein and will be able to use the kits for such uses.

In some embodiments of the kit, at least one (e.g., at least one, two, three, or four) antibody or fragment has a _KD of equal to or less than 8.59 x ^10-10 M for binding to human soluble ST2. In some embodiments of the kit, the kit is provided as an enzyme-linked immunosorbent assay. Some embodiments of the kit further contain recombinant human soluble ST2 isolated from human cells (e.g., human embryonic kidney cells). Some embodiments of the kit further contain fully glycosylated human soluble ST2 (e.g., present in a cell extract or provided as an isolated protein).

Example

The present invention is further described in the following examples, which are not to be construed as limiting the scope of the invention described in the claims.

Example 1: Generation and characterization of anti-ST2 monoclonal antibodies

Monoclonal antibodies against human soluble ST2 (sST2) protein were generated in mice vaccinated with recombinant protein produced from the human sST2 cDNA sequence.

Antigen production and confirmation: Human sST2 cDNA clone, GeneBank accession number NM_003856.2, was purchased from Origene Technologies, Inc. in Rockville, MD. Standard PCR techniques were used to construct an expression vector containing the entire human soluble ST2 sequence with a six-histidine purification tag incorporated into the amino-terminal region of the protein. The integrity of the expression clone was confirmed by DNA sequencing. The recombinant protein was produced by transient transfection and expression in human embryonic kidney cells (HEK293). The recombinant human soluble ST2 protein was purified by passing the cell lysate through a metal chelate column (which specifically binds to the histidine purification tag incorporated into the expressed protein). The purification of the recombinant human soluble ST2 was confirmed by Coomassie-stained polyacrylamide gel and Western blot analysis using commercially available anti-ST2 antibodies (monoclonal antibody D067 obtained from MBL International) and anti-His tag antibodies. See Figures 1, 2 and 3A-3C. The protein itself has a molecular weight of 36 kD based on the amino acid sequence, and Kuroiwa et al. (Biochem. Biophys. Res. Comm. 284: 1104–1108, 2001) showed that the native protein in human serum has a molecular weight of ∼58 kD. The purified recombinant protein produced from this expression system has a molecular weight corresponding to a fully glycosylated protein of ∼58 kD and can be recognized accordingly by commercially available anti-ST2 antibodies. Quantification was performed by Bradford total protein assay.

Hybridomas and Monoclonal Antibody Generation: Monoclonal antibodies were generated by immunizing mice with the recombinant proteins generated as described above. Three Balb/c mice were immunized as follows:

T1 20 μg/animal, with CFA (Freund's complete adjuvant)

T1+3 day: 20 μg/animal, with IFA (Freund's incomplete adjuvant)

T1+6 days 20 μg/animal in saline

T1+9 days 20 μg/animal in saline

The present invention relates to the present invention to the invention of the present invention.After final immunization, determine antibody titer by getting blood from each animal tail.Carry out spleen fusion and set up hybridoma with the animal with the highest antibody titer.After hybridoma is built into the stable cell culture in 96 well plates, screen itself and the combination of recombinant human soluble ST2 protein and with the combination of the universal protein (generic protein) that contains six his purification tags, to eliminate the special hybridoma of this marker.Select two hybridomas to carry out further characterization and product development: 7E4 and 9F8.Test these two kinds of monoclonal antibodies to the sensitivity of recombinant human soluble ST2 antigen, promptly use each antibody 9F8 and 7E4 of consistent amount to be coated with each well of 96 well microtiter plates, then test (referring to Fig. 4) at 3 times of serial dilutions (concentration range is 300 to 0.41ng/mL) of the recombinant human soluble ST2 of biotin coupling.

The two antibodies showed similar analyte sensitivity, with very strong absorbance values of 1.0 observed at the lowest analyte concentration of 0.41 ng/mL tested. In addition, both antibodies were coated in each well of a 96-well plate at a concentration ranging from 5 μg/ml to 0 and tested against a single concentration of biotin-coupled recombinant human soluble ST2 (see Figure 5). Both antibodies showed significant sensitivity at concentrations of ≥1.25 μg/ml, with antibody 9F8 showing slightly higher sensitivity.

9F8 and 7E4 antibody were also tested for the ability of monoclonal antibody sandwich enzyme immunoassay (EIA) together. Each monoclonal antibody was coated in a single well of a 96-well microtiter plate with a constant concentration, and measured for 3 times of serial dilutions (its concentration was 10 ng/ml to 0.01 ng/ml) of human soluble sST2, using another monoclonal antibody detection complex coupled to biotin. As shown in Figure 6, two antibody combinations all obtained similar sensitivity, and easily detected human soluble ST2 as low as 0.01 ng/mL.

Additional analysis using surface plasmon resonance (SPR) confirmed that antibodies 9F8 and 7E4 each recognize a unique epitope, as well as an epitope that is different from the epitopes recognized by commercially available antibodies D066 and D067 (monoclonal antibodies used by MBL International Corporation (MBL) in its ELISA). Table 7 illustrates the results of SPR analysis of antibodies 9F8, 7E4, a third novel anti-human soluble ST2 antibody (11A7) (included for reference), two commercially available monoclonal antibodies from MBL (D066 and D067), and an unrelated antibody (for baseline determination). A single coated SPR chip was prepared for each monoclonal antibody. Recombinant human soluble ST2 was flowed over the chip and allowed to bind. The test monoclonal antibody was then flowed over the primary antibody-human soluble ST2 complex to assess whether the secondary antibody could also bind to the complex via the human soluble ST2 protein. Only secondary antibodies that recognized a different epitope than the primary antibody would bind to the complex.

The results of the SPR analysis are shown in Figures 7A-F.

Figure A1 (Figure 7A): When 9F8 (A1) was used as the primary capture antibody, each test antibody exhibited at least the minimum detectable signal. Test antibody 7E4 (L2) had the highest signal, and an irrelevant antibody (L6) had the lowest signal. Based on this figure, it was concluded that antibody 9F8 recognized a different epitope than all the tested antibodies, and that the 9F8-7E4 pairing provided the strongest overall binding.

Figure A2 (Figure 7B): When 7E4 (A2) was used as the primary capture antibody, the test antibody 9F8 (L1) showed very good binding, the irrelevant antibody showed no detectable signal, and the remaining test antibodies showed lower but detectable signals. The conclusion from this figure is that the antibody 7E4 recognizes a different epitope than all the antibodies tested, and the 9F8-7E4 pairing provided the strongest overall binding.

Figure A3 (Figure 7C): When the novel antibody 11A7 (A3) was used as the primary capture antibody, the test antibody 9F8 (L1) showed very good binding, the irrelevant antibody showed no detectable signal, and the remaining test antibodies showed a lower but detectable signal. These results were almost identical to those generated using 7E4 as the capture antibody.

Figure A4 (Figure 7D): When using MBL antibody D066 (A4) as the primary capture antibody, the test antibody 9F8 (L1) shows very good binding, followed by antibody 7E4 (L2). An irrelevant antibody showed no detectable signal, while the remaining test antibodies showed lower but detectable signals. This figure suggests that antibody D066 recognizes a different epitope than all the antibodies tested and forms a binding pair with a second MBL antibody, D067, but the binding affinity of this pairing is much lower than that of the 9F8-7E4 pairing.

Figure A5 (Figure 7E): When using MBL antibody D067 (A5) as the primary capture antibody, the test antibody 9F8 (L1) exhibited very good binding, followed by antibodies 7E4 (L2) and D066 (L4). An irrelevant antibody exhibited no detectable signal, while antibody 11A7 exhibited very low signal. This figure suggests that antibody D067 recognizes a different epitope than all the antibodies tested and forms a binding pair with the secondary MBL antibody D066, but the binding affinity of this pairing is much lower than that of the 9F8-7E4 pairing.

Figure A6 (Figure 7F): When an irrelevant antibody was used as the primary capture antibody, no detectable signal was generated from any of the tested antibodies, confirming the specificity of the tested antibodies in terms of affinity for human soluble ST2.

This analysis confirmed that the novel anti-human soluble ST2 monoclonal antibodies 9F8 and 7E4 recognize different epitopes than either of the MBL monoclonal antibodies D066 and D067. In addition, this analysis also confirmed that the 9F8-7E4 pairing has higher binding affinity than the D066-D067 pairing.

The binding affinity of the increase observed for 9F8-7E4 pairing is confirmed in direct comparisons of two pairs of monoclonal antibodies to test human plasma samples. In the experiment summarized in Table 1, the MBL ELISA comprising antibody pair D066-D067 was compared to the new antibody pair 9F8-7E4. Four (4) plasma samples were tested in a 2-fold dilution series using EDTA plasma and heparin plasma matching pairs from donors with low concentrations of human soluble ST2, as well as EDTA plasma and heparin plasma samples from donors whose concentrations of human soluble ST2 increased.

Table 1: Comparison of sensitivity of D066-D067 and 9F8-7E4 to human plasma samples

LS = low sST2 concentration sample, HS = high sST2 concentration sample,

ND = Not Detected, EUL = Above the Upper Limit of Detection

The results in Table 1 were generated using each experiment optimized for its individual performance, and the results are expressed as ng/mL based on the calibrator optimized for each antibody pair. The masses reported here do not match because the calibrated proteins were not normalized to each other but were quantified independently of each other. As shown in Table 1, the limit of detection for the D066-D067 pairing was a 1:4 dilution of the LSEDTA sample, with poor precision, while 9F8-7E4 was able to accurately measure down to a 1:256 dilution with good precision of <10%. This difference in sensitivity is consistent with the situation when testing HS EDTA plasma samples. It is also worth noting that the D066-D067 pairing was unable to detect even the least diluted LS heparin plasma sample, while for HS samples, the heparin plasma sample had a much lower signal compared to the EDTA plasma sample when paired with D066-D067, indicating that this antibody pairing is sensitive to or inhibited by heparin. 9F8-7E4 did not exhibit this heparin sensitivity in the tested plasma samples with low and high concentrations of human soluble ST2, maintaining good precision and low CV.

Example 2: EIA Characterization of 9F8-7E4 Monoclonal Antibody Pairing

The characteristics of the 9F8-7E4 monoclonal antibody pair were analyzed in an enzyme immunoassay (EIA).

Functional Sensitivity (Limit of Quantitation): The functional sensitivity limit was determined by assaying various concentrations of diluted calibrants in 20 replicates. In addition to the buffer blank, the calibrant concentrations tested included 0.0625, 0.125, and 0.25 ng/mL. Functional sensitivity was defined as the lowest concentration resulting in a CV ≤ 20%. As shown in Table 2 below, all concentrations tested met this criterion, with the lowest concentration tested being 0.0625 ng/mL.

Table 2: Summary of functional sensitivity analysis

ST2 (ng/mL) Standard Deviation CV% 0.0 0.137 0.020 14.% 0.065 0.222 0.038 17.% 0.125 0.297 0.011 3.7% 0.25 0.471 0.026 5.5%

Also use SPR to determine the affinity of four kinds of antibodies (9F8, 7E4, 11A7 and 15D06) produced by the method described herein, and two kinds of antibodies (D066-3 and D067-3) produced by MBL International.Each experiment is carried out by detecting the combination of the recombinant human soluble ST2 protein for the preparation of 9F8 and 7E4 antibodies described herein of following concentration: 50nM, 25nM, 12.5nM, 6.25nM, 3.25nM and 0nM.The data of these experiments are shown in Figures 8 A-8F.The K that every kind of antibody is combined with the recombinant human soluble ST2 separated from human embryonic kidney cells _D calculated value is shown in Table 3.

Table 3: _KD of each antibody binding to human soluble ST2

Precision: The precision of the test was evaluated according to the Clinical and Laboratory Standards Institute (CLSI) guideline Evaluation of Precision Performance of Quantitative Measurement Methods: Approved Guideline-Second Edition. (InVitro Diagnostics) EP5-A2. Three pooled patient plasma samples were aliquoted into twenty 1.5-mL plastic tubes at each concentration level and stored frozen at -80°C. These samples were analyzed in duplicate daily for 20 days over a 2-month blood collection period. Intra-run and overall analytical imprecision ( _CVA ) were calculated using the CLSI single-run analytical precision evaluation test. The assay had an intra-run _CVA of 2.4% and a total _CVA of 4.0% at an average concentration of 11 ng/mL (Pool 1, low), an intra-run CVA of 2.0% and a total _CVA of 3.9% at an average concentration of 87 ng/mL (Pool 2, medium), and an intra-run _CVA of 2.2% and a total _CVA of 3.9% at an average concentration of 140 ng _/mL (Pool 3, high). See Table 4.

Table 4: Accuracy analysis summary

Collection Average value (ng/ml) Single wheel CV Total CV Low 10.56 2.42% 3.96% medium 87.00 2.31% 3.87% high 140.05 2.24% 3.86%

The assay did not exhibit any precision bias within the concentration range tested.

Interfering substances (sensitivity to anticoagulants) evaluation: in 30 apparently healthy volunteers, plasma samples were taken in the most common test tube form: serum, EDTA plasma, citrate plasma, and heparin plasma. Analysis was carried out in a single human soluble ST2 assay kit immediately after common centrifugation and sample processing. These volunteers consisted of 9 males and 21 females, aged 22 to 66 years old. The results of this analysis are summarized in Table 5. As shown in Table 5, the median value of the citric acid test tube was slightly lower than that of other test tube types, which was unexpected because the citric acid test tube had a small amount of liquid anticoagulant in the test tube, causing it to be compared with other test tube types that did not affect sample volume, and a little dilution was had to the sample collected. In order to test the consistency of these measurements, each plasma test tube type was compared with the serum test tube. Each comparison resulted in highly significant ^R values, ranging from 0.849 to 0.964. Therefore, except for a little dilution that may affect normal concentration measurement in the citric acid test tube, the test tube type did not cause measurable deviation.

Table 5: Summary of Anticoagulant Test Results

Determination of Normal Concentration Reference Intervals: A cohort of 490 donors was recruited for this analysis who were self-reported to be healthy, had no known serious illnesses, were not currently receiving treatment for any serious illnesses, and were evenly distributed in terms of sex and age representation ranging from 18 to 84 years.

Table 6 has been summed up individual quantity and the sex distribution of each age group in this healthy reference range grouping, and Figure 10 is the histogram of people's soluble ST2 concentration distribution.In Figure 10, bar represents real data, and the vertical line in the bar represents theoretical normal distribution.The concentration distribution in these healthy individuals is non-normal.Table 7 compares people's soluble ST2 concentration as the function of sex.In this normal, healthy colony, the median concentration of men is significantly higher than the median concentration (Kruskal-Wallis test of women; p＜0.0001).

Table 6: Health Reference Range Grouping

Table 7: Comparison of median values of different genders in each reference group

Median value (ng/mL) 95% CI IQR male 23.6 21.3-25.1 17.6-30.6 female 16.2 15.3-17.4 12.4-19.9

The soluble ST2 concentration of people is revealed by age stratification, and there is no significant difference between these groups. Figure 11 (Kruskal-Wallis test; Male p=0.501, Female p=0.056). Therefore, the value of gender specific reference value and whole group uses nonparametric percentile method (90% one-sided) to calculate. These results are summarized in Table 8.

Table 8: Summary of CCD healthy reference group

Parameter/Group The entire group male female N 490 245 245 Average sST2 (ng/mL) 20.9 24.9 16.9 Median sST2 (ng/mL) 18.8 23.6 16.2 95% confidence interval of the median 18.1–19.9 21.3-25.1 15.3-17.4 Interquartile range 14.5–25.3 17.6-30.6 12.4-19.9 Upper limit: 90% (95%CI) 34.4 (32.4-35.8) 37.4 (35.5-41.1) 23.7 (22.2-25.9)

Table 9 lists the human soluble ST2 concentrations at several specific thresholds.

Table 9: sST2 Concentrations at Specific Thresholds in Self-Reported Health Groups in the United States

Fasting is to non-fasting people's soluble ST2 concentration: 25 patients (19 males and 6 women) with different disease states (wherein 8 have type 2 diabetes) get plasma samples at 7:00 in the morning after fasting overnight.After this, the patient takes in standardized breakfast (the patient without diabetes is 730kcal, and the patient with diabetes is 522kcal). At 11:00 in the morning, carry out second time blood drawing to measure people's soluble ST2.After this, all patients take in standardized lunch (the patient without diabetes is 800kcal, and the patient with diabetes is 716kcal). The third time also is to finally draw blood and carry out at 2:00 in the afternoon.Calculate 25 individualities' average people's soluble ST2 concentration at all three time points.Use paired t test to determine, non-fasting people's soluble ST2 plasma concentration (i.e. the blood getting at 11:00 in the morning and 2:00 in the afternoon) compares whether there is difference with corresponding fasting value (i.e. the blood getting at 7:00 in the morning). The relative change in human soluble ST2 concentration over time was compared to the RCV to determine the effect of food on the analyte concentration.

The average fasting human soluble ST2 concentration was 18 U/mL (median value: 17 U/mL; range 9-26 U/mL) at 7:00 in the morning, the average human soluble ST2 concentration at 11:00 in the morning after breakfast was 19 U/mL (median value, 18 U/mL; range, 11-28 U/mL; p=0.025 compared to fasting ST2), and the average human soluble ST2 concentration at 2:00 in the afternoon after lunch was 18 U/mL (median value, 18 U/mL; range, 10-28 U/mL; p=0.014 compared to fasting human soluble ST2). Therefore, the average human soluble ST2 concentration at 11:00 in the morning and 2:00 in the afternoon was higher than the average fasting value of 7:00 in the morning by <5%.

The comparison of normal people's soluble ST2 concentration and disease state concentration: the people's soluble ST2 concentration of heart failure patient is obviously higher than that of normal healthy individual.In following analysis, the normal concentration determined from 490 healthy donors is compared with a plurality of different colonies: 528 confirm by biomarker screening that there is not concealed cardiovascular disease (CVD) or inflammatory disease (by BNP, PCT, CRP, and IL-6 screening) healthy volunteers, 709 diagnoses suffer from the patient of acute heart failure, 1159 diagnoses suffer from the patient of chronic, stable heart failure, 190 diagnoses suffer from the patient of pulmonary hypertension (PAH), 48 diagnoses suffer from the patient of asthma, 223 diagnoses suffer from the patient of asthma or COPD, 58 diagnoses suffer from the patient of pulmonary embolism (PE), 119 diagnoses have the patient of pneumonia (PNA), 109 diagnoses suffer from the patient of (ARDS) of late respiratory disease syndrome, 50 diagnoses suffer from the teenager of Kawasaki disease (KD), and 15 diagnoses suffer from the patient of septicemia. Table 10 lists the median values, 95% confidence intervals, and interquartile ranges (IQRs) for human soluble ST2 concentrations in each group.

Table 10: sST2 concentrations in various disease states

Comparison of human soluble ST2 concentrations and risk of death at 1 year. The concentration of human soluble ST2 was also measured in blood samples from the PRIDE group (Junuzzi et al., J. Am. Coll. Cardiol. 50: 607-613, 2007). Of the 599 subjects in the initial PRIDE group, 586 were able to provide blood samples for measurement of human soluble ST2 using the above-described method. The samples used were EDTA plasma aliquots frozen at -80°C. Receiver operating characteristic (ROC) plots were analyzed, with death at one year as the reference standard, and the area under the curve (AUC) was compared according to the method of Hanley et al. (Radiology 148: 839-843, 1983) to determine the ability of the test to predict all-cause mortality at one year in the PRIDE group.

In these experiments, the median concentration for the entire group for this analysis was 27 ng/mL (range, <2-393 ng/mL). In this group, nonparametric correlation analysis revealed a correlation coefficient ( _rs ) of 0.955 (95% CI, 0.947-0.962; p<0.001) between the two methods. Of the 586 patients, 92 (16%) had died at one year, while 494 (84%) survived. ROC curve analysis confirmed that the test had an AUC value of 0.803 (95% CI, 0.768-0.834) for predicting death at one year.

Other implementation plans

It is to be understood that while the invention has been described with reference to its specific description, the foregoing description is intended to illustrate and not to limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages and modifications are within the scope of the appended claims.

Claims (20)

1. Use of at least one isolated antibody or antigen-binding fragment in the preparation of a composition for selecting a therapeutic treatment for a subject, said antibody being generated from a hybridoma deposited at the American Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, said antigen-binding fragment being (i) an antigen-binding fragment of an antibody generated from a hybridoma deposited at ATTC with patent accession number PTA-10431 or (ii) an antigen-binding fragment of an antibody generated from a hybridoma deposited at ATTC with patent accession number PTA-10432. The selection includes determining the level of human soluble ST2 in a biological sample from the subject using the at least one isolated antibody or antigen-binding fragment, wherein an elevated level of human soluble ST2 in the sample relative to a human soluble ST2 reference level indicates that a therapeutic treatment should be selected for the subject. 2. The use of claim 1, wherein the therapeutic treatment comprises a renin-angiotensin-aldosterone system inhibitor. 3. The use of claim 2, wherein the renin-angiotensin-aldosterone system inhibitor is a β-adrenergic receptor blocker. 4. Use of at least two different antibodies or antigen-binding fragments in the preparation of a composition for a subject-selective therapeutic treatment, wherein the antibodies are generated from a hybridoma deposited at the American Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, wherein the antigen-binding fragment is selected from the group consisting of: (i) an antigen-binding fragment of an antibody generated from a hybridoma deposited at ATTC with patent accession number PTA-10431 and (ii) an antigen-binding fragment of an antibody generated from a hybridoma deposited at ATTC with patent accession number PTA-10432. The selection includes determining the level of human soluble ST2 in a biological sample from the subject using the at least two different antibodies or antigen-binding fragments, wherein an elevated level of human soluble ST2 in the sample relative to a human soluble ST2 reference level indicates that a therapeutic treatment should be selected for the subject. 5. The use of claim 4, wherein the therapeutic treatment comprises a renin-angiotensin-aldosterone system inhibitor. 6. The use of claim 5, wherein the renin-angiotensin-aldosterone system inhibitor is a β-adrenergic receptor blocker. 7. Use of at least one isolated antibody or antigen-binding fragment in the preparation of a composition for identifying a subject with an increased risk of developing cardiovascular disease, said antibody being produced by a hybridoma deposited at the American Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, said antigen-binding fragment being (i) an antigen-binding fragment of an antibody produced by a hybridoma deposited at ATTC with patent accession number PTA-10431 or (ii) an antigen-binding fragment of an antibody produced by a hybridoma deposited at ATTC with patent accession number PTA-10432. The identification process includes obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using the at least one isolated antibody or antigen-binding fragment. Furthermore, if the subject's human soluble ST2 level is elevated relative to the human soluble ST2 reference level, then the subject is identified as having an increased risk of developing cardiovascular disease. 8. The use of claim 7, wherein the cardiovascular disease is heart failure or coronary artery disease. 9. Use of at least two different antibodies or antigen-binding fragments in the preparation of a composition for identifying a subject at risk of developing cardiovascular disease, said antibody being produced by a hybridoma deposited at the American Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, said antigen-binding fragment being selected from the group consisting of: (i) an antigen-binding fragment of an antibody produced by a hybridoma deposited at ATTC with patent accession number PTA-10431 and (ii) an antigen-binding fragment of an antibody produced by a hybridoma deposited at ATTC with patent accession number PTA-10432. The identification process includes obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using the at least two different antibody or antigen-binding fragments. Furthermore, if the subject's human soluble ST2 level is elevated relative to the human soluble ST2 reference level, then the subject is identified as having an increased risk of developing cardiovascular disease. 10. The use of claim 9, wherein the cardiovascular disease is heart failure or coronary artery disease. 11. Use according to any one of claims 1-10, wherein the subject is undiagnosed or does not present with two or more symptoms of a disease. 12. Use according to any one of claims 1-6, wherein the subject has been diagnosed with a disease. 13. The use of claim 12, wherein the disease is renal insufficiency or stroke. 14. The use of claim 12, wherein the disease is a cardiovascular disease. 15. The use of claim 14, wherein the cardiovascular disease is heart failure or coronary artery disease. 16. Use according to any one of claims 1-6, wherein the subject has been identified as having an increased risk of developing the disease. 17. Use according to any one of claims 1-10, wherein the subject has one or more of the following: hypertriglyceridemia, hypercholesterolemia, hypertension, and a body mass index ≥30. 18. Use according to any one of claims 1-10, wherein the subject has one or more of the following symptoms: chest discomfort, shortness of breath, nausea, vomiting, belching, sweating, palpitations, dizziness, fatigue, and syncope. 19. Use of at least one isolated antibody or antigen-binding fragment in the preparation of a composition for determining whether a subject has normal human soluble ST2 levels, said antibody being produced from a hybridoma deposited at the United States Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, said antigen-binding fragment being (i) an antigen-binding fragment of an antibody produced from a hybridoma deposited at ATTC with patent accession number PTA-10431 or (ii) an antigen-binding fragment of an antibody produced from a hybridoma deposited at ATTC with patent accession number PTA-10432. The determination includes obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using the at least one isolated antibody or antigen-binding fragment, wherein if the level of human soluble ST2 falls within a specific range, then the subject is determined to have a normal level of human soluble ST2, wherein the specific range is 14.5 ng/mL to 25.3 ng/mL. 20. Use of at least two different antibodies or antigen-binding fragments in the preparation of a composition for determining whether a subject has normal human soluble ST2 levels, said antibodies being produced from a hybridoma deposited at the United States Center for Type Culture Collection (ATTC) with patent accession number PTA-10431 or PTA-10432, said antigen-binding fragment being selected from the group consisting of: (i) antigen-binding fragments of antibodies produced from hybridomas deposited at ATTC with patent accession number PTA-10431 and (ii) antigen-binding fragments of antibodies produced from hybridomas deposited at ATTC with patent accession number PTA-10432. The determination includes obtaining a sample from the subject and determining the level of human soluble ST2 in the sample using the at least two different antibody or antigen-binding fragments, wherein if the level of human soluble ST2 falls within a specific range, then the subject is determined to have a normal level of human soluble ST2, wherein the specific range is 14.5 ng/mL to 25.3 ng/mL.