Protein Biomarkers for Diseases Associated with Contact Activation System

Abstract

To provide a method and a kit for analyzing a biological sample obtained from a subject having, suspected to have, or at the risk for a disease associated with a contact activation system.SOLUTION: There is provided a method for analyzing a sample including: (i) preparing a biological sample (for example, serum sample or plasma sample) obtained from a subject (human subject) having, suspected to have, or at the risk for a disease associated with a contact activation system; and measuring the level of a set of biomarkers including at least one type of protein selected from a table 1, in which when the set of biomarkers consists of one type of protein, the protein is not C4, plasma prekallikrein, thrombin, a tissue-type plasminogen activator (tPA), and heat shock protein 90.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 518,492, filed Jun. 12, 2017, and U.S. Provisional Application No. 62 / 395,712, filed Sep. 16, 2016, under 35 U.S.C. § 119(e). The entire contents of each of the above - mentioned applications are hereby incorporated by reference into this specification.

Background Art

[0002] Background The plasma contact activation system is a pro - inflammatory and pro - coagulant system involving a group of plasma proteases. The plasma contact activation system is activated by factor XIIa upon exposure to foreign surfaces or negatively charged surfaces, or by prolyl carboxypeptidase on the endothelial cell surface (Sainz I.M. et al., Thromb.Haemost. (2007) 98, 77 - 83). Inappropriate or disordered activation of the contact system is involved in various diseases, including hereditary angioedema (HAE).

[0003] HAE is a disease that causes sudden attacks of swelling that can affect multiple parts of the body (such as the face, limbs, genitals, gastrointestinal tract, and upper airway). Since the symptoms of HAE often resemble those of allergies or intestinal colic, patients with HAE are often difficult to identify until they exhibit severe or life - threatening symptoms. Early diagnosis would enable better management of emergencies associated with acute HAE attacks, and early diagnosis would also help manage HAE patients to prevent or attenuate acute HAE episodes (e.g., by having HAE patients avoid exposure to stimuli that may trigger HAE episodes).

[0004] Accordingly, it is highly interesting to identify biomarkers for HAE and to develop reliable diagnostic and prognostic methods for identifying subjects having a particular type of HAE or subjects at risk of suffering an acute HAE attack. Such biomarkers would also be useful for research into the disease mechanism, which could facilitate the development of effective new therapies for this disease. SUMMARY OF THE INVENTION

[0005] SUMMARY OF THE DISCLOSURE The present disclosure is based on the identification of proteins that are differentially present in biological samples obtained from subjects having a disease associated with the contact activation system compared to healthy individuals, or that are differentially present in biological samples obtained from subjects at different stages of such a disease (e.g., attack versus quiescent state).

[0006] Accordingly, one aspect of the present disclosure provides a method of analyzing a sample comprising: (i) providing a biological sample (e.g., a serum sample or a plasma sample) obtained from a subject (such as a human subject) having, suspected of having, or at risk of having a disease associated with the contact activation system; and (ii) measuring the levels of a set of biomarkers comprising at least one protein selected from Table 1, wherein when the set of biomarkers consists of one protein, the protein is not C4, plasma prekallikrein, thrombin, tissue-type plasminogen activator (tPA), and heat shock protein 90. In some embodiments, the disease associated with the contact activation system is hereditary angioedema (HAE), such as type I HAE or type II HAE.

[0007] In some embodiments, the set of biomarkers consists of 2 to 10 proteins selected from Table 1. In some embodiments, at least one protein may be a mitochondrial protein, such as ATP synthase subunit O (ATPO), cyclophilin F, or mitochondrial heat shock protein 60 (HSP60). In some embodiments, at least one protein is 14-3-3 zeta / delta or 14-3-3 beta / alpha. In some embodiments, at least one protein may be a protein kinase, such as protein kinase YES, protein kinase LYN, or mitogen-activated protein kinase 14 (MAPK14). In some embodiments, at least one protein is glycogen synthase kinase 3 alpha / beta. In some embodiments, at least one protein is ATP-dependent RNA helicase DDX19B (DEAD box 19B). In some embodiments, at least one protein is eukaryotic translation initiation factor 5A-1 (eIF-5A-1).

[0008] In some embodiments, preparing a biological sample includes collecting the biological sample in a vacuum blood collection tube containing one or more protease inhibitors. In some embodiments, measuring the levels of the set of biomarkers is performed using an enzyme-linked immunosorbent assay (ELISA), an immunoblotting assay, or a lateral flow assay.

[0009] In some embodiments, the method further comprises identifying that a subject has a disease associated with the contact system when the level of a set of target biomarkers deviates from the level of the same set of biomarkers in a control subject. In some embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent (such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor inhibitor, and / or a C1 esterase inhibitor) for treating the disease when the subject is identified as having the disease. In some embodiments, the pKal inhibitor is an anti-pKal antibody (such as lanadelumab) or an inhibitory peptide (such as ecallantide). In some examples, the bradykinin 2 receptor inhibitor is an inhibitory peptide (such as icatibant). In some examples, the C1 esterase inhibitor is a human plasma-derived C1 esterase inhibitor.

[0010] In some embodiments, the subject is a human patient undergoing treatment for a disease, where the method further comprises evaluating the effectiveness of the treatment based on the level of the set of biomarkers, and deviation of the level of the set of biomarkers of the subject from that of a control subject indicates the effectiveness of the treatment. In some embodiments, the method further comprises identifying a treatment suitable for the subject based on the level of the set of biomarkers. In some embodiments, the method further comprises identifying the subject as a candidate for treatment of the disease based on the level of the set of biomarkers.

[0011] The present disclosure provides biomarkers capable of identifying patients having a disease associated with the contact activation system (such as HAE). Measuring the level of a set of biomarkers may also be useful in the evaluation and treatment of such diseases.

[0012] In another aspect, a kit for analyzing a sample of a subject having, suspected of having, or at risk of a disease associated with a contact system, the kit comprising a first binding substance specific for a first protein biomarker selected from Table 1 and a second binding substance specific for a second protein biomarker selected from Table 1, wherein the first protein biomarker and the second protein biomarker are different. In some examples, the first binding substance and / or the second binding substance is an antibody specific for the protein biomarker. In some embodiments, the kit may further comprise a first detection substance that binds to the first binding substance and a second detection substance that binds to the second binding substance. In some embodiments, the first binding substance and the second binding substance are immobilized on a support member.

[0013] Details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of some embodiments, and even from the appended claims.

[0014] The following drawings form a part of this specification and are included to further illustrate certain aspects of the present disclosure that can be better understood by referring to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

Brief Description of the Drawings

[0015]

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Mode for Carrying Out the Invention

[0016] Detailed Description The contact activation system initiates the intrinsic pathway of blood coagulation and promotes inflammation through the release of bradykinin, a pro-inflammatory peptide. Factor XII (FXII), also known as Hageman factor, is a serine protease that plays a role in the activation of the intrinsic pathway of blood coagulation as well as the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid) to yield the active form, FXIIa. Activated FXIIa has the ability to cleave prekallikrein to yield active pKal. Subsequently, activated pKal can cleave FXII to FXIIa, resulting in a positive feedback loop in which FXIIa generates more pKal, which further activates additional FXII to FXIIa. Activated pKal can also cleave high molecular weight kininogen (HMWK) to release bradykinin. In diseases associated with activation of the contact system, such as HAE, an increase in bradykinin levels can induce vasodilation and inflammation that result in edematous HAE attacks. For example, it is desirable to identify novel biomarkers that can be used to identify subjects having or at risk of having such diseases in order to identify such diseases mediated by the contact activation system.

[0017] The present disclosure is based at least in part on the identification of proteins that are differentially present in biological samples obtained from subjects having a disease associated with the contact activation system (e.g., in a basal state or an attack) compared to healthy individuals by proteomic analysis. It was unexpectedly observed that proteins belonging to specific cellular pathways or processes (e.g., proteins involved in mitochondrial function) and protein families (e.g., 7 member protein family) tend to have a similar trend (e.g., elevated or decreased levels) in samples from subjects having the disease compared to healthy individuals.

[0018] Accordingly, provided herein is a method for analyzing a biological sample from a subject having, suspected of having, or at risk for a disease associated with the contact activation system (e.g., HAE) by detecting the presence or measuring the level of a set of protein biomarkers. Such methods can be useful, for example, to identify patients at risk for a disease associated with the contact activation system (e.g., HAE), to select candidates for treatment, to monitor disease progression or condition, to evaluate the effectiveness of treatment for a disease, to determine the course of treatment, to assess whether a subject is at risk for an episode of a disease, to identify whether a disease or disorder is associated with the contact activation system, and / or for research purposes (including research into the mechanisms of the disease and / or the biological pathways / processes involved in the disease that may be utilized for the development of new therapies).

[0019] Protein biomarkers of the contact activation system The methods and kits described herein are based at least in part on the identification of proteins that have been found to be differentially present in samples from subjects with HAE compared to samples from healthy subjects and / or differentially present in samples from subjects at different stages of such a disease (e.g., baseline vs. attack). As used herein, the term "protein biomarker" or "set of protein biomarkers" refers to a protein or set of proteins that are present at different levels in samples from different groups of subjects (e.g., subjects having a disease associated with the contact system vs. healthy subjects (e.g., subjects without the disease), or subjects having a disease in a quiescent stage vs. subjects during an attack of the disease). Such biomarkers / sets of biomarkers may be used in both diagnostic / prognostic applications and non-clinical applications (e.g., for research purposes).

[0020] In some embodiments, a protein biomarker may be present at elevated levels in a sample from a subject having a disease associated with the contact activation system (e.g., HAE) compared to the levels of the same protein biomarker in a sample from a healthy subject. In some embodiments, a protein biomarker may be present at decreased levels in a sample from a subject having a disease associated with the contact activation system (e.g., HAE) compared to the levels of the biomarker in a sample from a healthy subject. In yet other examples, a protein biomarker may be present at elevated levels in a sample obtained from a subject during an episode of a disease as described herein compared to the subject during a quiescent state of the disease. Alternatively, a protein biomarker may be present at decreased levels in a sample obtained from a subject during an episode of a disease as described herein compared to the subject during a quiescent state of the disease.

[0021] In some embodiments, a set of protein biomarkers comprising one or more biomarkers may be analyzed by the methods described herein. If the set of protein biomarkers comprises two or more biomarkers, all of those biomarkers may be present at elevated or decreased levels in a subject having a disease compared to a healthy subject. Alternatively, the set of protein biomarkers may comprise at least one biomarker that is elevated in a subject having a disease compared to a healthy subject and at least one biomarker that is decreased in a subject having a disease compared to a healthy subject.

[0022] Similarly, a set of protein biomarkers for distinguishing between a subject during an episode of a disease and a subject in a quiescent state of the disease, the set of biomarkers may include a plurality of biomarkers all of which are elevated or decreased in a first disease stage (e.g., episode) compared to a second disease stage (e.g., quiescent state). Alternatively, the set of biomarkers may include at least one biomarker that is elevated in the first disease stage compared to the second disease stage and at least one biomarker that is decreased in the first disease stage compared to the second disease stage.

[0023] Table 1 below presents markers that can be evaluated by the methods described herein for evaluating a subject or a biological sample from a subject for diseases associated with the contact activation system.

[0024] In some embodiments, a set of biomarkers measured and analyzed by any of the methods described herein includes at least one (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or more) protein selected from Table 1. When the set of biomarkers includes a single protein, that protein may not be any of complement protein 4 (C4), C1 inhibitor, prekallikrein, heat shock protein 90, tissue-type plasminogen activator, and thrombin. In some examples, a set of protein biomarkers measured and analyzed by the methods described herein does not include any combination of complement protein 4 (C4), C1 inhibitor, prekallikrein, heat shock protein 90, tissue-type plasminogen activator, and thrombin.

[0025] As described in Example 1, it was unexpectedly found that several proteins involved in mitochondrial function are differentially present in samples from subjects with HAE compared to healthy subjects. In some embodiments, the set of biomarkers includes one or more mitochondrial proteins as listed in Table 1. In some embodiments, the set of mitochondrial protein biomarkers includes ATP synthase subunit O (ATPO), mitochondrial heat shock protein 60 (HSP60), cyclophilin F (also referred to as peptidyl-prolyl cis-trans isomerase F; EC: 5.2.1.8) or a combination thereof.

[0026] Also as described in Example 1, it was also found that several proteins belonging to related protein families are differentially present in samples from subjects with HAE compared to healthy subjects. In some embodiments, the biomarker is 14-3-3 zeta / delta or 14-3-3 beta / alpha. In some embodiments, the biomarker is a protein kinase such as tyrosine protein kinase YES, tyrosine protein kinase LYN or mitogen-activated protein kinase 14 (MAPK14) or a combination thereof. In some embodiments, the biomarker is glycogen synthase kinase 3 alpha / beta (GSK-3 alpha / beta). In some embodiments, the biomarker is ATP-dependent RNA helicase DDX19B (DEAD box protein DDX19B). In some embodiments, the biomarker is eukaryotic translation initiation factor 5A-1 (eIF-5A-1). Also, any combination of these protein biomarkers is within the scope of the present disclosure.

[0027] Table 1: Biomarkers for activation of the contact system

Table 1

[0028] Usefulness of Protein Biomarkers One aspect of the present disclosure relates to a method for analyzing a sample obtained from a subject (e.g., a human patient) having, suspected of having, or at risk of a disease associated with a contact activation system by measuring the levels of a set of biomarkers as described herein in the sample. Results obtained from such an assay would be useful for diagnostic and / or prognostic uses as well as other non-clinical uses (such as research uses).

[0029] (i) Analysis of Biological Samples The methods described herein involve preparing a biological sample obtained from a subject. As used herein, "biological sample" refers to a composition comprising tissue from a subject (e.g., blood, plasma, or protein). Samples include both the first untreated sample taken from the subject as well as later processed (e.g., partially purified or preserved) forms. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a body fluid sample such as a serum sample or a plasma sample. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or 6 or more) biological samples may be taken from the subject over time or at specific time intervals, for example, to assess disease progression or the effectiveness of treatment.

[0030] A biological sample can be obtained from a subject using any means known in the art. In some embodiments, the sample is obtained from the subject by collecting the sample (e.g., a blood sample) into a vacuum collection tube (e.g., a vacuum blood collection tube). In some embodiments, the vacuum collection tube contains one or more protease inhibitors, e.g., to reduce or prevent ex vivo activation of the contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitor includes at least one serine protease inhibitor and at least one cysteine protease inhibitor. Such vacuum collection tubes are known in the art. See, e.g., PCT Application No. US2016 / 046681. Optionally, the vacuum blood collection tube may further include one or more anticoagulants.

[0031] The terms "patient," "subject," or "individual" may be used interchangeably and refer to a subject in need of the assays described herein. In some embodiments, the subject is a human or a non-human mammal. In some embodiments, the subject is suspected of having or at risk for a disease or disorder associated with the contact activation system (e.g., HAE). Such a subject may exhibit one or more symptoms associated with the disease. Alternatively, or in addition, such a subject may possess one or more risk factors for the disease (e.g., a genetic factor associated with the disease (e.g., a genetic defect in CI-INH)).

[0032] Alternatively, the subject in need of the assays described herein may be a patient with the disease. Such a subject may currently be suffering an attack of the disease or may have suffered from the disease in the past (e.g., currently in a quiescent state of the disease). In some examples, the subject is a human patient who may have received treatment for the disease (e.g., treatment involving a C1-esterase inhibitor (C1-INH), a plasma kallikrein inhibitor, or a bradykinin inhibitor). In other examples, such a human patient may not have received such treatment.

[0033] Examples of diseases associated with the contact activation system include, but are not limited to, kallikrein-mediated disorders such as bradykinin-mediated disorders (e.g., hereditary angioedema (HAE)), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic diseases, and coronary heart disease with unstable angina, edema, eye diseases, gout, intestinal bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis - degenerative spinal disorders, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic diseases and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue injury (e.g., burns or chemical injury).

[0034] In some embodiments, the disease or condition associated with the contact activation system is hereditary angioedema (HAE). Hereditary angioedema (HAE) is also known as "Quincke's edema", C1-esterase inhibitor deficiency, C1-inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent episodes of severe swelling (angioedema) that can affect, for example, the extremities, face, genitalia, gastrointestinal tract, and airways. Symptoms of HAE can include, for example, swelling in the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction that may be accompanied by swelling of the larynx and sudden hoarseness; repeated episodes of abdominal cramps without an obvious cause; and / or swelling of the intestine that can be severe and lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with HAE develop a non-itchy rash called erythema marginatum during an attack.

[0035] Swelling of the airway can be life-threatening and can result in death in some patients. The mortality rate is estimated to be 15 - 33%. HAE results in approximately 15,000 - 30,000 emergency hospital visits per year.

[0036] Trauma or stress (e.g., dental procedures, illness (e.g., viral diseases such as colds and influenza), menstruation, and surgery) can trigger an attack of angioedema. To prevent acute attacks of HAE, patients can try to avoid certain stimuli that have previously triggered an attack. However, in many cases, attacks occur without a known precipitating factor. Typically, the symptoms of HAE first appear in childhood and worsen during puberty. On average, untreated individuals have an attack every 1 - 2 weeks, and most episodes last about 3 - 4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among people with hereditary angioedema, even among people in the same family.

[0037] There are three types of HAE known as type I, type II, and type III. One in 50,000 people has HAE. Type I accounts for approximately 85% of cases, type II accounts for approximately 15% of cases, and type III is thought to be very rare. Type III is the most recently described form and was initially thought to occur only in women, but families with affected males have been identified.

[0038] HAE is inherited in an autosomal dominant pattern, so an affected person may inherit the mutation from one affected parent. Also, new mutations in the gene can occur, so HAE can occur in people with no family history of this disorder. 20 - 25% of cases are estimated to arise from new spontaneous mutations.

[0039] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene gives instructions to produce C1 inhibitor protein, which is important for the suppression of inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema reduce the level of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of abnormally functioning C1 inhibitor. When the level of functional C1 inhibitor is not appropriate, an excessive amount of bradykinin is produced. Bradykinin promotes inflammation by increasing the leakage of body fluids into body tissues through the blood vessel wall. Excessive accumulation of body fluids in tissues causes the swelling episodes seen in individuals with types I and II hereditary angioedema.

[0040] Mutations in the F12 gene are associated with some cases of type III hereditary angioedema. The F12 gene provides instructions for producing coagulation factor XII. In addition to playing a crucial role in blood clotting (coagulation), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Specific mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is generated, increasing the leakiness of blood vessel walls, which leads to episodes of swelling. The cause of other cases of type III hereditary angioedema remains unknown. In these cases, mutations in one or more genes that have not yet been identified may be responsible for this disorder.

[0041] HAE can present similarly to other forms of angioedema that are caused by allergies or other medical conditions, but HAE differs significantly in terms of cause and treatment. If HAE is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are usually ineffective for HAE (although epinephrine can be used for life-threatening reactions). Misdiagnosis has also sometimes led to unnecessary exploratory laparotomies for patients with abdominal swelling, and in some HAE patients, abdominal pain has been incorrectly diagnosed as being psychogenic.

[0042] C1 inhibitor therapy and other therapies for HAE are described in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925.

[0043] The acute treatment of HAE attacks is carried out to stop the progression of swelling as quickly as possible. Intravenous administration of C1 inhibitor concentrate derived from donor blood is one of the acute treatments. However, this treatment is not available in many countries. In emergencies where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as an alternative because fresh frozen plasma (FFP) also contains C1 inhibitor.

[0044] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor therapies are currently available in the United States, and two C1 inhibitor products are currently available in Canada. Berinert P (CSL Behring), which is pasteurized, was approved by the FDA in 2009 for acute attacks. Cinryze (registered trademark), which is nanofiltrated, was approved by the FDA in 2008 for prophylaxis. Ruconest (Pharming) is a recombinant C1 inhibitor under development that does not carry the risk of infectious disease transmission due to human blood-derived pathogens.

[0045] Treatment of acute HAE attacks may also include medication and / or intravenous fluids for pain relief.

[0046] Other therapies may stimulate the synthesis of C1 inhibitor or reduce the consumption of C1 inhibitor. Androgenic drugs such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0047] Helicobacter pylori can cause abdominal attacks. Antibiotics for treating H. pylori will reduce abdominal attacks.

[0048] Newer therapies attack the contact cascade. Ecallantide (KALBITOR (registered trademark)) inhibits plasma kallikrein and is approved in the United States. Icatibant (Firazyr (registered trademark), Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0049] The diagnosis of HAE can rely on, for example, family history and / or blood tests. The laboratory findings associated with types I, II, and III HAE are described, for example, in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, the levels of C1 inhibitor, as well as C4, are decreased, while the level of C1q is normal. In type II HAE, the level of C1 inhibitor is normal or increased. However, the function of C1 inhibitor is abnormal. The level of C4 is decreased, and the level of C1q is normal. In type III, the levels of C1 inhibitor, C4, and C1q may all be normal. The present disclosure is based at least in part on the identification of additional proteins (Table 1) that have different levels in samples from HAE patients compared to healthy individuals. Measuring the levels of a set of biomarkers of these proteins can be used to identify whether a subject has a disease such as HAE. In some embodiments, the method may be utilized to determine whether a patient has had or is having an HAE attack.

[0050] The symptoms of HAE can be evaluated, for example, using a questionnaire (e.g., a questionnaire answered by the patient, clinician, or family). Such questionnaires are known in the art and include, for example, visual analog scales. See, for example, McMillan, C.V. et al. Patient. 2012;5(2):113-26.

[0051] The biological samples described herein may be subjected to analysis by measuring the levels of a set of biomarkers as described herein in the biological samples. The levels (e.g., amounts) of the biomarkers disclosed herein or changes in the levels of the biomarkers may be evaluated using the assays described herein and / or assays known in the art. One or more of the biomarkers described herein may be analyzed using conventional methods. In some embodiments, the level of a biomarker is evaluated or measured by directly detecting the protein in the biological sample. Alternatively, or in addition, the level of the protein may be indirectly evaluated or measured in the biological sample, for example, by detecting the level of the activity of that protein (e.g., an enzyme assay).

[0052] In some embodiments, the biomarker is measured using an immunoassay. Examples of immunoassays include, but are not limited to, immunoblotting assays (western blot), enzyme-linked immunosorbent assay (ELISA) (e.g., sandwich ELISA), radioimmunoassay, detection assays utilizing electrochemiluminescence, magnetic immunoassay, lateral flow assay, and related techniques. Further suitable immunoassays for detecting the biomarkers presented herein will be apparent to those skilled in the art. However, it will be apparent to those skilled in the art that the present disclosure is not limited to immunoassays, and that detection assays that do not utilize antibodies, or fragments of antibodies that bind to antigens (such as mass spectrometry) are also useful for detecting and / or quantifying the contact-based biomarkers as presented herein. Assays that rely on chromogenic substrates may also be useful for detecting and / or quantifying the contact-based biomarkers as presented herein.

[0053] The type of detection assay used for the detection and / or quantification of a contact biomarker (such as those presented herein) will depend on several parameters, including the specific situation in which the assay will be used (e.g., clinical or research use), the type and number of biomarkers to be detected, and the type and number of patient samples to be run simultaneously.

[0054] ELISA is known in the art (see, e.g., Crowther, John R (2009), "The ELISA Guidebook", 2nd ed., Humana Press and Lequin R (2005), "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)", Clin. Chem. 51(12):2415 - 8), and an exemplary ELISA is described herein. Kits for performing ELISA are also known in the art and are commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0055] In some embodiments, an immunoassay is used to measure the level of a protein biomarker(s). The immunoassay described herein may be in the format of a sandwich ELISA where a first binding substance that specifically binds to the protein of a set of biomarkers is immobilized on a support member. The support member may then be incubated with a biological sample as described herein for a suitable time under conditions that allow for the formation of a complex between the binding substance and the protein in the sample. The complex may then be detected using a detection substance that binds to the protein, the binding substance - protein complex, or the binding substance. The detection substance may be conjugated to a label that can emit a signal directly or indirectly. The intensity of the signal represents the level of the protein in the sample. In some embodiments, the detection substance is detected and its level represents the level of the protein in the sample.

[0056] Any binding substance that specifically binds to a desired protein may be used in the methods and kits described herein to measure the level of the protein in a biological sample. In some embodiments, the binding substance is an antibody that specifically binds to the desired protein. In some embodiments, the binding substance is an aptamer antibody that specifically binds to the desired protein. In some embodiments, the sample may be contacted with two or more binding substances that bind to different proteins, either simultaneously or sequentially (e.g., multiplex analysis; e.g., SOMAScan™ assay (SOMALogic)). The biological sample is contacted with the binding substance under appropriate conditions. Generally, the term "contact" refers to bringing the binding substance into contact with the biological sample or agent for a suitable time sufficient for the formation of a complex between the binding substance and the protein (if present) in the sample. In some embodiments, the contact is effected by capillary action across the surface of a support membrane by the biological sample or agent.

[0057] In some embodiments, the immunoassay may be performed on a low-throughput platform that includes a single immunoassay format. For example, using a low-throughput platform, the presence and amount of proteins in a biological sample (e.g., a biological tissue, a tissue extract) may be measured for diagnostic methods, for monitoring the progression of a disease and / or treatment, and / or for predicting whether a disease or disorder may benefit from a particular treatment.

[0058] In some embodiments, it may be necessary to immobilize the binding substance to a support member. The method for immobilizing the binding substance will depend on factors such as the nature of the binding substance and the material of the support member and may require a specific buffer. Such methods will be apparent to those of skill in the art. For example, a set of biomarkers in a biological sample as described herein may also be measured using any of the kits and / or detection devices described herein.

[0059] As used herein, the terms "measuring" or "measurement" or "detecting" or "detection" mean evaluating the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample (including deriving qualitative or quantitative concentration levels of such a substance), or evaluating a value or classification of a subject.

[0060] Assays (e.g., Western blot assays) may further involve the use of commercially available quantitative imaging systems (e.g., LI-COR imaging technology) (see, e.g., the Odyssey® CLx infrared imaging system from LI-COR Biosciences). In some embodiments, electrochemiluminescence detection assays, or assays that rely on a combination of electrochemiluminescence and patterned array technology, are used (e.g., ECL or MULTI-ARRAY technology assays from Meso Scale Discovery (MSD)).

[0061] In any of the methods described herein, the protein levels of a set of biomarkers may be compared to the protein levels in a control sample or a reference sample.

[0062] The methods and kits described herein (also with any of the sets of protein biomarkers described herein) may be applied to the evaluation of diseases associated with contact activation systems (such as those described herein).

[0063] (ii) Diagnostic and / or prognostic uses The levels of the proteins presented in Table 1 detected in a sample from a subject can be used as reliable biomarkers for diagnosing a disease related to the contact activation system (e.g., HAE), monitoring the progression of such a disease, evaluating the effectiveness of treatment for a disease, identifying patients suitable for a particular treatment, and / or predicting the onset of a disease in a subject.

[0064] Accordingly, methods for diagnosis and prognosis for diseases related to the contact activation system based on the levels of a set of biomarkers in a biological sample obtained from a subject are described herein. In some embodiments, the levels of biomarkers measured using any of the methods described herein can be utilized to assess whether a subject (e.g., a human patient) from whom the biological sample was obtained has or is at risk of having a disease related to the contact activation system, such as a disease related to plasma kallikrein (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease).

[0065] In some embodiments, the biomarker level may then be compared to a reference sample or control sample to determine a value indicative of the amount of protein in the sample. In some embodiments, the value for a biomarker may be obtained by comparing the level of the protein in the sample to the level of another protein in the sample (e.g., an internal control or internal standard). Such a biomarker value may be a value normalized against an internal control or internal standard. The biomarker value may be compared to a reference value to determine whether the subject has or is at risk of having a disease associated with the contact activation system. The reference value may represent the level of the corresponding biomarker in a subject (e.g., a human subject) without the target disease. In some embodiments, when the biomarker level or value is higher than the reference level or reference value, the subject may be identified as having or being at risk of having a disease associated with the contact activation system. In some embodiments, when the biomarker level or value is lower than the reference level or reference value, the subject may be identified as having or being at risk of having a disease associated with the contact activation system.

[0066] In some embodiments, the biomarker level may then be compared to a predetermined threshold for that protein, a deviation from which may indicate that the subject may have a disease associated with the contact system. The predetermined threshold may represent a biomarker value that distinguishes between the biomarker level in patients with the target disease and the biomarker level in patients without the target disease.

[0067] In some embodiments, the set of biomarkers comprises two or more proteins, and for at least one of them, an elevated level indicates that the subject has or is at risk of having a disease, and for at least one of them, a decreased level indicates that the subject has or is at risk of having a disease. In some embodiments, the set of biomarkers comprises two or more proteins, and for each of them, an elevated level indicates that the subject has or is at risk of having a disease. In some embodiments, the set of biomarkers comprises two or more proteins, and for each of them, a decreased level indicates that the subject has or is at risk of having a disease.

[0068] In some embodiments, the control sample or reference sample is a biological sample obtained from a healthy individual. In some embodiments, the control sample or reference sample contains a known amount of the protein to be evaluated. In some embodiments, the control sample or reference sample is a biological sample obtained from a subject to be used as a control.

[0069] As used herein, the subject to be used as a control may be a healthy individual (i.e., an individual who is clearly free of the target disease (e.g., a disease related to the contact system) or has no medical history of that disease at the time when the level(s) of the protein(s) is / are measured), or may represent a population of healthy subjects who preferably have characteristics (e.g., age, gender, ethnicity, etc.) that match those of the subject to be analyzed by the methods described herein.

[0070] The control level may be a predetermined level or threshold. Such a predetermined level may represent the level of that protein in a population of subjects who do not have or are not at risk of having the target disease (e.g., the average level in a population of healthy subjects). It may also represent the level of that protein in a population of subjects who have the target disease.

[0071] The predetermined level can take various forms. For example, it may be a single cutoff value such as a median or an average value. In some embodiments, such a predetermined level may be established based on a comparison group (such as a comparison group where one defined group is known to have the target disease and another defined group is known not to have the target disease). Alternatively, the predetermined level may be a range (e.g., a range representing the level of that protein in a control population).

[0072] The control level as described herein can be determined by conventional techniques. In some examples, the control level can be obtained by performing a conventional method (e.g., the same assay as used to obtain the level of that protein in a test sample as described herein) on a control sample as also described herein. In other examples, the level of that protein can be obtained from members of a control population and the results analyzed, for example, by a computational program, to obtain a control level (predetermined level) representing the level of that protein in the control population.

[0073] By comparing the level of a biomarker in a sample obtained from a candidate subject with a reference value as described herein, it can be determined whether the candidate subject has a disease associated with the contact system (e.g., HAE) or is at risk thereof. For example, if the level of the biomarker(s) in the sample of the candidate subject deviates from the reference value (e.g., is increased compared to the reference value), the candidate subject may be identified as having the disease or being at risk. If the reference value represents a range of values of the level of the biomarker in a population of subjects having the target disease, the fact that the value of the biomarker in the candidate's sample is within that range indicates that the candidate subject has the target disease or is at risk.

[0074] As used herein, "elevated level" or "level above a reference value" means that the level of a biomarker is higher than a reference value (such as a predetermined threshold of the level of the biomarker in a control sample). The control level is described in detail herein. Elevated levels of a biomarker include levels of the biomarker that exceed the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% or more. In some embodiments, the level of a biomarker in a test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or 10000-fold or more higher than the level of the biomarker in a reference sample.

[0075] As used herein, "decreased level" or "level below a reference value" means that the level of a biomarker is lower than a reference value (such as a predetermined threshold of the biomarker in a control sample). The control level is described in detail herein. Decreased levels of a biomarker include levels of the biomarker that are lower than the reference value by, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400% or 500% or more. In some embodiments, the level of a biomarker in a test sample is at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2-fold, 2.5-fold, 3-fold, 3.5-fold, 4-fold, 4.5-fold, 5-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 50-fold, 100-fold, 150-fold, 200-fold, 300-fold, 400-fold, 500-fold, 1000-fold or 10000-fold or more lower than the level of the biomarker in a reference sample.

[0076] In some embodiments, the candidate subject is a human patient having symptoms of a disease associated with the contact activation system, such as a pKal-mediated disorder (e.g., HAE) or an autoimmune disease (such as RA, UC, and Crohn's disease). For example, the subject has edema; swelling that is complete or primarily peripheral; urticaria; erythema, pain, and swelling in the absence of evidence of infection; non-histamine-mediated edema; recurrent attacks of swelling; or combinations thereof. In other embodiments, the subject does not have symptoms of a pKal-mediated disorder, does not have a history of symptoms of a pKal-mediated disorder, or does not have a history of a pKal-mediated disorder such as HAE at the time the sample is taken. In still other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0077] Subjects identified by the methods described herein may receive suitable treatment, such as treatment with a pKal inhibitor, as described herein.

[0078] Considering the correlation between the level of a biomarker and such a disease, the assay methods and kits described herein can also be applied to evaluate the effectiveness of treatment for diseases related to the contact system (such as those described herein). For example, a plurality of biological samples (e.g., blood samples or plasma samples) may be collected from a subject to be treated before and after treatment, or during the course of treatment. The level of the biomarker may be measured by any of the assay methods as described herein, and accordingly, the value (e.g., amount) of the biomarker may be determined. For example, if an elevated level of the biomarker indicates that the subject has the target disease and the level of the biomarker decreases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a subsequently collected sample), it indicates that the treatment is effective. As another example, if a decreased level of the biomarker indicates that the subject has the target disease and the level of the biomarker increases after treatment or over the course of treatment (compared to the level of the biomarker in a previously collected sample, the level of the biomarker in a subsequently collected sample), it indicates that the treatment is effective. In some examples, the treatment involves an effective amount of a therapeutic agent (such as a plasma kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1 esterase inhibitor (C1-INH)). Examples of therapeutic agents include, but are not limited to, lanadelumab, ecallantide, icatibant, and human plasma-derived C1-INH.

[0079] If a subject is identified as not responding to treatment, a higher dose and / or more frequent administration of the therapeutic agent is administered to the identified subject. In some embodiments, the dose or frequency of administration of the therapeutic agent is maintained, reduced, or discontinued in a subject identified as responding to treatment or not requiring further treatment. Alternatively, a different treatment may be applied to a subject found not to respond to the initial treatment.

[0080] In other embodiments, the value of a biomarker or set of biomarkers can also be utilized to identify that the disorder is associated with the contact system or that the disorder may be treatable, for example, by a pKal inhibitor. To carry out this method, the level of the biomarker in a sample (e.g., a blood sample or plasma sample) taken from a subject having the target disease may be measured by a suitable method (e.g., those described herein such as Western blot assay or ELISA assay). If the level of the biomarker deviates from a reference value (e.g., is elevated or decreased), it indicates that a pKal inhibitor may be effective in treating the disease. If the disease is identified as being sensitive to a pKal inhibitor (treatable by a pKal inhibitor), the method may further comprise administering to a subject having the disease an effective amount of a pKal inhibitor (such as an anti-pKal antibody or an inhibitory peptide (e.g., lanadelumab, ecallantide)), a bradykinin 2 receptor inhibitor (e.g., icatibant) and / or C1-INH (e.g., human plasma-derived C1-INH).

[0081] Also within the scope of the present disclosure is a method for assessing the severity or condition of a disorder associated with the contact system. For example, as described herein, HAE may be in a quiescent state (baseline state) during which the subject may not experience symptoms of the disease. HAE attacks are typically recurrent episodes that can last 2 - 5 days during which the subject may experience pain and swelling, for example, in the hands, feet, face, gastrointestinal tract, genitals and pharynx (throat). In some embodiments, the level of one or more biomarkers indicates whether a subject is going to experience an HAE attack, is experiencing an HAE attack, or is about to experience an HAE attack soon. In some embodiments, the method involves comparing the level of a biomarker in a sample obtained from a subject having HAE with the level of that biomarker in a sample obtained from the same subject (e.g., a sample obtained from the same subject in a baseline state or a sample obtained from the same subject during an HAE attack).

[0082] (iii) Non-clinical use Furthermore, the level of any of the sets of biomarkers described herein may be used for research purposes. A number of diseases associated with contact activation systems have been identified, but there is a possibility that other diseases are mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify a disease as being associated with a contact activation system or a component of a contact activation system. In some embodiments, the methods described herein may be used to study the mechanism (e.g., discovery of a novel biological pathway or process involved in the development of a disease) or progression of a disease.

[0083] In some embodiments, the levels of sets of biomarkers as described herein may be utilized in the development of new therapies for diseases associated with contact activation systems. For example, the levels of sets of biomarkers may be measured in samples obtained from subjects undergoing a new treatment (e.g., a clinical trial). In some embodiments, the levels of sets of biomarkers may indicate the effectiveness of a new therapy or the progression of a disease in a subject before, during, or after a new treatment.

[0084] Kits and detection devices for measuring sets of protein biomarkers The disclosure also provides kits and detection devices for use in measuring the levels of sets of biomarkers as described herein. Such a kit or detection device may include binding substances that specifically bind to protein biomarkers (such as those listed in Table 1). For example, such a kit or detection device may include at least two binding substances that are specific for two different protein biomarkers selected from Table 1. In some examples, the kit or detection device includes binding substances that are specific for all members of the set of protein biomarkers described herein.

[0085] In some embodiments, one or more of the binding substances are antibodies that specifically bind to the proteins of the set of biomarkers. In some embodiments, one or more binding substances are aptamers (such as peptide aptamers or oligonucleotide aptamers) that specifically bind to the proteins of the set of biomarkers.

[0086] In some embodiments, the kit further comprises a detection substance (e.g., an antibody that binds to the binding substance) for detecting the binding of the binding substance to the protein(s) of the set of biomarkers. The detection substance may be conjugated to a label. In some embodiments, the detection substance is an antibody that specifically binds to at least one of the binding substances. In some embodiments, the binding substance comprises a tag that can be recognized by the detection substance and to which the detection substance can bind directly or indirectly.

[0087] In some embodiments, the support member is a membrane such as a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, or a cellulose acetate membrane. In some examples, the immunoassay may be in the format of a Western blot assay or a lateral flow assay.

[0088] In some embodiments, the support member is a multi-well plate, such as an ELISA plate. In some embodiments, the immunoassays described herein may be performed on a high-throughput platform. In some embodiments, multi-well plates (e.g., 24-well, 48-well, 96-well or 384-well or greater plates) may be used for high-throughput immunoassays. Individual immunoassays may be performed simultaneously in each well. Thus, generally, it is desirable to use a plate reader to measure multiple wells simultaneously in order to increase the throughput of the assay. In some embodiments, a plate reader capable of imaging multiple wells (e.g., 4 wells, 16 wells, 24 wells, 48 wells, 96 wells or 384 or more wells) simultaneously may be used for this platform. For example, a commercially available plate reader (e.g., the plate::vision system available from Perkin Elmer (Waltham, MA)) may be used. This plate reader is capable of kinetic-based fluorescence analysis. The plate::vision system has an optical system with high light collection efficiency and a special optical system designed for simultaneous analysis of 96 wells. Further suitable parallel plate readers include, but are not limited to, SAFIRE (Tecan, San Jose, CA), FLIPRTETRA® (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ) and CellLux (Perkin Elmer, Waltham, MA).

[0089] In a kit or detection device, one or more of the binding substances may be immobilized on a support member (e.g., a membrane, beads, slide, or multi-well plate). The selection of an appropriate support member for an immunoassay will depend on various factors such as the number of samples and the method of detecting the signal released from the label conjugated to the second agent.

[0090] The kit may also include one or more buffers as described herein (including but not limited to coating buffer, blocking buffer, washing buffer, and / or stopping buffer).

[0091] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The instructions included may describe the method of use of the components included in the kit for measuring the protein levels of a set of biomarkers in a biological sample taken from a subject such as a human patient.

[0092] Instructions regarding the use of the kit generally include information about the amount of each component and the suitable conditions for performing the assay methods described herein. The components in the kit may be in unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions provided in the kits of the present disclosure are typically instructions described on a label or an insert (e.g., a sheet of paper included in the kit), although machine-readable instructions (e.g., instructions held on a magnetic or optical storage disk) are also acceptable.

[0093] The label or insert indicates that the kit is used to evaluate the protein levels of a set of biomarkers. The instructions may be provided to perform any of the methods described herein.

[0094] The kits of the present disclosure are contained within a suitable package. Suitable packages include, but are not limited to, vials, bottles, jars, flexible packages (e.g., sealed Mylar or plastic bags), etc. Also contemplated are packages for use in combination with certain devices such as inhalers, nasal administration devices (e.g., nebulizers) or infusion devices (such as mini-pumps). The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper penetrable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper penetrable by a hypodermic needle).

[0095] Optionally, the kit may provide additional components such as information for determination (such as control samples and / or standard or reference samples). Typically, the kit includes a container and a label or instructions (s) on or associated with the container. In some embodiments, the present disclosure provides a product comprising the contents of the above-described kit.

[0096] Treatment of diseases associated with the contact activation system Subjects at risk of or suffering from a disease associated with the contact activation system identified using the methods described herein may be treated with any suitable therapeutic agent. In some embodiments, the provided method includes selecting treatment for a subject based on the output of the described method (e.g., measurement of the levels of a set of biomarkers).

[0097] In some embodiments, the method includes one or both of selection or administration of a therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor and / or a C1 esterase inhibitor) for administration to a subject based on the output of an assay (e.g., detection of a biomarker).

[0098] In some embodiments, the therapeutic agent is administered to the subject one or more times. In some embodiments, a plasma kallikrein inhibitor is administered to the subject. In some embodiments, the kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein antibody or a fragment thereof. In some embodiments, an antagonist of the bradykinin B2 receptor is administered to the subject. In some embodiments, C1-INH is administered to the subject.

[0099] The therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor and / or C1-INH) may be given together with another therapy as part of a combination therapy for the treatment of a disease or condition associated with the contact activation system. The combination therapy (e.g., combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist or a C1-INH replacement agent, e.g., combination therapy with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist or a C1-INH replacement agent and another therapy) may be provided in a plurality of different configurations. The first drug may be administered before or after the administration of the other therapy. In some situations, the first drug and another therapy (e.g., a therapeutic agent) are given simultaneously or in close temporal proximity (e.g., during the same treatment session, at short injection intervals). The first drug and the other therapy may also be given at longer time intervals.

[0100] Therapeutic agent Plasma kallikrein-binding substances (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding substances (e.g., small molecules)) are useful therapeutic agents for various diseases and conditions (e.g., diseases and conditions associated with plasma kallikrein activity). For example, in some embodiments, the diseases and conditions associated with plasma kallikrein activity are hereditary angioedema (HAE). In some embodiments, plasma kallikrein-binding substances such as plasma kallikrein inhibitors are administered to subjects at risk of or suffering from diseases associated with the contact activation system.

[0101] Some useful proteinaceous inhibitors of kallikrein (either tissue kallikrein and / or plasma kallikrein) contain a Kunitz domain. As used herein, a “Kunitz domain” is a polypeptide domain having at least 51 amino acids and containing at least two (preferably three) disulfides. This domain is folded such that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a Kunitz domain having 58 amino acids, the cysteines may be at positions corresponding to amino acids 5, 14, 30, 38, 51 and 55 of the BPTI homology sequence presented below, and the disulfides may be formed between cysteines at positions 5 and 55, 14 and 38, and 30 and 51), or, if two disulfides are present, they may be formed between the corresponding subsets of those cysteines. The spacing between each pair of cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino acids of the spacing between the positions corresponding to 5-55, 14-38 and 30-51 according to the numbering of the BPTI sequence presented below. The BPTI sequence can be used as a reference for indicating specific positions in any common Kunitz domain. Comparison of a Kunitz domain of interest with BPTI can be performed by identifying the best aligned alignment that maximizes the number of matching cysteines.

[0102] The (high-resolution) 3D structure of the knotted domain of BPTI is known. One of the X-ray structures has been deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of several BPTI homologs (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are known. The sequences of at least 81 knotted domains are known. Known human homologs include the three knotted domains of LACI, also known as tissue factor pathway inhibitor (TFPI) (Wun et al., J. Biol. Chem. (1988) 263(13):6001-6004; Girard et al., Nature (1989) 338:518-20; Novotny et al, J. Biol. Chem. (1989) 264(31):18832-18837), the two knotted domains of inter-α-trypsin inhibitor APP-I (Kido et al. J. Biol. Chem. (1988) 263(34):18104-18107), the knotted domain of collagen, the three knotted domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91:3353-3357), the knotted domain of hepatocyte growth factor activator inhibitor type 1, the knotted domain of hepatocyte growth factor activator inhibitor type 2, and the knotted domain described in U.S. Patent Application Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa that contains three knotted domains (amino acid sequence in Table 2).

[0103] Table 2: Exemplary Natural Knotted Domains

Table 2

[0104] The above-mentioned knotted domains are designated as LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (213-270). The cDNA sequence of LACI was reported by Wun et al. (J. Biol. Chem. (1988) 263(13): 6001-6004). Girard et al. (Nature (1989) 338: 518-20) reported a mutagenesis study in which the P1 residue of each of the three knotted domains was altered. LACI-K1 inhibits factor VIIa (F.VIIa) when F.VIIa is complexed with tissue factor, and LACI-K2 inhibits factor Xa.

[0105] Proteins containing exemplary knotted domains include the following (the numbers in parentheses are SWISS-PROT accession numbers):

Chemical formula

[0106] Knitted domains can be identified from an array database using various methods. For example, a known amino acid sequence, consensus sequence, or motif of a knitted domain (e.g., ProSite Motif) can be searched against, for example, the Pfam database of HMMs (Hidden Markov Models) using BLAST (e.g., using default parameters for Pfam searches; against the SMART database; or against the ProDom database), against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda MD). For example, the Pfam accession number PF00014 of Pfam Release 9 provides numerous knitted domains, as well as HMMs for identifying knitted domains. The description of the Pfam database can be found in Sonhammer et al. Proteins (1997) 28(3):405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al. Meth.Enzymol. (1990) 183:146-159; Gribskov et al. Proc.Natl.Acad.Sci.USA (1987) 84:4355-4358; Krogh et al. J.Mol.Biol. (1994) 235:1501-1531; and Stultz et al. Protein Sci. (1993) 2:305-314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, DE) is as described in Schultz et al. Proc.Natl.Acad.Sci.USA (1998) 95:5857 and Schultz et al. Nucl.Acids Res(2000) 28:231.The SMART database contains domains identified by profile hidden Markov models of the HMMer2 search program (R. Durbin et al. (1998) "Biological sequence analysis: probabilistic models of proteins and nucleic acids", Cambridge University Press). This database is also annotated and monitored. The ProDom protein domain database consists of an automatic compilation of homologous domains (Corpet et al. Nucl. Acids Res. (1999) 27:263-267). The current version of ProDom was constructed using recursive PSI-BLAST searches of the SWISS-PROT 38 and TREMBL protein databases (Altschul et al. Nucleic Acids Res. (1997) 25:3389-3402; Gouzy et al. Computers and Chemistry (1999) 23:333-340). This database automatically generates a consensus sequence for each domain. Prosite catalogs knitted domains as motifs and identifies proteins containing such domains. See, for example, Falquet et al. Nucleic Acids Res. (2002) 30:235-238.

[0107] The knotted domain interacts with the target protease mainly using the amino acids within two loop regions (the "binding loops"). The first loop region is around the residues corresponding to amino acids 13 - 20 of BPTI. The second loop region is around the residues corresponding to amino acids 31 - 39 of BPTI. In an exemplary library of the knotted domain, one or more amino acid positions within the first and / or second loop regions are altered. When screening for knotted domains that interact with kallikrein or when selecting variants with improved affinity, particularly useful positions to be altered include positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 with respect to the sequence of BPTI. At least some of these positions are expected to be in close contact with the target protease. Also, it is useful to alter other positions (e.g., positions adjacent to the aforementioned positions in the three-dimensional structure).

[0108] The "framework region" of the knotted domain is defined as the residues that are part of the knotted domain, specifically excluding the residues within the first and second binding loop regions (i.e., around the residues corresponding to amino acids 13 - 20 of BPTI and amino acids 31 - 39 of BPTI). Conversely, residues not within the binding loops may allow a wider range of amino acid substitutions (e.g., conservative substitutions and / or non-conservative substitutions).

[0109] In one embodiment, these knotted domains are variant forms of a loop structure that includes knotted domain 1 of human lipoprotein-associated coagulation inhibitor (LACI). LACI contains three clearly defined internal peptide loop structures that are paradigmatic knotted domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of LACI's knotted domain 1 described herein have been screened and isolated and these bind to kallikrein with improved affinity and specificity (see, for example, U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to the framework of other knotted domains to obtain other knotted domains that interact with kallikrein (e.g., plasma kallikrein). A useful modulator of kallikrein function, as determined using kallikrein binding assays and kallikrein inhibition assays, typically binds to and / or inhibits kallikrein.

[0110] In some embodiments, the plasma kallikrein inhibitor binds to the active form of plasma kallikrein. In some embodiments, the plasma kallikrein inhibitor binds to and inhibits plasma kallikrein (e.g., human plasma kallikrein and / or mouse kallikrein). Exemplary polypeptide-based plasma kallikrein agents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983, and U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991, and U.S. Patent Application Publication No. 20110086801, the entire contents of each of which are incorporated herein by reference. In some embodiments, the plasma kallikrein inhibitor is an inhibitory polypeptide or an inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also referred to as DX-88 or KALBITOR®; SEQ ID NO: 3). In some embodiments, the kallikrein inhibitor comprises, or consists of, a sequence of about 58 amino acids of amino acids 3 to 60 of SEQ ID NO: 3, or the DX-88 polypeptide having the 60 amino acid sequence of SEQ ID NO: 3.

[0111] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 3).

[0112] The plasma kallikrein inhibitor may be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD and IgE), or may only contain an antigen-binding fragment (e.g., Fab fragment, F(ab’)2 fragment or scFv fragment). The binding protein may contain two heavy chain immunoglobulins and two light chain immunoglobulins, or may be a single-chain antibody. The plasma kallikrein inhibitor may be a recombinant protein such as a humanized antibody, CDR-grafted antibody, chimeric antibody, deimmunized antibody or in vitro generated antibody, and optionally may contain a constant region derived from the sequence of a human germline immunoglobulin. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0113] Exemplary plasma kallikrein-binding proteins are disclosed in U.S. Patent Application Publication No. 20120201756, the entire content of which is incorporated herein by reference. In some embodiments, the kallikrein-binding protein is an antibody (e.g., a human antibody) having a light chain and / or a heavy chain of an antibody selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or ranalizumab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein-binding protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein-binding protein is ranalizumab. See U.S. Patent Application Publication No. 20110200611 and U.S. Patent Application Publication No. 20120201756, which are incorporated herein by reference.

[0114] An example of a plasma kallikrein inhibitory antibody is ranalizumab. The amino acid sequences of the heavy chain variable region and the light chain variable region of ranalizumab are presented below, with the CDR regions identified in bold and underlined.

[0115] Sequence of the heavy chain variable region of ranalizumab (SEQ ID NO: 4) EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SS

[0116] The sequence of the light chain variable region of ranalizumab (SEQ ID NO: 5) DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPKLLIY KASTLESGVP SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI

[0117] In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the framework regions of the HC and / or LC (e.g., FR1, 2, 3 and / or 4 of the HC and / or LC) to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the CDRs of the HC and / or LC (e.g., CDR1, 2 and / or 3 of the HC and / or LC) to the plasma kallikrein inhibitors described herein. In some embodiments, the plasma kallikrein inhibitor may have at least about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity in the constant regions (e.g., CH1, CH2, CH3 and / or CL1) to the plasma kallikrein inhibitors described herein.

[0118] In one aspect, a small molecule binds to and inhibits the active form of plasma kallikrein.

[0119] Bradykinin B2 receptor inhibitor In some embodiments, a bradykinin B2 receptor inhibitor (e.g., an antagonist) is administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant (Firazyr®), a peptidomimetic that blocks the binding of native bradykinin to the bradykinin B2 receptor and contains 10 amino acids.

[0120] C1-INH replacement drug In some embodiments, a C1-esterase inhibitor (C1-INH), such as a C1-INH replacement drug, is administered to a subject. Exemplary C1-INH replacement drugs are publicly available and include, for example, human plasma-derived C1-INH (e.g., Berinert® and Cinryze®).

[0121] Without further elaboration, based on the above description, those skilled in the art should be able to make the fullest use of the present disclosure. Accordingly, the following specific embodiments should be construed as merely illustrative and not as limiting the other parts of the present disclosure in any way. All publications cited herein are hereby incorporated by reference for the purposes or subject matter referred to herein.

Examples

[0122] Examples Example 1: Identification of Proteins That Differentially Exist in Samples from HAE Patients Compared to Healthy Individuals Plasma samples were collected from healthy individuals (N = 22; referred to as the "normal" sample), as well as from patients with HAE (type I / II) during the quiescent state of the disease (N = 33; referred to as "baseline") and patients with HAE (type I / II) during an attack (N = 33; referred to as "attack"). Blood was collected according to a strict blood collection protocol by venipuncture using a winged needle, plastic catheter, and plastic collection tube. The first collection tube was a serum tube, which was discarded. A second collection tube (P100 tube) containing a protease inhibitor cocktail and an anticoagulant was used for proteomic analysis. The blood collected in the P100 tube was processed into plasma within 1 hour of collection, aliquoted, and frozen at less than -70°C.

[0123] Plasma samples were analyzed using a multiplex assay (SOMAscan™ assay; SomaLogic; Boulder, CO) capable of detecting the relative abundance of 1,310 different human proteins. In this assay, the signal levels of each of the 1,310 proteins were compared for three different sample types (healthy individuals, patients with HAE in the quiescent state, and patients experiencing an HAE attack).

[0124] Statistical analysis of the data was performed using the Kruskal-Wallis analysis of variance. This is a nonparametric method for testing the distribution of three or more groups, which may have the same or different sample sizes. Under the assumption that the scaled distribution is the same for all groups, except for any differences in the medians, the null hypothesis is that the medians of all groups are equal, and the alternative hypothesis is that the population median of one group is different from the population median of at least one other group. If the statistic does not indicate significance, no probabilistic superiority is recognized between the samples. However, if there is significance in the medians, at least one sample probabilistically superior to another sample.

[0125] Protein levels that differ between HAE patients (in the basal state or during an attack) compared to healthy individuals with a false positive rate (q-value) of less than 0.01 and t-tests (p-value < 0.05, means of pooled variance) are listed in Table 1. Proteins are ranked according to the C-statistic from receiver operating characteristic (ROC) analysis, where C-statistics approaching 1.0 have the highest specificity and sensitivity for the positive detection of HAE (I / II). Patients with type I HAE are identified as having at least 50% (usually less than 30%) of the normal amount of total C1 inhibitor protein (C1-INH) (David-Lorton, M. J. Drugs Dermatol. (2015) 14:151-157). Patients with type II HAE have mutations in the SERPING1 gene that result in dysfunctional C1-INH protein and are identified as having at least 50% of the normal amount of functional C1-INH. In this study, it was not determined whether the HAE patients had type I HAE or type II HAE.

[0126] As shown in Table 1, 152 proteins were found to have statistically different levels (P < 0.05) between plasma samples obtained from HAE patients (during an attack or in the basal state) and plasma samples obtained from healthy individuals, and to be biomarkers that can be evaluated to distinguish individuals with HAE from those without the disease. By proteome analysis, 58 proteins with levels more than twice as high (P < 0.050) and 12 proteins with levels more than twice as low (P < 0.05) in plasma samples from HAE patients compared to samples from healthy individuals were identified. 10 proteins with C-statistics greater than 0.93 were identified. These proteins (e.g., those with high C-statistics (e.g., > 0.9)) can be used alone or in combination as reliable biomarkers for HAE and other diseases related to the contact system.

[0127] Plasma samples from HAE patients contained significantly lower amounts of complement protein 4 ("C4") than plasma from healthy individuals (Panel A of Figure 1). Low C4 levels are used in the clinical diagnosis of HAE (I / II) (Davis-Lorton, M. J. Drugs Dermatol. (2015) 14:151-157). Furthermore, a slight decrease in the amount of prekallikrein was observed in samples from HAE patients compared to samples from healthy individuals (Panel B of Figure 1). The amount of prekallikrein has also been previously shown to be decreased in HAE patients compared to normal levels. The changes in the abundance of C4 and pKal in HAE patients relative to those in healthy individuals, observed using the methods described herein, indicate that these methods can detect changes in protein levels associated with the onset of the disease.

[0128] Plasma samples from HAE patients and healthy individuals were also evaluated for plasma kallikrein generation. Briefly, citrated plasma samples were activated with FXIIa and then FXIIa was quenched with corn trypsin inhibitor. Plasma kallikrein rates were slightly decreased in samples from HAE patients compared to normal levels (Figure 2).

[0129] In this study, 15 out of 33 subjects with HAE were receiving prophylactic treatment with C1-INH (Cinryze®), which would increase the amount of plasma C1-INH detected. However, in HAE patients not receiving prophylactic treatment with C1-INH, total C1-INH was decreased compared to normal plasma samples (Panel A of Figure 3). As indicated by the arrow in Panel A of Figure 3, plasma samples from one subject contained elevated levels of C1-INH in both the basal and attack states. Excluding this outlier sample, the data showed a clear decrease in plasma C1-INH in HAE patients (Panel B of Figure 3).

[0130] Proteomic data also provided new insights into the pathobiology of HAE. For example, a subset of proteins identified as being elevated in plasma samples from HAE patients is associated with mitochondrial function (Panels A–C of FIG. 4). ATP synthase subunit O (ATPO) is an essential mitochondrial membrane protein that produces ATP from ADP in the presence of a proton gradient across the mitochondrial membrane generated by the electron transport complexes of the respiratory chain (also known as F1F0 ATP synthase or complex V). Similarly, cyclophilin F (also known as cyclophilin D or mitochondrial peptidyl-prolyl cis-trans isomerase F; EC: 5.2.1.8) is also a mitochondrial membrane protein. The level of 60 kDa mitochondrial heat shock protein (HSP60) was also found to be elevated in plasma samples from HAE patients.

[0131] A further protein identified in proteomic analysis that can be used as a biomarker for HAE is the 14-3-3 zeta / delta (14-3-3ζ) protein. As shown in Figure 5, the level of the 14-3-3 zeta / delta protein was elevated in plasma from HAE patients compared to healthy individuals. The 14-3-3 zeta / delta protein is one of seven members of a protein family, and other members of the seven-member protein family, including 14-3-3 beta / alpha, were also found to be elevated in plasma from HAE patients (Table 1). 14-3-3 proteins are ubiquitously expressed and highly conserved between plants and mammals, and are involved in the regulation of signaling pathways involved in metabolism, transcription, apoptosis, protein transport, and cell cycle regulation (Aghazadeh et al. Drug Discov. Today (2015)). Changes in the plasma or serum levels of these proteins have been associated with the development of diseases such as rheumatoid arthritis (Maksymowych et al. Clin. Exp. Rheumatol. (2014) 32:S35-S39), large vessel vasculitis including Takayasu arteritis and giant cell arteritis (Chakravarti et al. Arthritis Rheumatol. (2015) 67:1913-1921), cancer (Matta et al. Exper Opin. Ther. Targets (2012) 16:515-523), Parkinson's disease (Slone et al. Neurobiol. Dis. (2015) 79:1-13), and Alzheimer's disease (Steinacker et al. Semin. Cell Dev. Biol. (2011) 22:696-704). The results described herein are the first to identify that the level of the 14-3-3 zeta / delta protein is elevated in plasma from HAE patients compared to plasma from healthy volunteers.

[0132] Additional proteins that have been identified as deviating in plasma from patients with HAE compared to healthy individuals include IL-1F6 (also known as interleukin 36 alpha); protein kinases: tyrosine protein kinase YES, tyrosine protein kinase LYN, and mitogen-activated protein kinase 14 (MAPK14); glycogen synthase kinase 3 alpha / beta (GSK3 alpha / beta); ATP-dependent RNA helicase DDX19B (DEAD box protein 19B); and eukaryotic translation initiation factor 5A-1 (eIF-5A-1) (Table 1). As shown in Figure 6, levels of IL-1F6 were significantly lower in plasma samples from HAE patients, whereas, as shown in Figures 7-10, tyrosine protein kinase YES, tyrosine protein kinase LYN, MAPK14, GSK3 alpha / beta, DEAD box protein 19B, and eIF-5A-1 were each significantly elevated in plasma samples from HAE patients.

[0133] Proteomic analysis has identified over 150 proteins that are present at different levels between patients with HAE and healthy individuals. Any of the proteins identified herein may be used as (individually or in combination (as a set of biomarkers)) biomarkers for diseases related to the contact activation system, e.g., in methods for identifying patients at risk of diseases related to the contact activation system (e.g., HAE), selecting candidates for treatment, monitoring disease progression or condition, assessing the effectiveness of treatment for a disease, determining the course of treatment, identifying whether a disease or disorder is related to the contact activation system, and / or for research purposes (including studying the mechanisms of diseases that may be utilized for the development of new therapies).

[0134] Other embodiments All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Accordingly, unless otherwise specified, each disclosed feature is merely an example of a general series of equivalent or similar features.

[0135] From the above description, those skilled in the art can easily identify the essential features of this disclosure and make various changes and modifications to this disclosure to adapt to various applications and conditions without departing from their spirit and scope. Accordingly, other embodiments also fall within the scope of the claims.

[0136] Equivalents and Scope Those skilled in the art will recognize many equivalents to the specific embodiments of this disclosure described herein or will be able to confirm them using nothing more than routine experimentation. The scope of this disclosure is not intended to be limited to the above description but rather is as set forth in the appended claims.

[0137] In the claims, articles such as "a," "an," and "the" may mean one or more unless otherwise indicated or clear from the context. A claim or description that includes "or" between one or more members of a group is considered to be satisfied when, unless otherwise indicated or clear from the context, one, two or more, or all of the members of the group are present in, used in, or related to a given product or process. This disclosure includes embodiments where exactly one member of the group is present in, used in, or related to a given product or process. This disclosure includes embodiments where two or more or all of the members of the group are present in, used in, or related to a given product or process.

[0138] Furthermore, the present disclosure encompasses all variations, combinations, and substitutions in which one or more limitations, elements, clauses, and descriptive terms from one or more of the recited claims are introduced into another claim. For example, any claim that depends on another claim may be modified to include one or more limitations found in any other claim that depends on the same base claim. When elements are presented as a list (e.g., in Markush group format), each subgroup of the elements is also disclosed, and any element(s) may be deleted from the group. Generally, when the present disclosure or an aspect of the present disclosure is referred to as including a particular element and / or feature, it should be understood that a particular embodiment of the present disclosure consists of or consists essentially of such element and / or feature. For the sake of brevity, those embodiments are not specifically described herein by those words. It should also be noted that the terms "comprising" and "containing" are intended to be open and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Further, unless otherwise indicated or not apparent from the context and the understanding of one of ordinary skill in the art, values expressed as ranges may assume any specific value or sub-range within the recited range, to the tenth of the unit of the lower limit of the range, in various embodiments of the present disclosure, unless the context clearly indicates otherwise.

[0139] This application refers to various issued patents, published patent applications, academic papers and other publications, all of which are hereby incorporated by reference into this specification. In case of any conflict between any of the incorporated references and this specification, this specification shall prevail. Further, any particular embodiment of the present disclosure that is included in the prior art may be explicitly excluded from one or more of the claims. Such embodiments are considered to be known to those skilled in the art, and thus they may be excluded even if such exclusion is not explicitly stated in this specification. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of the prior art.

[0140] Those skilled in the art will recognize many equivalents to the particular embodiments described herein or will be able to confirm them using nothing more than routine experimentation. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is as set forth in the appended claims. Those skilled in the art will understand that various changes and modifications may be made to this description without departing from the spirit or scope of the present disclosure as defined by the following claims.

Claims

1. (i) preparing a biological sample obtained from a subject having, suspected of having, or at risk of hereditary angioedema (HAE); (ii) measuring the levels of a set of biomarkers comprising at least one protein selected from the group consisting of 14-3-3 zeta / delta and 14-3-3 beta / alpha; A method for analyzing a sample, comprising:

2. The method according to claim 1, wherein the set of biomarkers consists of 2 to 10 proteins selected from Table 1.

3. The method according to claim 1 or 2, wherein the biological sample is a serum sample or a plasma sample.

4. The method according to any one of claims 1 to 3, wherein the HAE is type I HAE or type II HAE.

5. The set of biomarkers further comprises: (i) interleukin 36 alpha (IL-1F6); (ii) a mitochondrial protein selected from the group consisting of ATP synthase subunit O (ATPO), cyclophilin F, and mitochondrial heat shock protein 60 (HSP60); (iii) a protein kinase selected from the group consisting of protein kinase YES, protein kinase LYN, and mitogen-activated protein kinase 14 (MAPK14); and / or (iv) a protein selected from the group consisting of glycogen synthase kinase 3 alpha / beta, ATP-dependent RNA helicase DDX19B, and eukaryotic translation initiation factor 5A-1. The method according to any one of claims 1 to 4.

6. The biological sample is a biological sample collected in a vacuum blood collection tube containing one or more protease inhibitors, and / or Step (ii) is carried out using an enzyme-linked immunosorbent assay (ELISA), an immunoblotting assay, or a lateral flow assay, The method according to any one of claims 1 to 5. **Claim 7** The method according to any one of claims 1 to 6, wherein the subject is a human patient. **Claim 8** The method according to any one of claims 1 to 7, wherein deviation of the level of the set of biomarkers of the subject from the level of the same set of biomarkers of a control subject indicates that the subject has HAE. **Claim 9** The method according to any one of claims 1 to 7, wherein the subject is a human patient undergoing treatment for HAE, and the method further comprises evaluating the effectiveness of the treatment based on the level of the set of biomarkers, and deviation of the level of the set of biomarkers of the subject from that of a control subject indicates the effectiveness of the treatment. **Claim 10** The method according to any one of claims 1 to 7, wherein deviation of the level of the set of biomarkers of the subject from the level of the same set of biomarkers of a control subject indicates that the subject is a candidate for treatment of HAE. The method according to any one of claims 1 to 7. **Claim 11** A kit for analyzing a sample from a subject having, suspected of having, or at risk of having hereditary angioedema (HAE), comprising: (i) a first binding substance specific for a first protein biomarker that is 14-3-3 zeta / delta or 14-3-3 beta / alpha; (ii) a second binding substance specific for a second protein biomarker selected from Table 1; wherein the first protein biomarker and the second protein biomarker are different. The kit. **Claim 12** ​The kit according to claim 11, further comprising a first detection substance that binds to the first binding substance and a second detection substance that binds to the second binding substance.

13. The first binding substance is an antibody specific to the first protein biomarker, and / or the second binding substance is an antibody specific to the second protein biomarker, and / or the first binding substance and the second binding substance are immobilized on a support member. The kit according to claim 11 or 12.

14. The HAE is type I HAE or type II HAE. The kit according to claims 11 to 13.

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