Protein Biomarkers for Ranibizumab Treatment

Abstract

Provided herein are a method for determining whether a disorder is susceptible to treatment with a plasma kallikrein inhibitor, and a method for identifying a subject as a candidate for treatment with a plasma kallikrein inhibitor. Also provided herein are a method for identifying that a subject has or is at risk of having a disorder, and a method for treating a disorder of a subject. Further provided herein is a method for evaluating the effectiveness of treatment in a subject.

Description

Technical Field

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 63 / 357,596, filed Jun. 30, 2022, and U.S. Provisional Application No. 63 / 458,740, filed Apr. 12, 2023, under 35 U.S.C. § 119(e), the entire contents of each of which are incorporated herein by reference.

[0002] Reference to Electronically Submitted Sequence Listing The contents of the electronically submitted sequence listing (D061770139WO00-SEQ-JRV.xml; size: 13,585 bytes; and created on Jun. 29, 2023) are incorporated herein by reference in their entirety.

Background Art

[0003] Lanadelumab (TAKHZYRO® / Takeda) is a human monoclonal antibody that inhibits active plasma kallikrein and has been approved by multiple regulatory authorities for the treatment of hereditary angioedema (HAE) in human patients. HAE is a disease that causes transient episodes of swelling, which can affect multiple parts of the body, such as the face, limbs, genitals, gastrointestinal tract, and upper airway. Because HAE symptoms often resemble those of allergies or abdominal pain, it is often difficult to identify HAE attacks until the patient exhibits severe or life-threatening symptoms. Early diagnosis allows for better management of emergencies associated with acute HAE attacks. Also, for example, by enabling HAE patients to avoid exposure to stimuli that can trigger HAE attacks, it would be useful for the management of HAE patients to prevent or suppress acute HAE attacks.

Summary of the Invention

[0004] The plasma contact activation system is an inflammation-inducing and coagulation-promoting system involving a group of plasma proteases. The plasma contact activation system is activated by activated factor XII (FXIIa) when exposed to exogenous 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 uncontrolled activation of the contact system is involved in various diseases including hereditary angioedema (HAE). Therefore, it is of great interest to identify biomarkers for HAE and other diseases associated with the contact activation system in order to develop reliable diagnostic and prognostic methods for identifying subjects with or at risk of having HAE. Such biomarkers are also useful for research to understand the underlying disease mechanisms, which will thereby facilitate the development of effective new therapies.

[0005] The present disclosure is at least partially based on the identification of protein biomarkers that are differentially present in biological samples obtained from subjects with HAE compared to healthy individuals, or that are differentially present in biological samples obtained from subjects after treatment with a plasma kallikrein (pKal) inhibitor (i.e., lanadelumab) that normalize (revert to control levels) after treatment. The protein biomarkers identified herein may be used, for example, to determine whether a disorder is susceptible to treatment with a pKal inhibitor (e.g., lanadelumab), to identify diseases associated with the contact activation system, to identify patients with a disorder or at risk of a disorder, to identify a subject as a candidate for treatment, to monitor disease progression or disease state, and / or to evaluate the efficacy of disorder treatment.

[0006] Accordingly, aspects of the present disclosure are methods for determining whether a disorder is likely to respond to treatment with a pKal inhibitor, comprising measuring the level of a biomarker set comprising at least one protein selected from Table 1 in a biological sample from a subject having the disorder, and identifying that the disorder is likely to respond to treatment with a pKal inhibitor if the level of the biomarker set deviates from a reference value. In some embodiments, if it is identified that the disorder is likely to respond to treatment with a pKal inhibitor, the method further comprises administering a pKal inhibitor to the subject.

[0007] Aspects of the present disclosure are methods for identifying a subject as a candidate for treatment with a pKal inhibitor, comprising providing a biological sample from a subject having, suspected of having, or at risk of having a disorder, and measuring the level of a biomarker set comprising at least one protein selected from Table 1 in the biological sample, wherein the subject is identified as a candidate for treatment with a pKal inhibitor if the level of the biomarker set in the biological sample deviates from a reference value. In some embodiments, the method further comprises administering a pKal inhibitor to the subject identified as a candidate for treatment.

[0008] Aspects of the present disclosure are methods for identifying that a subject has or is at risk of having a disorder, comprising providing a biological sample from the subject, and measuring the level of a biomarker set comprising at least one protein selected from Table 1 in the biological sample, wherein the subject is identified as having or being at risk of having the disorder if the level of the biomarker set in the biological sample deviates from the level of the biomarker set in a control sample. In some embodiments, if it is identified that the subject has or is at risk of having the disorder, the method further comprises administering an effective amount of a pKal inhibitor to the subject.

[0009] Aspects of the present disclosure are methods of treating a disorder in a subject, comprising administering to the subject an effective amount of a plasma kallikrein (pKal) inhibitor, wherein the subject has a biomarker set at a level that deviates from the level of the biomarker set in a control sample, and the biomarker set comprises at least one protein selected from Table 1. In some embodiments, the disorder is not hereditary angioedema (HAE).

[0010] Aspects of the present disclosure are compositions for use in treating a subject having a disorder, the composition comprising a plasma kallikrein (pKal) inhibitor, wherein the subject has a biomarker set at a level that deviates from the level of the biomarker set in a control sample, and the biomarker set comprises at least one protein selected from Table 1.

[0011] In some embodiments, the pKal inhibitor is lanadelumab.

[0012] In some embodiments, the biomarker set consists of 2 to 10 proteins selected from Table 1. In some embodiments, the biological sample is a serum sample or a plasma sample.

[0013] In some embodiments, the disorder is a disease associated with the contact activation system. In some embodiments, the disorder is hereditary angioedema (HAE). In some embodiments, the disorder is type I HAE or type II HAE. In some embodiments, the disorder is not hereditary angioedema (HAE).

[0014] In some embodiments, at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-stranded high molecular weight kininogen (KNG1), and kininostatin. In some embodiments, at least one protein is a blood coagulation protein selected from the group consisting of alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5). In some embodiments, at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15: cytoplasmic domain (CDH15), ephrin type-A receptor 2 (EPHA2), multimerin-2 (MMRN2), olfactorymedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3). In some embodiments, at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta type-6 (PSMB6), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4 A (UBE4A), and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3). In some embodiments, at least one protein is a complement activation protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0015] In some embodiments, at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0016] In some embodiments, the biological sample is provided in a vacuum blood collection tube that contains one or more protease inhibitors. In some embodiments, the levels of the biomarker set are measured by enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay.

[0017] In some embodiments, the subject is a human patient.

[0018] Aspects of the present disclosure are methods for evaluating treatment in a subject, including measuring the levels of a biomarker set comprising at least one protein selected from Table 1 in a biological sample obtained from the subject before and after treatment or during a course of treatment, and evaluating the effectiveness of the treatment based on the levels of the biomarker set, and providing a method in which a deviation level of the biomarker set after treatment or throughout a course of treatment, compared to before treatment, indicates that the treatment is effective for the subject.

[0019] In some embodiments, the present treatment includes administering a plasma kallikrein (pKal) inhibitor to the subject. In some embodiments, the pKal inhibitor is lanadelumab.

[0020] In some embodiments, the biomarker set consists of 2 to 10 proteins selected from Table 1. In some embodiments, the biological sample is a serum sample or a plasma sample.

[0021] In some embodiments, at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-stranded high molecular weight kininogen (KNG1), and kininostatin. In some embodiments, at least one protein is a blood coagulation protein selected from the group consisting of alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5). In some embodiments, at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15: cytoplasmic domain (CDH15), ephrin type-A receptor 2 (EPHA2), multimerin-2 (MMRN2), olfactory modulin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3). In some embodiments, at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta type-6 (PSMB6), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4 A (UBE4A), and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3). In some embodiments, at least one protein is a complement activation protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0022] In some embodiments, at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0023] In some embodiments, the biological sample is obtained in a vacuum blood collection tube that contains one or more protease inhibitors. In some embodiments, the levels of the biomarker set are measured by enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay.

[0024] In some embodiments, the subject is a human patient. In some embodiments, the subject has, is suspected of having, or is at risk of having a disease associated with the contact activation system. In some embodiments, the disease associated with the contact activation system is hereditary angioedema (HAE). In some embodiments, the HAE is type I HAE or type II HAE. In some embodiments, the disease associated with the contact activation system is not HAE.

[0025] Aspects of the present disclosure are methods of analyzing a sample, comprising: (i) providing a biological sample (e.g., a serum sample or a plasma sample) obtained from a subject having, suspected of having, or at risk of having a disease associated with the contact activation system, such as a human subject; and (ii) measuring the levels of a biomarker set comprising at least one protein selected from Table 1, wherein when the biomarker set consists of one protein, the protein is not double-stranded high molecular weight kininogen (KNG1), α-2-macroglobulin (A2M), complement C1q, and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or interleukin-21 (IL-21).

[0026] In some embodiments, the biomarker set consists of 2 to 10 proteins selected from Table 1. In some embodiments, the biological sample is a serum sample or a plasma sample. In some embodiments, the disease associated with the contact activation system is hereditary angioedema (HAE). In some embodiments, the HAE is type I HAE or type II HAE.

[0027] In some embodiments, at least one protein of the biomarker set is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-stranded high molecular weight kininogen (KNG1), and kininostatin. In some embodiments, at least one protein of the biomarker set is a coagulation protein selected from the group consisting of alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5). In some embodiments, at least one protein of the biomarker set is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15: cytoplasmic domain (CDH15), ephrin type-A receptor 2 (EPHA2), multimerin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3). In some embodiments, at least one protein of the biomarker set is a proteolysis-related protein selected from the group consisting of proteasome subunit beta type-6 (PSMB6), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4 A (UBE4A), and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3). In some embodiments, at least one protein of the biomarker set is a complement activation protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0028] In some embodiments, at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), alpha-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0029] In some embodiments, providing a biological sample includes collecting the biological sample in a vacuum blood collection tube that contains one or more protease inhibitors. In some embodiments, measuring the levels of the biomarker set is performed by an enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay.

[0030] In some embodiments, the subject is a human patient. In some embodiments, if the levels of the subject's biomarker set deviate from the levels of the same biomarker set of a control subject, the method further includes identifying that the subject has a disease associated with the contact system. In some embodiments, if the subject is identified as having a disease, the method further includes administering to the subject a therapeutically effective amount of a therapeutic agent that treats the disease.

[0031] In some embodiments, the subject is a human patient undergoing treatment for a disease. In some embodiments, the method further comprises evaluating the efficacy of treatment by comparing the levels of a biomarker set measured in a biological sample obtained from the subject after treatment or during a course of treatment to the levels of the same biomarker set measured in a biological sample obtained from the subject before treatment, and determining that the treatment is effective if the levels of the biomarker set in the sample obtained after treatment or during a course of treatment deviate from the levels of the biomarker set in the sample obtained before treatment. In some embodiments, the method further comprises evaluating the efficacy of treatment by comparing the levels of a biomarker set measured in a biological sample obtained from the subject after treatment or during a course of treatment to the levels of the same biomarker set measured in a control sample obtained from a healthy subject, and determining that the treatment is effective if the levels of the biomarker set in the sample obtained after treatment or during a course of treatment do not deviate from the levels of the biomarker set in the control sample. In some embodiments, if it is determined that the treatment is not effective, the method further comprises administering to the subject a therapeutically effective amount of a therapeutic agent that treats the disease. In some embodiments, if the subject has previously been administered a therapeutic agent as part of a course of treatment, the subject is administered an increased dose of the therapeutic agent.

[0032] In some embodiments, the therapeutic agent administered to the subject is a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor inhibitor, and / or a C1 esterase inhibitor. In some embodiments, the pKal inhibitor is an anti-pKal antibody (e.g., lanadelumab) or an inhibitory peptide (e.g., ecallantide). In some embodiments, the bradykinin 2 receptor inhibitor is an inhibitory peptide (e.g., icatibant). In some embodiments, the C1 esterase inhibitor is a C1 esterase inhibitor derived from human plasma.

[0033] Aspects of the present disclosure provide a kit for analyzing a sample from a subject having, suspected of having, or at risk of having a disease associated with a contact system, the kit including a first binder specific for a first protein biomarker selected from Table 1 and a second binder 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 and / or second binder is an antibody specific for the protein biomarker. In some embodiments, the kit may further include a first detection agent that binds to the first binder and a second detection agent that binds to the second binder. In some embodiments, the first binder and the second binder are immobilized on a support member.

[0034] 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 several embodiments, as well as from the appended claims.

[0035] The following drawings form a part of this specification and are included to further demonstrate specific aspects of the present disclosure. These drawings, when considered in conjunction with the detailed description of specific embodiments presented herein, can be better understood.

Brief Description of the Drawings

[0036]

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[0037] The contact activation system initiates the intrinsic pathway of coagulation and promotes inflammation through the release of bradykinin, an 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 coagulation and the kallikrein-kinin system. FXII is activated by negatively charged surfaces (e.g., polyanionic surfaces, glass, polyphosphate, ellagic acid), resulting in activated FXIIa. Activated FXIIa has the ability to cleave prekallikrein, generating activated pKal. Subsequently, activated pKal can cleave FXII to FXIIa, and FXIIa generates more pKal, forming a positive feedback loop that 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, elevated levels of bradykinin may induce vasodilation and inflammation that result in edematous HAE attacks. For example, to identify diseases mediated by the contact activation system, to identify that a subject has or is at risk of having such a disease, and in addition, to identify additional diseases that may benefit from treatment with a plasma kallikrein inhibitor (e.g., lanadelumab / TAKHZYRO®), it is desirable to identify novel biomarkers that can be used.

[0038] The present disclosure is at least partially based, in part, on the identification via proteasome profiling of proteins that are differentially present in biological samples obtained from subjects with HAE as compared to healthy individuals. A number of proteins have been found and observed to deviate in their levels from those of healthy (control) subjects in subjects with HAE. Interestingly, many proteins were unexpectedly observed to normalize (return to control levels) following treatment with lanadelumab, a pKal inhibitor. Groups of proteins predicted to belong to specific cellular pathways or processes (e.g., proteins involved in mitochondrial function, proteolysis, blood coagulation, etc.) and proteins belonging to protein families (e.g., interleukins) were found to have similar trends (e.g., increases or decreases in levels) in samples from diseased subjects as compared to healthy individuals.

[0039] Accordingly, provided herein are methods for determining whether a disorder is likely to respond to treatment with a plasma kallikrein (pKal) inhibitor and for identifying a subject as a candidate for treatment with a pKal inhibitor. Also provided herein are methods of analyzing a biological sample from a subject suspected of having, having a risk of having, or having a disorder in which one or more protein biomarkers deviate from control levels, based on detecting the presence of or measuring the levels of a protein biomarker or set of protein biomarkers. Such methods can be useful, for example, for identifying patients at risk of a disease, selecting candidates for treatment, monitoring disease progression or disease state, assessing the efficacy of treatment for a disease, determining a course of treatment, evaluating whether a subject is at risk of disease onset, and / or for research purposes such as studying the mechanism of a disease and / or the biological pathways and / or processes involved in a disease, which can serve as a basis for the development of new therapies.

[0040] Protein biomarker 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 proteins that have been found to be differentially present in samples from subjects with HAE and that returned to (normalized to) the levels of healthy subjects after treatment with lanadelumab, i.e., that are differentially present in samples from subjects at different stages of such a disease (e.g., at baseline compared to after treatment with lanadelumab). As used herein, the term "protein biomarker" or "protein biomarker set" means a protein or set of proteins that are present at different levels in samples from different groups of subjects, such as subjects with a disease versus healthy subjects (e.g., subjects without the disease), or subjects with a disease and in a quiescent phase versus subjects experiencing an acute phase of the disease. In some embodiments, the protein or set of proteins are present at different levels in samples from different groups of subjects, such as subjects with a disease (e.g., at baseline, i.e., prior to treatment) versus subjects to whom one or more treatments for the disease have been administered.

[0041] Such biomarkers / biomarker sets may be used for clinical and non-clinical applications (e.g., research purposes).

[0042] In some embodiments, a protein biomarker may be present at an elevated level in a sample from a subject having a disease as compared to the level of the same protein biomarker in a sample from a healthy subject. In some embodiments, a protein biomarker may be present at a decreased level in a sample from a subject having a disease as compared to the level of the protein biomarker in a sample from a healthy subject. In still other examples, a protein biomarker may be present at an elevated level in a sample obtained from a subject experiencing an acute phase of a disease described herein as compared to a subject during a quiescent state (baseline) of the disease. Alternatively, a protein biomarker may be present at a decreased level in a sample obtained from a subject experiencing an acute phase of a disease described herein as compared to a subject during a quiescent state (baseline) of the disease.

[0043] In some embodiments, a protein biomarker set containing one or more biomarkers can be analyzed by the methods described herein. If the protein biomarker set contains more than one biomarker, all of the biomarkers may be present at elevated or decreased levels in a subject having a disease as compared to a healthy subject. Alternatively, the protein biomarker set may contain at least one biomarker that increases in a subject having a disease as compared to a healthy subject and at least one biomarker that decreases in a subject having a disease as compared to a healthy subject.

[0044] Similarly, a protein biomarker set for evaluating whether a treatment is effective in treating a target disorder may include a plurality of biomarkers that all increase or decrease in a sample obtained at baseline (before treatment) compared to their levels in a sample obtained after treatment. Alternatively, the biomarker set may include at least one biomarker that increases in a sample obtained at baseline (before treatment) compared to its level in a sample obtained after treatment, and at least one biomarker that decreases in a sample obtained at baseline (before treatment) compared to its level in a sample obtained after treatment.

[0045] Table 1 below provides biomarkers that can be used to evaluate a subject or a biological sample from a subject by the methods described herein, for example, to identify and / or treat a subject having, suspected of having, or at risk of having a disorder, to determine whether the disorder is susceptible to treatment with a plasma kallikrein inhibitor, and / or to evaluate the efficacy of treatment of the disorder.

[0046] In some embodiments, a biomarker set 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 more) protein selected from Table 1. In some embodiments, when the biomarker set contains a single protein, that protein is not double-stranded high molecular weight kininogen (HMWK; KNG1), α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or interleukin-21 (IL-21). In some examples, a protein biomarker set measured and analyzed by the methods described herein does not include any combination of two or more of double-stranded high molecular weight kininogen (HMWK; KNG1), α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and / or interleukin-21 (IL-21).

[0047] As described in Example 1, several proteins involved in the kallikrein-kinin system were found to be differentially present in samples from subjects with pre-treatment HAE compared to healthy subjects and / or to be normalized (e.g., returned to levels corresponding to healthy subjects) in samples from subjects with post-treatment HAE compared to healthy subjects. In some embodiments, the biomarker set contains one or more kallikrein-kinin system proteins listed in Table 1. In some embodiments, the kallikrein-kinin system proteins of the biomarker set include kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-stranded high molecular weight kininogen (KNG1), or kininostatin, or combinations thereof.

[0048] As described in Example 1, several proteins involved in the blood coagulation pathway were found to be differentially present in samples from subjects with pre-treatment HAE compared to healthy subjects and / or to be normalized (e.g., returned to levels corresponding to healthy subjects) in samples from subjects with post-treatment HAE compared to healthy subjects. In some embodiments, the biomarker set contains one or more blood coagulation proteins listed in Table 1. In some embodiments, the blood coagulation proteins of the biomarker set include α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or plasma serine protease inhibitor (SERPINA5), or combinations thereof.

[0049] As described in Example 1, several proteins involved in cell adhesion were found to be differentially present in samples from subjects with pre-treatment HAE compared to healthy subjects and / or to be normalized (e.g., returned to levels corresponding to healthy subjects) in samples from subjects with post-treatment HAE compared to healthy subjects. In some embodiments, the biomarker set contains one or more proteins involved in cell adhesion listed in Table 1. In some embodiments, proteins involved in cell adhesion of the biomarker set include cadherin-1 (CDH-1), cadherin-15: cytoplasmic domain (CDH15), ephrin type-A receptor 2 (EPHA2), multimerin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), or protocadherin gamma-C3 (PCDHGC3), or combinations thereof.

[0050] In some embodiments, the biomarker set contains one or more proteins involved in cardiovascular function listed in Table 1. In some embodiments, a protein involved in cardiovascular function of the biomarker set includes apolipoprotein B (APOB).

[0051] As described in Example 1, several proteins involved in proteolysis, including proteases and protease inhibitors, were found to be differentially present in samples from subjects with pre-treatment HAE compared to healthy subjects and / or to be normalized (e.g., returned to levels corresponding to healthy subjects) in samples from subjects with post-treatment HAE compared to healthy subjects. In some embodiments, the biomarker set contains one or more proteins involved in proteolysis listed in Table 1. In some embodiments, proteins related to proteolysis in the biomarker set include proteasome subunit beta type-6 (PSMB6), ubiquitin-conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-conjugating factor E4 A (UBE4A), or E3 ubiquitin-protein ligase ZNRF3 (ZNRF3), or combinations thereof. In some embodiments, proteins related to proteolysis in the biomarker set include thrombin (F2), tissue kallikrein 14 (KLK14), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), or alpha-macroglobulin (A2M), or combinations thereof.

[0052] As described in Example 1, several proteins involved in complement activity were found to be differentially present in samples from subjects with pre-treatment HAE compared to healthy subjects and / or to be normalized (e.g., returned to levels corresponding to healthy subjects) in samples from subjects with post-treatment HAE compared to healthy subjects. In some embodiments, the biomarker set contains one or more proteins involved in complement activity listed in Table 1. In some embodiments, the complement activity proteins in the biomarker set are complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0053] In some embodiments, the biomarker set contains one or more proteins that are the cytokines listed in Table 1. In some embodiments, the cytokines of the biomarker set include interleukin-12 (IL-12A / IL-12B), interleukin 21 (IL-21) or interleukin 7 (IL-7), or combinations thereof.

[0054]

Table 1

[0055] Usefulness of Protein Biomarkers Aspects of the present disclosure relate to a method of analyzing a sample obtained from a subject (e.g., a human patient) having, suspected of having, or at risk of having a disease or disorder, such as a disease or disorder associated with a contact activation system, by measuring in the sample the levels of the biomarker sets described herein. In some aspects, the methods described herein relate to identifying a subject having or at risk of having a disorder associated with any of the proteins shown in Table 1 and, optionally, treating the subject with a plasma kallikrein inhibitor. Still other aspects of the present disclosure relate to determining whether a disorder is susceptible to treatment with a pKal inhibitor and evaluating the efficacy of treatment of the disorder in a subject. The results obtained from the methods described herein can be useful for diagnostic and / or prognostic purposes, as well as for other non-clinical purposes such as research purposes.

[0056] (i) Analysis of a biological sample The methods described herein involve providing a biological sample obtained from a subject. As used herein, "biological sample" means a composition containing tissue from a subject, such as blood, plasma, or protein. Samples include the initial untreated sample taken from the subject, as well as samples that have been subsequently processed, such as in a partially purified or preserved form. 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 types (e.g., at least 2, 3, 4, 5 or more) of biological samples may be taken from the subject over time or at specific time intervals, e.g., to evaluate disease progression or to evaluate the efficacy of treatment. In some embodiments, multiple types (e.g., at least 2, 3, 4, 5 or more) of biological samples may be taken from the subject, e.g., before treatment, during treatment, and / or after treatment, to evaluate disease progression or to evaluate the efficacy of treatment. In some embodiments, the biological sample is plasma.

[0057] 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 test tube (e.g., a vacuum blood collection tube). In some embodiments, the vacuum test tube contains one or more protease inhibitors to reduce or prevent ex vivo activation of the contact system during sample collection, for example. Such protease inhibitors may be contained in a liquid formulation. In some embodiments, the protease inhibitor comprises at least one serine protease inhibitor and at least one cysteine protease inhibitor. Such vacuum test tubes are known in the art. See, for example, PCT Application No. PCT / US2016 / 046681 (WO2017 / 027771), which is incorporated herein by reference in its entirety. Optionally, the vacuum blood collection tube may further contain one or more anticoagulants.

[0058] The terms "patient", "subject" or "individual" may be used interchangeably and refer to the subject described herein. In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is suspected of or at risk for a disease or disorder, such as a disease or disorder involving or mediated by any of the proteins in Table 1. In some embodiments, the subject is suspected of 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 above disease. Alternatively, or in addition, such a subject may have one or more risk factors for the above disease, such as a genetic factor associated with the disease (e.g., a gene defect in CI-INH).

[0059] Alternatively, the subject described herein may be a patient with the above disease. Such a subject may currently be in the acute phase of the above disease (e.g., showing an HAE attack), or may have suffered from the above disease in the past (e.g., currently in a quiescent state of the disease (baseline)). In some examples, the subject is a human patient who may be undergoing treatment for the above disease, such as treatment with a therapeutic agent targeting the kallikrein-kinin system (KKS), such as a C1-esterase inhibitor (C1-INH), a plasma kallikrein inhibitor, or a bradykinin inhibitor. In other cases, such a human patient may not have received such treatment.

[0060] The methods described herein relate to determining whether a disorder is susceptible to treatment with a pKal inhibitor. In such embodiments, the disease or disorder may not have previously been recognized as being associated with pKal activity and / or the contact activation system. Such methods may be used to identify additional diseases or disorders for which treatment with a pKal inhibitor may be effective. In some embodiments, the disease or disorder is related to or mediated by abnormal expression and / or activity of any one or more of the proteins shown in Table 1. In some embodiments, the disease or disorder is not associated with the contact activation system. In some embodiments, the disease or disorder is not hereditary angioedema.

[0061] In some embodiments, the disease or disorder is a disease associated with the contact activation system. Examples of diseases associated with the contact activation system include kallikrein-mediated disorders, such as bradykinin-mediated disorders like 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 diseases, 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, nervous system 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), as well as tissue damage (e.g., burns or chemical injuries), but are not limited thereto.

[0062] In some embodiments, the disease or disorder associated with the contact activation system is hereditary angioedema (HAE). In some embodiments, the methods described herein may be used to identify whether a subject has or is at risk of having HAE. Alternatively, or in addition, the methods described herein may be used to evaluate the efficacy of treatment and / or the progression of the disease. Alternatively, or in addition, the methods described herein may be used to evaluate whether a subject has or is at risk of having an HAE attack. In some embodiments, the methods described herein may be used to determine whether to administer an additional dose of one or more therapeutic agents, such as a pKal inhibitor, or to adjust the dosage of a therapeutic agent, e.g., by increasing or decreasing the dosage and / or frequency of HAE treatment, or to initiate or terminate treatment. 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 hands, feet, face, genitals, gastrointestinal tract, and airways. Symptoms of HAE can include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat, airway obstruction with swelling of the throat and sudden hoarseness, recurrent episodes of abdominal cramps without an apparent cause, and / or swelling of the intestines that can be severe and result in abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of individuals with HAE develop a non-itchy rash called angioedema marginatum during an attack.

[0063] Swelling of the airway can be life-threatening and can cause death in some patients. The mortality rate is estimated to be between 15% and 33%. HAE results in approximately 15,000 to 30,000 emergency department visits per year.

[0064] Trauma or stress, such as dental procedures, illnesses (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 may try to avoid certain stimuli that have previously caused attacks. However, often attacks occur without a known trigger. Typically, the symptoms of HAE first appear in childhood and worsen during puberty. On average, untreated individuals have attacks 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.

[0065] There are three types of HAE known as type I, type II, and type III. HAE is estimated to affect 1 in 50,000 people, with type I accounting for about 85% of cases, type II accounting for about 15% of cases, and type III being very rare. Type III is the most recently described form and was originally thought to occur only in women, but families with affected men have been identified.

[0066] Since HAE is inherited in an autosomal dominant pattern, an affected person may have inherited the mutation from one affected parent. New mutations can also occur in the gene, so HAE can occur in people with no family history of the disorder. 20-25% of cases are estimated to be due to new spontaneous mutations.

[0067] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene gives instructions for making the C1 inhibitor protein, which is important for controlling inflammation. The C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause hereditary angioedema type I result in reduced levels of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of an abnormally functioning C1 inhibitor. When the functioning C1 inhibitor is not at appropriate levels, excessive amounts of bradykinin are produced. Bradykinin promotes inflammation by increasing the leakage of body fluids from blood vessel walls into body tissues. Excessive accumulation of body fluids in tissues causes the swelling episodes seen in individuals with hereditary angioedema types I and II.

[0068] Mutations in the F12 gene are associated with some cases of hereditary angioedema type III. The F12 gene gives instructions for making coagulation factor XII. Factor XII plays an important role in blood clotting (coagulation) and 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 produced, making the blood vessel walls more leaky and leading to swelling episodes. The cause of other cases of hereditary angioedema type III remains unknown. Mutations in one or more yet-to-be-identified genes may be involved in the disorder in these cases.

[0069] HAE may appear similar to other forms of angioedema caused by allergies or other medical conditions, but the causes and treatments are quite different. If HAE is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine. Epinephrine may be used in life-threatening reactions, but these are typically not effective for HAE. Patients with abdominal swelling may undergo unnecessary diagnostic procedures due to misdiagnosis, and there are also HAE patients whose abdominal pain is misdiagnosed as psychosomatic.

[0070] C1 inhibitor therapy, and other therapies for HAE, are described in Kaplan, A.P., J Allergy Clin Immunol, 2010, 126(5):918-925.

[0071] The acute treatment of HAE attacks is carried out to stop the progression of edema as soon as possible. Intravenous administration of C1 inhibitor concentrate from donor blood is one acute treatment. 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 it also contains C1 inhibitor.

[0072] 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. Pasteurized Berinert P (CSL Behring) was approved by the FDA for acute attacks in 2009. Nanofiltrated CINRYZE® (Takeda) was approved by the FDA for prophylaxis in 2008. Ruconest (Pharming) is a recombinant C1 inhibitor under development that does not carry the risk of infection transmission by human blood-borne pathogens.

[0073] The treatment of acute HAE attacks may also include drug therapy and / or intravenous infusion for analgesia.

[0074] Other treatment modalities can stimulate the synthesis of C1 inhibitor or reduce the consumption of C1 inhibitor. Androgen drug therapy such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0075] Helicobacter pylori can be a trigger for abdominal attacks. Antibiotics that treat H. pylori are expected to reduce abdominal attacks.

[0076] Newer therapies attack the contact cascade. Ecallantide (KALBITOR®, Takeda) and lanadelumab (TAKHZYRO®, Takeda) inhibit plasma kallikrein and are approved in the United States and in the United States and Europe, respectively. Icatibant (FIRAZYR®, Takeda) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0077] The diagnosis of HAE can depend, for example, on family history and / or blood tests. 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 level of C1 inhibitor is decreased as is the level of C4, whereas the level of C1q is normal. In type II HAE, the level of C1 inhibitor is normal or increased, but the function of the C1 inhibitor is abnormal. The C4 level is decreased and the C1q level is normal. In type III, the levels of C1 inhibitor, C4, and C1q can 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. Measurement of the levels of a biomarker set of these proteins can be used to identify whether a subject has a disease such as HAE. In some embodiments, the method can be used to determine whether a patient has had or is having an HAE attack. In some embodiments, the method can be used to determine whether a treatment is or has been effective in treating HAE.

[0078] The symptoms of a disease or disorder such as HAE can be evaluated, for example, using a questionnaire (e.g., a questionnaire filled out by a patient, clinician, or family member). 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.

[0079] The biological samples described herein may be subjected to analysis by measuring the levels of the biomarker sets described herein in the biological samples. The levels (e.g., amounts) of the biomarkers disclosed herein, or changes in biomarker levels, can 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 is evaluated or measured indirectly in the biological sample, for example, by detecting the activity level of the protein (e.g., an enzyme assay).

[0080] 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 provided herein will be apparent to those skilled in the art. The present disclosure is not limited to immunoassays, and it will be apparent to those skilled in the art that detection assays not based on antibodies or antigen-binding antibody fragments, such as mass spectrometry, are also useful for the detection and / or quantification of the biomarkers provided herein. Assays that rely on chromogenic substrates may also be useful for the detection and / or quantification of the biomarkers provided herein.

[0081] The type of detection assay used for the detection and / or quantification of biomarkers, such as the biomarkers provided in this specification, will depend on several parameters, such as the specific situation in which the assay is utilized (e.g., clinical use or research use), as well as the type and number of biomarkers to be detected, and as well as the type and number of patient samples to be run in parallel.

[0082] 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 exemplary ELISAs are described herein. Kits for performing ELISA are known in the art and are commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0083] In some embodiments, an immunoassay is utilized to measure the level of protein biomarker(s). The immunoassay described herein may be in the form of a sandwich ELISA, in which a first binder that specifically binds to the protein of the biomarker set is immobilized on a support member. Subsequently, the support member can be incubated with the biological sample described herein for a suitable time under conditions that allow for the formation of a complex between the binder and the protein in the sample. Subsequently, such a complex can be detected using a protein, a binder-protein complex, or a detection agent that binds to the binder. The detection agent can be conjugated to a label that can directly or indirectly emit a signal. The intensity of the signal indicates the level of protein in the sample. In some embodiments, the detection agent is detected and the level of the detection agent indicates the level of protein in the sample.

[0084] In the methods and kits described herein, the level of a protein in a biological sample may be measured using any binding agent that specifically binds to a desired protein. In some embodiments, the binding agent is an antibody that specifically binds to the desired protein. In some embodiments, the binding agent is an aptamer that specifically binds to the desired protein. In some embodiments, the sample may be contacted with more than one binding agent that binds to different proteins, simultaneously or sequentially (e.g., multiplex analysis, such as the SOMASCAN® assay (SOMALogic)). The biological sample is contacted with the binding agent under suitable conditions. Generally, the term "contacting" means bringing the binding agent into contact with the protein in the sample, if present, for a suitable time sufficient to form a complex between the binding agent and the protein in the biological sample or agent. In some embodiments, contacting is effected by capillary action across the surface of a support membrane by the biological sample or agent.

[0085] In some embodiments, the immunoassay may be performed on a low-throughput platform, such as a single immunoassay format. For example, a low-throughput platform may be utilized to measure the presence and amount of proteins in a biological sample (e.g., biological tissue, tissue extract) as a diagnostic method for monitoring the progression of a disease and / or treatment and / or predicting whether a disease or disorder may benefit from a particular treatment.

[0086] In some embodiments, it may be necessary to immobilize the binding agent to a support member. The method for immobilizing the binding agent will depend on factors such as the nature of the binding agent and the material of the support member, and specific buffers may be required. Such methods will be apparent to those skilled in the art. For example, the biomarker set in a biological sample described herein may also be measured using any of the kits and / or detection devices described herein.

[0087] 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 the qualitative or quantitative concentration level of such a substance), or evaluating the value or classification of an object.

[0088] Assays, such as Western blot assays, may further involve the use of a quantitative imaging system, such as commercially available LI-COR imaging technology (e.g., see the CLx infrared imaging system manufactured by LI-COR Biosciences, Odyssey®). In some embodiments, an electrochemiluminescence detection assay, or an assay that relies on a combination of electrochemiluminescence and patterned array technology is used (e.g., ECL or MULTI-ARRAY technology assays manufactured by Meso Scale Discovery (MSD)).

[0089] In any of the methods described herein, the level of a protein in a biomarker set can be compared to the level of a protein in a control sample or a reference sample. Alternatively, or in addition, in any of the methods described herein, the level of a protein in a biomarker set can be compared to a reference level, a predetermined reference level, etc.

[0090] (ii) Clinical uses The level of a protein presented in Table 1 detected in a sample from a subject can be used as a biomarker for diagnosing a disease, such as a disease associated with a contact activation system (e.g., HAE), for monitoring the progression of a disease, for evaluating the efficacy of a disease treatment, for identifying a patient suitable for a particular treatment, or for identifying a subject as having or being at risk of having a disease or disorder.

[0091] Accordingly, described herein are methods for diagnosing and prognosing a disease or disorder, such as a disease or disorder associated with the expression of any of the proteins shown in Table 1, based on the levels of a biomarker set in a biological sample obtained from a subject. In some embodiments, the disease or disorder associated with the expression of any of the proteins shown in Table 1 may be treated with an agent such as lanadelumab. In some embodiments, the method is a method for diagnosing and prognosing a disease associated with the contact activation system based on the levels of a biomarker set in a biological sample obtained from a subject.

[0092] In some embodiments, the level of a biomarker 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. In some embodiments, the level of a biomarker 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 associated with the contact activation system, such as a disease associated with plasma kallikrein, e.g., HAE, or an autoimmune disease such as UC and Crohn's disease. In some embodiments, the method is a method for diagnosing and prognosing a disease that can be mediated by or associated with the kallikrein-kinin system (contact activation system), and thus a disease that can be treated with an agent (e.g., lanadelumab) that targets the kallikrein-kinin system.

[0093] In some embodiments, the level of the biomarker is subsequently compared to a reference sample or a control sample, and a value indicating the amount of protein in the sample can be determined. In some embodiments, the value of the biomarker is obtained by comparing the level of the protein in the sample to the level of another protein (e.g., an internal control or an internal standard) in the sample. Such a value of the biomarker may be a value normalized against an internal control or an internal standard. The value of the biomarker can be compared to a reference value to determine whether the subject has or is at risk of having a disease. The reference value may indicate the level of the corresponding biomarker in a subject (e.g., a human subject) without the target disease (e.g., a healthy subject). In some embodiments, if the level or value of the biomarker is higher than the reference level or value, the subject can be determined to have or be at risk of having a disease. In some embodiments, if the level or value of the biomarker is lower than the reference level or value, the subject can be determined to have or be at risk of having a disease.

[0094] In some embodiments, the level of the biomarker can be compared to a predetermined threshold of that protein, and deviation therefrom may indicate that the subject has a disease. The predetermined threshold may indicate a value of the biomarker that distinguishes the level of the biomarker in patients with the target disease from the level of the biomarker in patients without the target disease.

[0095] In some embodiments, the biomarker set contains more than one protein, and at least one elevated level of which indicates that the subject has or is at risk of having a disease, and at least one decreased level of the protein indicates that the subject has or is at risk of having a disease. In some embodiments, the biomarker set contains more than one protein, and each elevated level indicates that the subject has or is at risk of having a disease. In some embodiments, the biomarker set contains more than one protein, and each decreased level indicates that the subject has or is at risk of having a disease.

[0096] 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 serving as a control.

[0097] As used herein, a subject serving as a control may be a healthy individual, i.e., an individual who does not have an obvious target disease (e.g., a disease associated with an exposure system) when the level of the protein(s) is measured, or an individual without a medical history of the disease. The subject serving as a control may preferably represent a population of healthy subjects having characteristics (e.g., age, gender, ethnic group) that match those of the subject to be analyzed by the method described herein.

[0098] The control level may be a predetermined level or threshold. Such a predetermined level can represent the level of the protein in a population of subjects without the target disease or at no risk (e.g., the average level in a population of healthy subjects). The predetermined level may also indicate the level of the protein in a population of subjects having the target disease.

[0099] The predefined level can take various forms. For example, the predefined level can be a single cutoff value such as a median or an average. In some embodiments, such a predefined level can be established based on a comparison group where, for example, one defined group is known to have the target disease and the other defined group is known not to have the target disease. Alternatively, the predefined level can be a range, for example, a range indicating the protein level in a control population.

[0100] The control levels 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 described herein for obtaining the protein level of a test sample) on a control sample also described herein. In other examples, the protein level can be obtained from members of a control population and the results analyzed, for example, by a computational program, to obtain a control level (predefined level) indicating the protein level in the control population.

[0101] By comparing the level of a biomarker in a sample obtained from a subject who is a candidate with the reference values described herein, it can be determined whether the subject who is a candidate has a disease or is at risk. For example, if the level of the biomarker(s) in the sample of a subject who is a candidate deviates from the reference value (e.g., is higher or lower compared to the reference value), the subject who is a candidate may be identified as having a disease or being at risk. If the reference value indicates a range of values of the biomarker level 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 subject who is a candidate has the target disease or is at risk.

[0102] As used herein, "elevated level" or "level above the reference value" means a biomarker level that is higher than a reference value, such as a predetermined threshold of the biomarker level in a control sample. The control level is described in detail herein. Examples of 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%, 500% or more. In some embodiments, the level of the biomarker in the 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, 5.5-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, 10000-fold or more higher than the level of the biomarker in the reference sample.

[0103] As used herein, "decreased level" or "level below the reference value" means a biomarker level that is lower than a reference value, such as a predetermined threshold of the biomarker level in a control sample. The control level is described in detail herein. Examples of 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%, 500% or more. In some embodiments, the level of the biomarker in the 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, 5.5-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, 10000-fold or more lower than the level of the biomarker in the reference sample.

[0104] In some embodiments, the methods described herein are for identifying a subject as a candidate for a particular treatment, such as treatment with a pKal inhibitor. In some embodiments, the subject who is a candidate is a human patient having disease symptoms of a disease associated with a contact activation system, such as a pKal-mediated disorder, such as HAE, or an autoimmune disease such as RA, UC, and Crohn's disease. For example, the subject has edema; swelling that is completely peripheral or primarily peripheral; urticaria; erythema, pain, and swelling in the absence of evidence of infection; non-histamine-mediated edema; recurrent episodes 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.

[0105] A subject identified by the methods described herein may be administered a suitable treatment, such as treatment with a pKal inhibitor, as described herein.

[0106] 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 the evaluation of the efficacy of treatment with a pKal inhibitor, such as the treatment described herein. For example, a plurality of biological samples (e.g., blood samples or plasma samples) can be taken from a subject to be treated before and after treatment, or during a course of treatment. The level of a biomarker can be measured by any of the assay methods described herein, and accordingly, the value (e.g., amount) of the biomarker can be determined. For example, if an elevated level of a biomarker indicates that the subject has a target disease and the level of the biomarker decreases after treatment or throughout a course of treatment (the level of the biomarker in a sample taken later compared to the level of the biomarker in a sample taken earlier), this indicates that the treatment is effective. As another example, if a decreased level of a biomarker indicates that the subject has a target disease and the level of the biomarker increases after treatment or throughout a course of treatment (the level of the biomarker in a sample taken later compared to the level of the biomarker in a sample taken earlier), this indicates that the treatment is effective. Also, a treatment may be determined to be effective if the level of a biomarker or biomarker set (e.g., a biomarker or biomarker set indicative of a disease) in a subject normalizes (e.g., does not deviate from the level of the same biomarker or biomarker set in a control subject, e.g., a healthy subject, or a subject having the disease in a quiescent state) during or after a course of treatment. In some embodiments, the level of a biomarker or biomarker set does not substantially deviate from the level of the same biomarker or biomarker set in a control subject if there is no statistically significant difference between the level of the biomarker or biomarker set and the level in the control subject. As an example, an elevated biomarker indicative of a disease before treatment may decrease to a level similar to (substantially not deviating from) the level of the biomarker in a subject without the disease, in which case the treatment is determined to be effective.In another example, a decreased biomarker indicative of a pre-treatment disease may increase to a level similar to (not substantially deviating from) the level of the biomarker in a subject without the disease, in which case the treatment is determined to be effective. In some examples, the treatment involves administration of one or more plasma kallikrein inhibitors such as lanadelumab.

[0107] If a subject is identified as not responding to treatment or if the treatment is determined to be ineffective, the therapeutic agent (e.g., a pKal inhibitor such as lanadelumab) is administered to the identified subject at a higher dose and / or more frequent dosing of the therapeutic agent. In some embodiments, the dose or dosing frequency of the therapeutic agent (e.g., a pKal inhibitor such as lanadelumab) is maintained, reduced, or discontinued in a subject identified as responding to treatment or not requiring further treatment. Alternatively, a different treatment can be applied to a subject found not to respond to the initial treatment.

[0108] In other embodiments, the value of a biomarker or biomarker set can also be used to identify whether a disorder is susceptible to treatment with a pKal inhibitor (is treatable). To practice 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 can be measured by a suitable method, such as a method described herein, e.g., Western blot or ELISA. If the level of the biomarker deviates from a reference value (e.g., is high or low), this indicates that a pKal inhibitor may be effective in treating the disease. If the disease is identified as being susceptible to the action of a pKal inhibitor (and thereby treatable), this method can further comprise administering to the subject having the disease an effective amount of a pKal inhibitor such as an anti-pKal antibody (e.g., lanadelumab) or an inhibitory peptide (e.g., ecallantide); a bradykinin 2 receptor inhibitor (e.g., icatibant); and / or C1-INH (e.g., human plasma-derived C1-INH).

[0109] (iii) Non-clinical uses Furthermore, any level of the biomarker sets described herein may be used for research purposes. A number of diseases associated with the contact activation system have been identified, but there is a possibility that other diseases are mediated by similar mechanisms or involve similar components, such as proteins whose levels are affected by ranadelumab treatment. In some embodiments, the methods described herein may be used to identify that a disease is associated with the contact activation system or has a component of the contact activation system (e.g., pKal activity). In some embodiments, the methods described herein may be used to study the mechanism of a disease (e.g., discovery of a new biological pathway or process involved in the development of a disease) or the progression of a disease.

[0110] In some embodiments, the levels of the biomarker sets described herein may be used in the development of new therapies for diseases. For example, the levels of the biomarker set may be measured in a sample obtained from a subject being administered a new therapy (e.g., a clinical trial). In some embodiments, the levels of the biomarker set may indicate the efficacy of a new therapy or the progression of a disease in a subject before, during, or after a new treatment.

[0111] Treatment methods Also within the scope of the present disclosure are methods of identifying that a subject has or is at risk of having a disorder, methods of identifying a subject as a candidate for treatment, and methods of treating a subject and the subject's disorder. A subject at risk of having or suffering from a disorder, such as a disorder mediated by or associated with any of the biomarkers in Table 1, may be identified using the methods described herein and further treated with any suitable therapeutic agent. In some embodiments, the methods provided include treatment selection for a subject based on the output of the methods described, e.g., the measurement of the levels of a biomarker set.

[0112] In some embodiments, the method includes either selecting, or administering, or both, a therapeutic agent, such as a plasma kallikrein (pKal) inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1 esterase inhibitor, for administration to a subject based on an output of an assay, such as biomarker detection / level.

[0113] In some embodiments, the therapeutic agent is administered to the subject one or more times. In some embodiments, the plasma kallikrein inhibitor is administered to the subject one or more times. In some embodiments, the pKal 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, a C1 esterase inhibitor (C1-INH) is administered to the subject.

[0114] The therapeutic agent, such as a pKal inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1-INH, may be administered together with another therapy as part of a combination therapy for the treatment of a disease or condition involving the contact activation system. The combination therapy, such as a combination therapy with one or more of a pKal inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH replacement agent, such as a combination therapy with one or more of a pKal 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 agent may be administered before or after the administration of the other therapy. In some cases, the first agent and another therapy (e.g., a therapeutic agent) are administered simultaneously or in close temporal proximity (e.g., with a short injection interval such as during the same treatment session). The first agent and the other therapy may be administered at a longer time interval.

[0115] Therapeutic agent Plasma kallikrein binders (such as binding proteins, such as polypeptides, such as inhibitory polypeptides, such as antibodies, such as inhibitory antibodies, or other binders, such as small molecules) are useful therapeutic agents for various disorders and conditions, such as diseases and conditions associated with the expression of the biomarkers in Table 1. In some embodiments, the disease or disorder is associated with the contact activation system, such as involvement of pKal activity. In some embodiments, the disease or condition in which plasma kallikrein activity is involved is hereditary angioedema (HAE). In some embodiments, the disease or condition is not hereditary angioedema. In some embodiments, the plasma kallikrein inhibitor is administered to a subject at risk of, or suffering from, a disease associated with the contact activation system.

[0116] A number of useful protein inhibitors of kallikrein, which are 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 the 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 general 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.

[0117] 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. At least 81 knotted domain sequences 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 US Patent 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).

[0118]

Table 2

[0119] Examples of proteins containing knotted domains include the following (the numbers in parentheses are SWISS-PROT accession numbers):

Chemical formula

[0120] The knotted domain interacts with the target protease mainly using the amino acids within two loop regions ("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 a knotted domain that interacts with kallikrein or selecting a variant 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).

[0121] The "framework region" of the knotted domain is defined as the residues that are part of the knotted domain, but specifically 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) are excluded. Conversely, residues not within the binding loops may tolerate a wider range of amino acid substitutions (e.g., conservative substitutions and / or non-conservative substitutions).

[0122] In one embodiment, these knitted domains are variant forms of a loop structure that includes Knitted Domain 1 of human lipoprotein-associated coagulation inhibitor (LACI). LACI contains three clearly defined internal peptide loop structures that are paradigmatic knitted domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of LACI's Knitted Domain 1 described herein have been screened and isolated, and these bind to kallikrein with improved affinity and specificity (see, e.g., U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to the frameworks of other knitted domains to obtain other knitted domains that interact with kallikrein, such as 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.

[0123] In some embodiments, the plasma kallikrein inhibitor binds to the active form of plasma kallikrein. In some forms, the plasma kallikrein inhibitor binds to, for example, human plasma kallikrein and / or mouse kallikrein and inhibits it. Exemplary polypeptide plasma kallikrein inhibitors are described 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, 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 plasma kallikrein inhibitor comprises, or consists of, a sequence of about 58 amino acids from 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.

[0124]

Chemical formula

[0125] 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, F(ab’)2, or scFv fragment). The plasma kallikrein-binding antibody may contain two heavy chains and two light chains, 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 an antibody produced in vitro, and may optionally contain a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0126] Exemplary plasma kallikrein-binding proteins are described in U.S. Patent Application Publication No. 2012 / 0201756, the entire contents of which are incorporated herein by reference. In some embodiments, the plasma 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 lanadelumab), 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 lanadelumab. Reference is made to U.S. Patent Publication No. 2011 / 0200611 and U.S. Patent Publication No. 2012 / 0201756, which are incorporated herein by reference.

[0127] An example of a plasma kallikrein inhibitory antibody is lanadelumab (TAKHZYRO®, which may also be referred to as TAK-743, SHP943, or DX-2930). The amino acid sequences of the heavy chain variable region and the light chain variable region of lanadelumab are provided below, with the CDR regions identified in bold and underlined.

[0128] Sequence of the heavy chain variable region of ranibizumab (SEQ ID NO: 4)

Chemical formula

Chemical formula

[0129] Ranibizumab heavy chain amino acid sequence (451 amino acids, 49439.02 Da)

Chemical formula

Chemical formula

Table 3

[0130] In some embodiments, the plasma kallikrein inhibitor can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the plasma kallikrein inhibitor described herein. In some embodiments, the plasma kallikrein inhibitor can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC framework regions (e.g., HC and / or LC FR1, 2, 3 and / or 4) to the plasma kallikrein inhibitor described herein. In some embodiments, the plasma kallikrein inhibitor can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC CDRs (e.g., HC and / or LC CDR1, 2, and / or 3) to the plasma kallikrein inhibitor described herein. In some embodiments, the plasma kallikrein inhibitor can have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the constant regions (e.g., CH1, CH2, CH3 and / or CL1) to the plasma kallikrein inhibitor described herein.

[0131] In some aspects, the small molecule binds to and inhibits the active form of plasma kallikrein.

[0132] 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®), which is a peptidomimetic containing 10 amino acids that blocks the binding of native bradykinin to the bradykinin B2 receptor.

[0133] C1-INH supplement 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 commercially available and include, for example, C1-INH derived from human plasma, such as BERINERT® and CINRYZE®.

[0134] Kit and detection device for measuring a protein biomarker set The present disclosure also provides a kit and a detection device for use in measuring the levels of the biomarker sets described herein. Such a kit or detection device may include a binding agent that specifically binds to a protein biomarker, such as those listed in Table 1. For example, such a kit or detection device may include at least two binding agents that are specific for two different protein biomarkers selected from Table 1. In some cases, the kit or detection device includes a binding agent that is specific for all members of the protein biomarker set described herein.

[0135] In some embodiments, one or more of the binding agents are antibodies that specifically bind to the proteins of the biomarker set. In some embodiments, one or more of the binding agents are aptamers, such as peptide aptamers or oligonucleotide aptamers, that specifically bind to the proteins of the biomarker set.

[0136] In some embodiments, the kit further includes a detection agent (e.g., an antibody that binds to the binding agent) for detecting the binding of the binding agent to the protein(s) of the biomarker set. The detection agent can be conjugated to a label. In some embodiments, the detection agent is an antibody that specifically binds to at least one of the binding agents. In some embodiments, the binding agent includes a tag that can be identified by the detection agent and to which the detection agent can bind directly or indirectly.

[0137] In some embodiments, the kit may further comprise a support member for performing any of the methods described herein, for example. 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 form of a Western blot assay or a lateral flow assay.

[0138] In some embodiments, the support member is a multi-well plate, such as an ELISA plate. In some embodiments, the immunoassays described herein can be performed on a high-throughput platform. In some embodiments, multi-well plates, such as 24-well, 48-well, 96-well, 384-well or greater plates, may be used for high-throughput immunoassays. Individual immunoassays can be performed simultaneously in each well. Thus, in order to increase assay throughput, it is generally desirable to use a plate reader to measure multiple wells simultaneously. In some embodiments, a plate reader capable of simultaneously imaging multiple wells (e.g., 4 wells, 16 wells, 24 wells, 48 wells, 96 wells, 384 wells or greater) can 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 a high light collection efficiency optical system 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).

[0139] In a kit or detection device, one or more of the binding agents may be immobilized on a support member, such as a membrane, beads, slide or multi-well plate. The selection of a suitable 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.

[0140] The kit can include one or more buffering agents described herein, such as, but not limited to, a coating buffering agent, a blocking buffering agent, a washing buffering agent, and / or a stopping buffering agent.

[0141] In some embodiments, the kit can include instructions for use for use according to any of the methods described herein. The included instructions can include a description of how to use the components included in the kit to measure the levels of proteins in a biomarker set in a biological sample taken from a subject such as a human patient.

[0142] Instructions for use of the kit generally include information about the amounts of each component and suitable conditions for performing the assay methods described herein. The components of the kit can be in unit doses, bulk packages (e.g., multi-dose packages), or sub-unit doses. The instructions supplied within the kits of the present disclosure are typically instructions by a document on a label or insert (e.g., a paper sheet included within the kit), although machine-readable instructions (e.g., instructions distributed on a magnetic or optical storage disk) are also acceptable.

[0143] The label or insert indicates that the kit is used to evaluate the levels of proteins in a biomarker set. The instructions for use can be provided to perform any of the methods described herein.

[0144] The kits of the present disclosure are contained in suitable packaging. Suitable packagings include, but are not limited to, vials, bottles, flasks, flexible packagings (e.g., sealed mylar or plastic bags), etc. Also contemplated are packages for use in combination with certain devices such as inhalers, nasal delivery devices (e.g., atomizers) or infusion devices such as minipumps. The kit may have a sterile access port (e.g., the container may be a vial having a stopper pierceable by an intravenous infusion bag or a hypodermic needle). The container may also have a sterile access port (e.g., the container may be a vial having a stopper pierceable by an intravenous infusion bag or a hypodermic needle).

[0145] The kit may optionally provide additional components, such as control samples and / or information for determination, such as standard samples or reference samples. Usually, the kit includes a container and a label or an attached document(s) on or associated with the container. In some embodiments, the present disclosure provides a product comprising the contents of the above-described kit.

[0146] Even without further details, those skilled in the art will be able to make the most of the present disclosure based on the above description. Accordingly, the following specific embodiments should be construed merely illustratively and should in no way limit the rest of the present disclosure. All publications cited herein are incorporated by reference for the purposes or subject matter mentioned herein.

Examples

[0147] Example 1: Identification of proteins that are differentially present in samples from HAE patients compared to healthy individuals In hereditary angioedema (HAE) with C1-esterase inhibitor (C1-INH) pathophysiology (HAE-C1-INH), despite the clinical demonstration of the role of the kallikrein-kinin system (KKS), no new disease state biomarkers useful for diagnosing and evaluating HAE patients have been identified. Such biomarkers may be useful for further elucidating the disease ecology of HAE and other plasma kallikrein (pKal)-mediated diseases, for developing improved diagnostic assays, and in addition for identifying further indications beyond HAE that may be susceptible to treatment with established therapies such as lanadelumab. To identify previously unrecognized HAE biomarkers, the plasma proteome of HAE subjects was analyzed before (baseline) and 6 months (26 weeks) after lanadelumab treatment and compared to analyte levels present in plasma samples from healthy control subjects (subjects without HAE).

[0148] Plasma samples were collected from the subjects and positive diagnosis of HAE in the patients was performed by measuring plasma levels of complement component 4 (C4) and functional C1-INH via ELISA and chromogenic assays, respectively, known in the art. Plasma proteasome analysis was performed by comparing the mean levels measured in plasma samples obtained from healthy control subjects (n = 30), plasma samples obtained from HAE patients before lanadelumab treatment (HAE C1-INH) (baseline, n = 125), and plasma samples obtained from HAE patients after lanadelumab treatment receiving 300 mg of lanadelumab every 2 weeks (26 weeks, post-treatment, n = 114) using a multiplex analysis technique that can evaluate the relative levels of 7,113 proteins using DNA aptamers (SOMALogic, Denver, CO, USA) specific for each protein being evaluated. Of the 114 HAE-C1-INH subjects for whom baseline / pre-dose plasma was available, post-dose samples that matched were available for 112 subjects.

[0149] Plasma collection Citrate-blood plasma samples from healthy controls were age- and sex-matched (n = 30; BioIVT, Westbury, NY, USA). Citrate-blood plasma samples from HAE-C1-INH subjects (n = 125) before lanadelumab treatment were collected as part of the Phase III trial (HELP trial) and also as non-rollover from the open-label extension (open-label extension portion of the HELP trial) (National Institutes of Health identifier numbers NCT02586805 and NCT02741596, respectively). Citrate-blood plasma samples from HAE-C1-INH subjects after 26 weeks of lanadelumab treatment (lanadelumab 300 mg administered every 2 weeks; n = 114) were collected, and pre-treatment and post-treatment samples that were patient-matched were available for 112 subjects. Plasma (sodium citrate) from age- and sex-matched healthy controls (n = 30) was obtained from BioIVT (Westbury, NY). To minimize ex vivo activation of the contact pathway system during blood collection, venipuncture was performed using a winged needle / catheter kit (BD Biosciences) on subjects with HAE-C1-INH and healthy control subjects, and plasma was collected by removing the tourniquet from the blood flow to reduce stasis. The blood placed in the first tube was discarded, and blood was collected in polypropylene vacuum tubes (BD Biosciences, San Jose, CA, USA) containing 3.8% sodium citrate. Blood samples were centrifuged within 1 hour, plasma was aliquoted, and stored at -80°C until processing.

[0150] ELISA For validation of differentially expressed HAE biomarkers, protein levels were evaluated by ELISA. A2M in plasma samples was measured using a commercially available ELISA kit (Raybiotech, catalog number ELH-A2M). APOB in plasma samples was measured using a commercially available ELISA kit (Raybiotech, catalog number ELH-ApoB). IL21 in plasma samples was measured using a commercially available ELISA kit (Raybiotech, catalog number ELH-IL21).

[0151] Results Of the 7,113 proteins evaluated, 1,041 proteins were determined to be differentially present at statistically significant levels in samples obtained from baseline HAE patients compared to healthy controls (Figure 1). As a validation of this approach, various biomarkers already known to be differentially present in HAE were identified in this set. For example, C1-INH and C4 are biomarkers utilized in the laboratory diagnosis of HAE-C1-INH, respectively (Grumach et al. (2021) Front Immunol, 12:785736), and were found to be decreased in HAE baseline samples (Figure 2 and Figure 3, respectively). Cleaved high molecular weight kininogen (cHMWK), which is known to be increased in HAE-C1-INH and decreased after lanadelumab treatment, was also observed during the analysis (Figure 4A and Figure 4B). See, for example, Suffritti et al. (2014) Clin Exp Allergy, 44:1503-14; Hofman et al. (2017) J Allergy Clin Immunol. 140:1700-1703; and Banerji et al. (2017) N Engl J Med., 376:717-728. Several additional proteins that have already been reported to be differentially present during the HAE-C1-INH disease state, such as thrombin, ITH4, IL-36A, and IL-21, were also observed during the analysis (see Figures 5-8). See, for example, van Geffen et al. (2012) Clin Exper Immunol, 167:472-478; Larrauri et al. (2020) Mol Immunol, 119:27-34; Sexton et al. (2017) Allergy, 0207; and Arcoleo et al. (2018) Clin Exp Med, 18:355-361.

[0152] Statistical analyses were performed to identify proteins that differed between samples from HAE-C1-INH patients compared to healthy subjects, and similarly proteins that did not differ (i.e., returned to control levels) after lanadelumab treatment. As shown in Table 1, levels of 120 proteins were further found to be significantly different in baseline HAE-C1-INH plasma compared to healthy controls, but there were no significant differences (normalized) after 26 weeks of treatment with lanadelumab. These proteins included several biomarkers previously known to be significantly different in HAE-C1-INH, namely cleaved HMWK (cHMWK; Figure 4A), thrombin (F2; Figure 5), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4; Figure 6), IL-21 (Figure 8), and similarly a number of proteins that have not previously been recognized to be differentially present in HAE-C1-INH and / or after treatment with plasma kallikrein inhibitors such as lanadelumab. Additional proteins identified included alpha-macroglobulin (A2M; Figure 10A), kininogen (KNG1; Figures 9B, 10D, and 13A - 13H), complement component C3 (C3; Figures 14A - 14D), and plasminogen (PLG; data not shown).

[0153] As an example of the method by which these 120 proteins were identified, there are DNA aptamers (ID15343-337 and ID19631-13; Table 1; FIGS. 13A-13D) that specifically bind to cHMWK. These indicate that cHMWK levels were elevated in HAE-C1-INH baseline plasma but there was no difference from healthy control plasma levels after HAE-C1-INH subjects received 26 weeks of lanadelumab treatment. DNA aptamers for which no information regarding other specificities was provided by SOMALogic were elevated against untreated HMWK and showed similar relative expression levels in plasma samples from healthy controls, baseline HAE-C1-INH patients, and HAE-C1-INH patients after 26 weeks of lanadelumab treatment (ID4918-21; Table 1; FIGS. 13E-13F). A second selective DNA aptamer for untreated HMWK showed binding to cHMWK and low molecular weight kininogen with at least 10-fold lower affinity compared to untreated HMWK, which showed decreased expression levels compared to healthy controls in HAE-C1-INH baseline plasma and increased expression levels in HAE-C1-INH subjects who received 26 weeks of lanadelumab treatment (ID7784-1; Table 1; FIGS. 13G-13H).

[0154] Complement C3 (C3) is another example identified by proteomics using HELP plasma samples among the list of potential biomarkers for the 120 proteins. Elevated expression levels of C3 and C3 fragments were observed in baseline plasma samples from HAE-C1-INH subjects compared to healthy controls (FIGS. 14A-14D). The expression level of C3b (ID4480-59; Table 1; FIG. 14B) was elevated in baseline plasma samples from HAE-C1-INH subjects and decreased after they received 300 mg of lanadelumab every 4 weeks for 26 weeks.

[0155] These proteins also include many biomarkers that have not previously been recognized as being significantly different in HAE-C1-INH or normalizing to levels equivalent to those of healthy subjects after treatment with a plasma kallikrein inhibitor such as lanadelumab. Examples of such proteins include apolipoprotein B (APOB; Figure 9A), G antigen 2 (GAGE2B; Figure 9C), mortality factor 4-like protein 2 (MORF4L2; Figure 9D), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9; Figure 9E), plasma serine protease inhibitor (SERPINA5; Figure 9F), and cadherin-15: cytoplasmic domain (CDH15; Figure 9G), alpha-2-macroglobulin (A2M; Figure 10A), interleukin-12 (IL12A-IL12B; Figure 10B), and liver-expressed antimicrobial peptide 2 (LEAP2; Figure 10C) (Figure 9A-9G, Figure 10A-10C; Table 1). Table 1 also provides the gene names, UniProt identification numbers, and the identification numbers of the SomaScan® aptamers used for the expression level analysis of each protein.

[0156] To determine whether these 120 proteins represented components of pathways functionally related to the HAE pathology, pathway enrichment analysis was performed using the Metabase process network (bio.tools / metabase; Bolser et al., (2012) Nucleic Acids Res, 40:D1250-D1254), and the Known Knowledge Network approach called CASnet (an ever-active subnetwork of known protein-protein interactions). Proteins functionally involved in the kallikrein-kinin system were among the most significantly enriched. However, pathways of proteins involved in coagulation, cell adhesion, cytokines, cardiovascular proteins and proteolysis (including proteases and protease inhibitors) were found to be significantly enriched, suggesting that these pathways were dysregulated in HAE-C1-INH and normalized after lanadelumab treatment. The summarized results of the Metabase pathway enrichment analysis are provided in Tables 3 and 5 and Figures 11 and 12.

[0157] CASnet incorporates the directionality of protein level changes, where red proteins increase and blue proteins decrease when the HAE baseline is compared to healthy control plasma (Figure 12). The proteins shown were added to the active subnetworks via known pathway associations. Proteins identified as potential disease state biomarkers affected by 26 weeks of lanadelumab treatment by CASnet analysis include proteases and protease inhibitors (e.g., thrombin, tissue kallikrein 14, inter-alpha-trypsin inhibitor heavy chain 4, α2-macroglobulin), apolipoproteins, and complement system proteins.

[0158] [Table 4]

[0159] [Table 5]

[0160] Proteasome analysis identified 120 proteins that were present at different levels between samples from patients with HAE and healthy individuals (controls), but normalized after 6 months (26 weeks) of lanadelumab treatment.

[0161] For the three proteins (A2M, APOB, and IL21) identified herein, commercially available ELISA kits (Raybiotech, catalog number ELH-A2M, Raybiotech, catalog number ELH-IL21, and Raybiotech, catalog number ELH-ApoB) were used as orthogonal assays to compare levels in different groups of healthy controls (n = 50), HAE-C1-INH controls from the HELP trial (NCT02586805) baseline (before receiving lanadelumab), and those receiving 150 mg or 300 mg of lanadelumab once monthly, 98 days after the first dose (Figures 15A–15D).

[0162] The analysis results are shown in Figure 15A (A2M), Figure 15B (APOB), and Figure 15C (IL21). The plasma concentration of A2M in HAE-C1-INH subjects was significantly different from that in healthy controls (Kruskal-Wallis test, p < 0.05), but in contrast to the SomaScan measurement of A2M, there was no difference between day 0 and day 98 after lanadelumab treatment. Also, in contrast to the SomaScan measurement, the concentrations of APOB and IL21 measured by ELISA were not significantly different from those in healthy controls, or between day 0 and day 98 for each dose of lanadelumab.

[0163] To determine whether the observed increase in A2M is due to an increase in either free A2M protein or its complex with protease, further studies may reveal whether Somamers against A2M also bind to the A2M-pKa covalent complex. A significant increase in plasma A2M concentration was observed in plasma before dosing of HAE-C1-INH compared to that in healthy control plasma. However, according to ELISA, the concentration of A2M did not decrease even with ranalizumab treatment in plasma before dosing of HAE-C1-INH. In ELISA, the lack of observed decrease in A2M plasma levels due to ranalizumab treatment is due to several considerations: 1) the results of different immunoassays may not be consistent with each other, so the difference between the ELISA result and the SomaScan result does not clearly indicate which is superior; 2) differences in assay characteristics (e.g., target immobilization and washing conditions) can affect the comparison between platforms; 3) assay specificity for different forms of the target (e.g., free A2M or protease-A2M covalent complex); 4) SomaScan analysis was performed on plasma from subjects who received higher doses of ranalizumab (300 mg every two weeks) for a longer period (6 months) compared to the plasma samples used in ELISA analysis (300 mg or 150 mg every four weeks for 98 days), which may suggest that a higher dose and longer exposure to ranalizumab are necessary to decrease A2M. The same plasma samples used to measure A2M by ELISA were also used for the measurement of IL21 and APOB. However, elevated plasma levels from HAE-C1-INH subjects were not observed in ELISA, which may have been due to the reasons described above for A2M.

[0164] Any of the proteins identified herein may be used as a biomarker (individually or in combination as a biomarker set) for research purposes, for example, in methods for identifying patients at risk of a disease associated with the contact activation system (e.g., HAE), such as in methods for identifying patients at risk of a disease associated with the contact activation system, selecting candidates for treatment, monitoring disease progression or disease state, evaluating the efficacy of treatment for a disease, determining a treatment course, and determining whether a disease or disorder is associated with the contact activation system, and / or for example, for research into the mechanisms of diseases that can serve as a basis for the development of new therapies.

[0165] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature that serves the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a general series of equivalent or similar features.

[0166] From the above description, those skilled in the art can readily identify the essential features of the present disclosure and make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from its spirit and scope. Accordingly, other embodiments are also within the scope of the claims.

[0167] Equivalents and scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. The scope of the disclosure is not intended to be limited to the above description, but rather is as set forth in the appended claims.

[0168] In the claims, articles such as "a", "an", and "the" may mean one or more than one unless the contrary is indicated or is otherwise clear from the context. A claim or description that includes "or" between one or more members of a group is considered satisfied if, unless otherwise indicated to the contrary or otherwise clear from the context, one, more than one, or all of the members of the group are present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which exactly one element of a group is present in, used in, or otherwise related to a given product or process. The present disclosure includes embodiments in which more than one or all of the group elements present, are used in, or are otherwise related to a given product or process.

[0169] Furthermore, the present disclosure encompasses all variations, combinations, and permutations in which one or more limitations, elements, clauses, descriptive terms, etc. from one or more of the recited claims are introduced into another claim. For example, any claim that depends on another claim can be modified to include one or more limitations found in any other claim that depends on the same base claim. If elements are presented as a list, for example, in Markush group form, each subgroup of the elements is also disclosed and any element(s) can be removed from this group. Generally, when the present disclosure or an aspect of the present disclosure is referred to as including a particular element and / or feature, a particular embodiment of the present disclosure or an aspect of the present disclosure should be understood to consist of or consist essentially of such element and / or feature. For the sake of brevity, those embodiments are not specifically described herein by those words. Also, note that the terms "comprising" and "containing" are intended to be unrestricted and allow the inclusion of additional elements or steps. When a range is given, the endpoints are included. Furthermore, unless otherwise indicated or otherwise apparent from the context and the understanding of one of ordinary skill in the art, values expressed as a range can take on any specific value or sub-range within the scope of the different embodiments of the present disclosure, down to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0170] This application references various issued patents, published patent applications, academic papers, and other publications, all of which are hereby incorporated by reference into this specification. In the event of any conflict between any of the incorporated references and this specification, this specification shall control. Moreover, any particular embodiment of the present disclosure within the prior art scope can be clearly excluded from one or more of the claims. Such embodiments are considered to be known to one of ordinary skill in the art and can be excluded even if the exclusion is not explicitly described herein. Any particular embodiment of the present disclosure can be excluded from any claim for any reason, regardless of the existence of the prior art.

[0171] One of ordinary skill in the art will recognize many equivalents to the specific embodiments described herein or will be able to ascertain such equivalents using only routine experimental methods. 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. One of ordinary skill 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 disclosure as defined by the following claims.

Claims

1. A method comprising the step of measuring the level of a biomarker set containing at least one protein selected from Table 1 in a biological sample, (a) The biological sample is obtained from a subject having a disorder, and the method further includes the step of identifying the disorder as being susceptible to treatment with a plasma kallikrein (pKal) inhibitor if the levels of the biomarker set deviate from reference values; (b) The biological sample is obtained from a subject who has, is suspected of having, or is at risk of having, a disorder, and the method further includes the step of identifying the subject as a candidate for treatment with a pKal inhibitor if the levels of the set of biomarkers in the biological sample deviate from reference values; or (c) The biological sample is obtained from a subject who has or is at risk of having a disorder, and the method further includes the step of identifying the subject as having or being at risk of having the disorder if the levels of the set of biomarkers in the biological sample deviate from the levels of the set of biomarkers in a control sample; method.

2. (a) The pKal inhibitor is lanadermab; (b) The biomarker set consists of 2 to 10 proteins selected from Table 1; (c) The biological sample is a serum sample or a plasma sample; (d) The disorder is a disease related to the contact activation system; (e) The at least one protein is a kallikrein-kinin protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-chain high molecular weight kininogen (KNG1), and kininogen; (f) The at least one protein is a blood coagulation protein selected from the group consisting of α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5); (g) The at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15:cytoplasmic domain (CDH15), ephrin type A receptor 2 (EPHA2), multimelin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3); (h) The at least one protein is a proteolytic protein selected from the group consisting of proteasome subunit β-6 (PSMB6), ubiquitin-binding enzyme E2R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4A (UBE4A), and E3 ubiquitin-protein ligase ZnRF3 (ZnRF3); (i) The at least one protein is a complement-activating protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (j) The at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), α-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (k) The biological sample is provided in a vacuum blood collection tube containing one or more protease inhibitors; (l) The level of the biomarker set is measured by enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay; and / or (m) The subject is a human patient; The method according to claim 1.

3. The method according to claim 1 or 2, wherein the disorder is (a) hereditary angioedema (HAE) and / or (b) type I HAE or type II HAE.

4. A composition for use in the treatment of a person with a disorder, comprising a plasma kallikrein (pKal) inhibitor, The level of the target biomarker set deviates from the level of the biomarker set in the control sample. The biomarker set includes at least one protein selected from Table 1. composition.

5. (a) The pKal inhibitor is lanadermab; (b) The biomarker set consists of 2 to 10 proteins selected from Table 1; (c) The disorder is a disease related to the contact activation system; (d) The disorder is hereditary angioedema (HAE), and the disorder may be type I HAE or type II HAE; (e) The at least one protein is a kallikrein-kinin protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-chain high molecular weight kininogen (KNG1), and kininostatin; (f) The at least one protein is a blood coagulation protein selected from the group consisting of α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5); (g) The at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15:cytoplasmic domain (CDH15), ephrin type A receptor 2 (EPHA2), multimelin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3); (h) The at least one protein is a proteolytic protein selected from the group consisting of proteasome subunit β-6 (PSMB6), ubiquitin-binding enzyme E2R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4A (UBE4A), and E3 ubiquitin-protein ligase ZnRF3 (ZnRF3); (i) The at least one protein is a complement-activating protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (j) The at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), α-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (k) The levels of the biomarker set are measured by enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay; and / or (l) The subject is a human patient; The composition according to claim 4.

6. A method for evaluating treatment in a subject, The steps include measuring the level of a biomarker set containing at least one protein selected from Table 1 in a biological sample obtained from the subject before, during, or after treatment, and A step of evaluating the effectiveness of the treatment based on the level of the biomarker set, Includes, A deviation in the levels of the biomarker set after or during the course of the treatment compared to before the treatment indicates that the treatment is effective for the subject. method.

7. (a) The treatment comprises administering a plasma kallikrein (pKal) inhibitor to the subject, wherein the pKal inhibitor may be lanadermab; (b) The biomarker set consists of 2 to 10 proteins selected from Table 1; (c) The biological sample is a serum sample or a plasma sample; (d) The at least one protein is a kallikrein-kinin protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-chain high molecular weight kininogen (KNG1), and kininostatin; (e) The at least one protein is a blood coagulation protein selected from the group consisting of α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5); (f) The at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15:cytoplasmic domain (CDH15), ephrin type A receptor 2 (EPHA2), multimelin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3); (g) The at least one protein is a proteolytic protein selected from the group consisting of proteasome subunit β-6 (PSMB6), ubiquitin-binding enzyme E2R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4A (UBE4A), and E3 ubiquitin-protein ligase ZnRF3 (ZnRF3); (h) The at least one of the proteins is a complement-activating protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (i) The at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), α-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (j) The biological sample is provided in a vacuum blood collection tube containing one or more protease inhibitors; (k) The levels of the biomarker set are measured by enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay; (l) The subject is a human patient; (m) The subject has, is suspected of having, or is at risk of having a disease associated with the contact activation system, and the disease associated with the contact activation system may be hereditary angioedema (HAE), and the HAE may be type I HAE or type II HAE; and / or (n) The disease associated with the contact activation system is not hereditary angioedema (HAE); The method according to claim 6.

8. A method for analyzing a sample, (i) Providing a set of biological samples obtained from subjects who have, are suspected of having, or are at risk of having, a disease associated with the contact activation system; (ii) A step of measuring the level of a biomarker set containing at least one protein selected from Table 1, Includes, If the biomarker set consists of one protein, the protein is not high molecular weight kininogen (KNG1), α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or interleukin-21 (IL-21). method.

9. (a) The biomarker set consists of 2 to 10 proteins selected from Table 1; (b) The biological sample is a serum sample or a plasma sample; (c) The disease associated with the contact activation system is hereditary angioedema (HAE), and the HAE may be type I HAE or type II HAE; (d) The at least one protein is a kallikrein-kinin protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), double-chain high molecular weight kininogen (KNG1), and kininostatin; (e) The at least one protein is a blood coagulation protein selected from the group consisting of α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and plasma serine protease inhibitor (SERPINA5); (f) The at least one protein is a cell adhesion protein selected from the group consisting of cadherin-1 (CDH-1), cadherin-15:cytoplasmic domain (CDH15), ephrin type A receptor 2 (EPHA2), multimelin-2 (MMRN2), olfactomedin-like protein 3 (OLFML3), and protocadherin gamma-C3 (PCDHGC3); (g) The at least one protein is a proteolytic protein selected from the group consisting of proteasome subunit β-6 (PSMB6), ubiquitin-binding enzyme E2R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin-binding factor E4A (UBE4A), and E3 ubiquitin-protein ligase ZnRF3 (ZnRF3); (h) The at least one of the proteins is a complement-activating protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (i) The at least one protein is selected from the group consisting of thrombin (F2), tissue kallikrein 14 (KLK4), tissue kallikrein 13 (KLK13), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), α-macroglobulin, apolipoprotein B (APOB), interleukin-21 (IL-21), complement component C3 (C3), kininogen (KNG1), protein arginine N-methyltransferase 1 (PRMT1), and complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9); (j) Step (i) includes collecting the biological sample in a vacuum blood collection tube containing one or more protease inhibitors; (k) Step (ii) is performed using an enzyme-linked antibody immunosorbent assay (ELISA), immunoblotting, or lateral flow assay; (l) The subject is a human patient; (m) If the level of the biomarker set in the subject deviates from the level of the same biomarker set in a control subject, further includes identifying the subject as a patient having a disease associated with the contact activation system; (n) The subject is a human patient who has been administered treatment for the disease, and the method further includes evaluating the efficacy of the treatment by comparing the levels of the set of biomarkers measured in a biological sample obtained from the subject after or during the treatment with the levels of the same set of biomarkers measured in a biological sample obtained from the subject before the treatment, If the levels of the biomarker set in the sample obtained after or during the treatment process deviate from the levels of the biomarker set in the sample obtained before the treatment, the treatment is determined to be effective; and / or (o) The subject is a human patient who has been administered treatment for the disease, and the method further includes evaluating the efficacy of the treatment by comparing the levels of the biomarker set measured in a biological sample obtained from the subject after or during the treatment with the levels of the same biomarker set measured in a control sample obtained from a healthy subject, The treatment is determined to be effective if the levels of the biomarker set in the sample obtained after or during the treatment process do not substantially deviate from the levels of the biomarker set in the control sample; The method according to claim 8.

10. A kit for analyzing samples from subjects who have, are suspected of having, or are at risk of having, diseases associated with the contact activation system, (i) A first binder specific to a first protein biomarker selected from Table 1, (ii) A second binder specific to a second protein biomarker selected from Table 1, Includes, The first protein biomarker and the second protein biomarker are different, and neither the first nor the second protein biomarker is selected from the group consisting of double-chain high molecular weight kininogen (KNG1), α-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and interleukin-21 (IL-21). kit.

11. (a) further comprising a first detection agent bound to the first binder and a second detection agent bound to the second binder; (b) The first binder is an antibody specific to the first protein biomarker, and / or the second binder is an antibody specific to the second protein biomarker; and / or the first binder is an aptamer specific to the first protein biomarker, and / or the second binder is an aptamer specific to the second protein biomarker; and / or (c) The first binder and the second binder are fixed on the support member; The kit according to claim 10.

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