Protein biomarkers for ranallinumin anti treatment

Abstract

Provided herein are methods for determining whether a condition is susceptible to treatment with a plasma kallikrein inhibitor and methods for identifying a subject as a candidate for treatment with a plasma kallikrein inhibitor. Provided herein are methods of identifying a subject as having a condition or at risk of a condition and methods of treating a condition in a subject. Also provided herein are methods of assessing the effectiveness of treatment in a subject.

Description

[0001] Related Applications

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 357,596, filed on June 30, 2022, and U.S. Provisional Application No. 63 / 458,740, filed on April 12, 2023, under 35 U.S.C. 119(e), each of which is incorporated by reference in its entirety.

[0003] References to Electronic Sequence Listings

[0004] The contents of the electronic Sequence Listing (D061770139WO00-SEQ-JRV.xml; size: 13,585 bytes; and creation date: June 29, 2023) are incorporated herein by reference in their entirety. Background Art

[0005] Lanadelumab / Takeda) is a human monoclonal antibody that inhibits active plasma kallikrein and has been approved by several regulatory agencies for the treatment of hereditary angioedema (HAE) in human patients. HAE is a disease that causes sporadic swelling attacks that can affect multiple parts of the body, such as the face, limbs, genitals, gastrointestinal tract, and upper airway. Because HAE symptoms are often similar to those of allergies or colic, it is often difficult to identify HAE attacks before patients exhibit severe or life-threatening symptoms. Early diagnosis will allow better management of emergencies involving acute HAE attacks, and will also help manage HAE patients to prevent or inhibit acute HAE attacks, for example, by allowing HAE patients to avoid exposure to stimuli that may trigger HAE attacks. Summary of the invention

[0006] The plasma contact activation system is a pro-inflammatory and procoagulant system involving a group of plasma proteases. It is activated by active factor XII (FXIIa) when exposed to an external or negatively charged surface, or by prolyl carboxypeptidase on the surface of endothelial cells (Sainz IM et al., Thromb. Haemost. (2007) 98, 77-83). Inappropriate or unregulated activation of the contact system is associated with a variety of diseases, including hereditary angioedema (HAE). Therefore, it is of great interest to identify biomarkers of HAE and other diseases associated with the contact activation system, so as to develop reliable diagnosis and prognosis methods for identifying subjects suffering from HAE or at risk of suffering from HAE. Such biomarkers will also be conducive to the study of understanding potential disease mechanisms, thereby promoting the development of effective new therapies.

[0007] The present disclosure is based, at least in part, 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., lanarizumab), including protein biomarkers that become normalized (restored to control levels) after treatment. The protein biomarkers identified herein can be used, for example, to determine whether a condition is sensitive to treatment with a pKal inhibitor (e.g., lanarizumab), to identify diseases associated with contact activation systems, to identify patients with or at risk for a condition, to identify subjects as candidates for treatment, to monitor disease progression or disease state, and / or to evaluate the efficacy of a treatment for a condition.

[0008] Thus, aspects of the present disclosure provide methods for determining whether a condition is sensitive to treatment with a pKal inhibitor, the methods comprising measuring the level of a biomarker set comprising at least one protein selected from Table 1 in a biological sample of a subject having the condition, and if the level of the biomarker set deviates from a reference value, then identifying the condition as sensitive to treatment with a pKal inhibitor. In some embodiments, the method further comprises administering a pKal inhibitor to the subject if the condition is identified as sensitive to treatment with a pKal inhibitor.

[0009] Aspects of the present disclosure provide methods for identifying a subject as a candidate for treatment with a pKal inhibitor, the methods comprising providing a biological sample from a subject having a condition, suspected of having a condition, or at risk for a condition, and measuring the level of a biomarker set comprising at least one protein selected from Table 1 in the biological sample, wherein if the level of the biomarker set in the biological sample deviates from a reference value, the subject is identified as a candidate for treatment with a pKal inhibitor. In some embodiments, the method further comprises administering a pKal inhibitor to the subject identified as a candidate for treatment.

[0010] Aspects of the present disclosure provide methods for identifying a subject as having a disorder or being at risk of a disorder, the methods 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 a disorder or being at risk of a 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, the method further comprises administering an effective amount of a pKal inhibitor to the subject if the subject is identified as having a disorder or being at risk of a disorder.

[0011] Aspects of the present disclosure provide methods for treating a disorder in a subject, the methods comprising administering to the subject an effective amount of a plasma kallikrein (pKal) inhibitor, wherein the level of a biomarker set in the subject deviates from the level of the biomarker set in a control sample, and wherein the biomarker set comprises at least one protein selected from Table 1. In some embodiments, the disorder is not hereditary angioedema (HAE).

[0012] Aspects of the present disclosure provide compositions for treating a subject having a disorder, the compositions comprising a plasma kallikrein (pKal) inhibitor, wherein the subject's level of a biomarker set deviates from the level of the biomarker set in a control sample, wherein the biomarker set comprises at least one protein selected from Table 1.

[0013] In some embodiments, the pKal inhibitor is lanaritumomab.

[0014] In some embodiments, the biomarker set consists of 2-10 proteins selected from Table 1. In some embodiments, the biological sample is a serum sample or a plasma sample.

[0015] In some embodiments, the disease is a disease associated with a contact activation system. In some embodiments, the disease is hereditary angioedema (HAE). In some embodiments, the disease is type I HAE or type II HAE. In some embodiments, the disease is not hereditary angioedema (HAE).

[0016] In some embodiments, the at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1) and kinin inhibitor (kininostatin). In some embodiments, the 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, 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), polyprotein-2 (MMRN2), olfactory protein-like protein 3 (OLFML3) and protocadherin γ-C3 (PCDHGC3). In some embodiments, the at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3). In some embodiments, 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).

[0017] In some embodiments, 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), 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).

[0018] In some embodiments, the biological sample is provided in a vacuumed blood collection tube that contains one or more protease inhibitors. In some embodiments, the level of the biomarker panel is measured by enzyme-linked immunosorbent assay (ELISA), immunoblot assay, or lateral flow assay.

[0019] In some embodiments, the subject is a human patient.

[0020] Aspects of the present disclosure provide methods for evaluating a treatment in a subject, the methods comprising measuring the level 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 the course of treatment, and evaluating the effectiveness of the treatment based on the level of the biomarker set, wherein a deviation in the level of the biomarker set after treatment or during treatment compared to before treatment indicates that the treatment is effective for the subject.

[0021] In some embodiments, the treatment comprises administering to the subject a plasma kallikrein (pKal) inhibitor. In some embodiments, the pKal inhibitor is lanariumab.

[0022] In some embodiments, the biomarker set consists of 2-10 proteins selected from Table 1. In some embodiments, the biological sample is a serum sample or a plasma sample.

[0023] 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), 2-chain high molecular weight kininogen (KNG1) and kallikrein. 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), polyprotein-2 (MMRN2), olfactory protein-like protein 3 (OLFML3) and protocadherin γ-C3 (PCDHGC3). In some embodiments, at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3). In some embodiments, 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).

[0024] In some embodiments, 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), 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).

[0025] In some embodiments, the biological sample is obtained in a vacuumed blood collection tube that contains one or more protease inhibitors. In some embodiments, the level of the biomarker panel is measured by enzyme-linked immunosorbent assay (ELISA), immunoblot assay, or lateral flow assay.

[0026] In some embodiments, the subject is a human patient. In some embodiments, the subject suffers from a disease associated with the contact activation system, is suspected of suffering from a disease associated with the contact activation system, or is at risk of 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, HAE is type I HAE or type II HAE. In some embodiments, the disease associated with the contact activation system is not HAE.

[0027] Aspects of the present disclosure provide methods for analyzing samples, the methods comprising (i) providing a biological sample (e.g., a serum sample or a plasma sample) obtained from a subject (such as a human subject) having, suspected of having, or at risk of a disease associated with exposure to an activated system; and (ii) measuring the level of a biomarker set comprising at least one protein selected from Table 1, wherein if the biomarker set consists of one protein, the protein is not 2-chain high molecular weight kininogen (KNG1), alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or interleukin-21 (IL-21).

[0028] In some embodiments, the biomarker set consists of 2-10 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, HAE is type I HAE or type II HAE.

[0029] 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), 2-chain high molecular weight kininogen (KNG1) and kallikrein. 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), polyprotein-2 (MMRN2), olfactory protein-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 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating 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 activating protein selected from the group consisting of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0030] In some embodiments, 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), 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).

[0031] In some embodiments, providing the biological sample comprises collecting the biological sample into a vacuumed blood collection tube comprising one or more protease inhibitors. In some embodiments, the measurement of the level of the biomarker set is performed using an enzyme-linked immunosorbent assay (ELISA), an immunoblot assay, or a lateral flow assay.

[0032] In some embodiments, the subject is a human patient. In some embodiments, the method further comprises identifying the subject as having a disease associated with the contact system if the level of the subject's biomarker set deviates from the level of the same biomarker set of a control subject. In some embodiments, the method further comprises administering to the subject an effective amount of a therapeutic agent for treating the disease if the subject is identified as having the disease.

[0033] In some embodiments, the subject is a human patient who is receiving treatment for the disease. In some embodiments, the method also includes comparing the level of the biomarker set measured in the biological sample obtained from the subject after treatment or during the course of treatment with the level of the same biomarker set measured in the biological sample obtained from the subject before treatment to evaluate the efficacy of the treatment, wherein if the level of the biomarker set in the sample obtained after treatment or during the course of treatment deviates from the level of the biomarker set in the sample obtained before treatment, then the treatment is determined to be effective. In some embodiments, the method also includes comparing the level of the biomarker set measured in the biological sample obtained from the subject after treatment or during the course of treatment with the level of the same biomarker set measured in the control sample obtained from a healthy subject to evaluate the efficacy of the treatment, wherein if the level of the biomarker set in the sample obtained after treatment or during the course of treatment does not deviate from the level of the biomarker set in the control sample, then the treatment is determined to be effective. In some embodiments, the method also includes administering an effective amount of a therapeutic agent for treating the disease to the subject if the treatment is not determined to be effective. In some embodiments, if the subject has been previously administered a therapeutic agent as part of a treatment process, an increased dose of the therapeutic agent is administered to the subject.

[0034] 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., lanariumab) 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 human plasma-derived C1 esterase inhibitor.

[0035] Aspects of the present disclosure provide kits for analyzing samples of subjects suffering from, suspected of suffering from, or at risk of a disease associated with a contact system, the kits comprising a first binding agent specific for a first protein biomarker selected from Table 1; and a second binding agent 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 binding agent is an antibody specific for a protein marker. In some embodiments, the kit may also include a first detection agent that binds to the first binding agent and a second detection agent that binds to the second binding agent. In some embodiments, the first binding agent and the second binding agent are fixed to a support member.

[0036] The details of one or more embodiments of the present disclosure are set forth in the description below. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of several embodiments and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0038] Figure 1 The use of DNA aptamers specific for protein analytes is shown Volcano plot of plasma proteins analyzed by multiplex methods. The plot represents the comparison of analyte levels in citrated plasma from patients with hereditary angioedema (HAE) (baseline) with analyte levels in citrated plasma from healthy subjects (control). Proteins previously known to be differentially present in the plasma of HAE patients are annotated. Statistical significance is indicated by dashed lines (p>0.05).

[0039] Figure 2 The expression analysis of C1 esterase inhibitor (C1-INH) in plasma samples from healthy controls and HAE patients at baseline is shown as Determined by aptamer binding.

[0040] Figure 3 Figure 2 shows the expression analysis of complement component C4 in plasma samples from healthy controls and HAE patients at baseline, as measured by Determined by aptamer binding.

[0041] Figure 4A and 4BFigure 2 shows the expression analysis of cleaved HMWK (cHMWK) in the plasma of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanaritumab treatment, as measured by Determined by aptamer binding. Figure 4A Comparison of expression between healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanariumab treatment is shown. Figure 4B cHMWK expression in patient-matched samples before (baseline) and after treatment (week 26) is shown.

[0042] Figure 5 Figure 2 shows the expression analysis of F2 (thrombin) in plasma samples of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanariumab treatment (week 26), as measured by Determined by aptamer binding.

[0043] Figure 6 Figure 2 shows the expression analysis of intermediate α-trypsin inhibitor heavy chain H4 (ITH4) in plasma samples of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanaritumab treatment (week 26), as shown by Determined by aptamer binding.

[0044] Figure 7 Figure 2 shows the expression analysis of interleukin-36A (IL-36A) in plasma samples of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanaritumab treatment (week 26), as shown by Determined by aptamer binding.

[0045] Figure 8 Figure 2 shows the expression analysis of interleukin-21 (IL-21) in plasma samples of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanariumab treatment (week 26), as shown by Determined by aptamer binding.

[0046] Figures 9A-9G Shown by Figure 2. Expression analysis of protein biomarkers identified by aptamer binding. Protein levels in plasma samples of healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanaritumab treatment (week 26) are shown. Fig.9A Levels of apolipoprotein B (APOB) are shown. Fig. 9B Levels of kallikrein (KNG1) are shown. Fig. 9C Levels of G antigen 2 (GAGE2B) are shown. Fig.9D Levels of mortality factor 4-like protein 2 (MORF4L2) are shown. Fig.9ELevels of complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9) are shown. Fig.9F Shown are levels of plasma serine protease inhibitor, serpina5. Figure 9G Levels of Cadherin-15: cytoplasmic domain (CDH15) are shown.

[0047] Figures 10A-10F Shown by Figure 2. Expression analysis of protein biomarkers identified by aptamer binding. Protein levels in plasma samples of healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanaritumab treatment (week 26) are shown. Fig. 10A Levels of alpha-2-macroglobulin (A2M) are shown. Fig. 10B Interleukin-12 (IL-12A / IL-12B) levels are shown. Fig. 10C Shown are levels of liver expressed antimicrobial peptide 2 (LEAP2). Fig. 10D Levels of kininogen (KNG1) are shown. Fig.10E Levels of tissue kallikrein-13 (KLK13) are shown. Fig.10F Levels of tissue kallikrein-14 (KLK14) are shown.

[0048] Fig.11 Schematic illustration of local network analysis using the causal association network (CASNET) approach, which identified 120 disease state biomarkers affected by lanarizumab treatment.

[0049] Fig.12 is a schematic diagram of local network analysis using the causal association network (CASNET) method, which has been incorporated into the known knowledge network. The study found that the proteins increased in the plasma samples of HAE patients at baseline compared with healthy controls are indicated by "+" and include thrombin (F2), apolipoprotein D (APOD), alpha-macroglobulin (A2M), apolipoprotein B (APOB), complement component C3 (C3), and kininogen (KNG1). The study found that the proteins decreased in the plasma samples of HAE patients at baseline compared with healthy controls are indicated by "*" and include protein arginine N-methyltransferase 1 (PRMT1), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), tissue kallikrein 14 (KLK14), tissue kallikrein 13 (KLK13), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), and GTP-binding protein SAR1b (SAR1B). Other proteins were included based on pathway associations.

[0050] Figures 13A-13HFigure 2 shows the expression analysis of kininogen (KNG1) in the plasma of healthy controls, HAE patients at baseline and HAE patients after 26 weeks of lanaritumab treatment, as shown by Determined by aptamer binding. Fig.13A Shown is a comparison of KNG1 (ID 15343-337, Table 1) expression between healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanariumab treatment. Fig. 13B KNG1 (ID 15343-337, Table 1) expression in patient-matched samples before (baseline) and after (week 26) treatment is shown. Fig. 13C Comparison of KNG1 (ID 19631-13, Table 1) expression between healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanaritumab treatment is shown. Fig.13D KNG1 (ID 19631-13, Table 1) expression in patient-matched samples before (baseline) and after (week 26) treatment is shown. Fig.13E Shown is a comparison of KNG1 (ID 4918-21, Table 1) expression between healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanariumab treatment. Fig.13F KNG1 (ID 4918-21, Table 1) expression in patient-matched samples before (baseline) and after (week 26) treatment is shown. Figure 13G Shown is a comparison of KNG1 (ID 7784-1, Table 1) expression between healthy controls, HAE patients at baseline, and HAE patients after 26 weeks of lanariumab treatment. Fig.13H KNG1 (ID 7784-1, Table 1) expression in patient-matched samples before (baseline) and after (week 26) treatment is shown.

[0051] Figures 14A-14D Figure 4 shows the expression analysis of complement C3 (C3) in the plasma of healthy controls, HAE-C1-INH patients at baseline and HAE-C1-INH patients after 26 weeks of lanaritumab treatment, as shown by Determined by aptamer binding. Fig.14A Comparison of C3 (ID 2754-50, Table 1) expression between healthy controls, HAE-C1-INH patients at baseline, and HAE-C1-INH patients after 26 weeks of lanarumab treatment is shown. Fig. 14B Comparison of C3b (ID 4480-59, Table 1) expression between healthy controls, HAE-C1-INH patients at baseline, and HAE-C1-INH patients after 26 weeks of lanarumab treatment is shown. Fig. 14CShown is a comparison of inactivated C3b (ID 2683-1, Table 1) expression between healthy controls, HAE-C1-INH patients at baseline, and HAE-C1-INH patients after 26 weeks of lanarumab treatment. Fig.14D Comparison of C3d (ID 5803-24, Table 1) expression between healthy controls, HAE-C1-INH patients at baseline, and HAE-C1-INH patients after 26 weeks of lanarumab treatment is shown.

[0052] Figures 15A-15C Protein analysis of protein biomarkers determined by ELISA in plasma samples of HAE-C1-INH subjects treated with 150 mg or 300 mg of lanarumab every 4 weeks and plasma samples of healthy controls is shown. Plasma samples from HAE-C1-INH subjects were collected at baseline (before dosing; day 0) and after 98 days of lanarumab treatment. Fig.15A Protein levels of alpha-2-macroglobulin (A2M) are shown. Fig. 15B Protein levels of apolipoprotein B (APOB) are shown. Fig. 15C The protein levels of interleukin-21 (IL-21) are shown. DETAILED DESCRIPTION

[0053] The contact activation system starts the intrinsic pathway of blood coagulation and promotes inflammation by releasing the proinflammatory peptide bradykinin. Factor XII (FXII), also known as Hageman factor, is a serine protease that plays a role in the intrinsic pathway of activated blood coagulation and the kallikrein-kinin system. FXII is activated by a negatively charged surface (e.g., polyanionic surface, glass, polyphosphate, ellagic acid) to produce an active form, FXIIa. Activated FXIIa has the ability to cut prekallikrein, thereby generating active pKal. Subsequently, activated pKal can cut FXII into FXIIa, thereby generating a positive feedback loop, wherein FXIIa generates even more pKal, which further activates other FXII into FXIIa. Activated pKal can also cut high molecular weight kininogen (HMWK), to release bradykinin. In diseases (such as HAE) associated with contact system activation, the increase in bradykinin levels can induce vasodilation and inflammation, thereby causing edematous HAE attacks. There is a need to identify novel biomarkers that can be used, for example, to identify diseases mediated by contact activation systems, to identify subjects suffering from or at risk for such diseases, and to identify subjects who may benefit from treatment with plasma kallikrein inhibitors (e.g., lanarumab / ) for the treatment of other diseases.

[0054] The present disclosure is based at least in part on the identification of proteins that are differentially present in biological samples obtained from subjects with HAE compared to healthy individuals by proteomic analysis. It has been observed that many proteins are found in subjects with HAE that deviate from the levels in healthy (control) subjects. Interestingly, it was unexpectedly observed that many proteins normalized (restored to control levels) after treatment with lanariumab (a pKal inhibitor). The study found that compared to healthy individuals, protein groups 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) have similar trends (e.g., increased or decreased levels) in samples from subjects with the disease.

[0055] Therefore, provided herein are methods for determining whether a condition is sensitive to treatment with a plasma kalinogen (pKal) inhibitor and methods for identifying a subject as a candidate for treatment with a pKal inhibitor. Also provided herein are methods for analyzing biological samples from subjects suffering from a disease or condition in which one or more protein biomarkers deviate from a control level, suspected of having the disease or condition, or at risk of the disease or condition, based on detecting the presence of a protein biomarker or protein biomarker set or measuring the level of a protein biomarker or protein biomarker set. Such methods can be used, for example, to identify patients at risk of disease, select candidates for treatment, monitor disease progression or disease state, evaluate the efficacy of treatment for a disease, determine the course of treatment, evaluate whether a subject is at risk of disease onset, and / or for research purposes, including, for example, studying the mechanism of the disease and / or the biological pathways / processes involved in the disease, which may be the basis for developing new therapies.

[0056] Protein Biomarkers

[0057] The methods and kits described herein are based, at least in part, on the identification of proteins that are differentially present in samples from subjects with HAE compared to samples from healthy subjects and / or proteins that are differentially present in samples from subjects at different stages of such diseases (e.g., at baseline versus after treatment with lanariumab), including proteins that are differentially present in samples from subjects with HAE compared to samples from healthy subjects, which are restored (normalized) to the levels of healthy subjects after treatment with lanariumab. As used herein, the term "protein biomarker" or "protein biomarker set" refers to a protein or protein collection that is present at different levels in samples from different subject groups (e.g., subjects with a disease versus healthy subjects (e.g., subjects without a disease), or subjects with a disease and in a quiescent phase versus subjects experiencing an acute phase of the disease). In some embodiments, a protein or protein collection that is present at different levels in samples from different subject groups (such as subjects with a disease (e.g., at baseline, before treatment) versus subjects to whom one or more treatments for the disease have been administered).

[0058] Such biomarkers / biomarker sets can be used both in clinical applications and in non-clinical applications (e.g., for research purposes).

[0059] In some embodiments, the same protein biomarker in a sample from a subject with a disease may be present at elevated levels compared to the level of the protein biomarker in a sample from a healthy subject. In some embodiments, the biomarker in a sample from a subject with a disease may be present at reduced levels compared to the level of the protein biomarker in a sample from a healthy subject. In other cases, the protein biomarker may be present at elevated levels in a sample obtained from a subject experiencing an acute phase of a disease as described herein compared to a subject during the quiescent phase (baseline) of the disease. Alternatively, the protein biomarker may be present at reduced levels in a sample obtained from a subject experiencing an acute phase of a disease as described herein compared to a subject during the quiescent phase (baseline) of the disease.

[0060] In some embodiments, a protein biomarker set containing one or more biomarkers can be analyzed in the methods described herein. When the protein biomarker set contains more than one biomarker, all biomarkers can be present at elevated levels or reduced levels in subjects with the disease compared to healthy subjects. Alternatively, the protein biomarker set can contain at least one biomarker that is elevated in subjects with the disease compared to healthy subjects and at least one biomarker that is reduced in subjects with the disease compared to healthy subjects.

[0061] Similarly, a protein biomarker set for evaluating whether a treatment is effective in treating a condition in a subject may contain a plurality of biomarkers that are either elevated or decreased in samples obtained at baseline (before treatment) compared to levels in samples obtained after treatment. Alternatively, a biomarker set may contain at least one biomarker elevated in samples obtained at baseline (before treatment) and at least one biomarker decreased in samples obtained at baseline (before treatment) compared to levels in samples obtained after treatment.

[0062] Table 1 below provides biomarkers that can be evaluated by the methods described herein for evaluating a subject or a biological sample from a subject, e.g., for identifying and / or treating a subject having or suspected of having or being at risk for a disorder, determining whether a disorder is sensitive to treatment with a plasma kallikrein inhibitor, and / or for evaluating the efficacy of a treatment for a disorder.

[0063] In some embodiments, the biomarker set to be measured and analyzed in 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 includes a single protein, the protein is not 2-chain high molecular weight kininogen (HMWK; KNG1), alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, or interleukin-21 (IL-21). In some examples, the protein biomarker set to be measured and analyzed in the methods described herein does not include a combination of any two or more of 2-chain high molecular weight kininogen (HMWK; KNG1), alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin, and / or interleukin-21 (IL-21).

[0064] As described in Example 1, it was found that several proteins involved in the kallikrein-kinin system were differentially present in samples from subjects with HAE before treatment compared to healthy subjects, and / or these proteins were normalized (e.g., restored to levels comparable to healthy subjects) in samples from subjects with HAE after treatment compared to healthy subjects. In some embodiments, the biomarker set includes one or more kallikrein-kinin system proteins as listed in Table 1. In some embodiments, the kallikrein-kinin system proteins of the biomarker set include kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1) or kinin inhibitors, or a combination thereof.

[0065] As described in Example 1, it was found that several proteins involved in the blood coagulation pathway were differentially present in samples from subjects with HAE before treatment compared to healthy subjects, and / or these proteins were normalized (e.g., restored to levels comparable to healthy subjects) in samples from subjects with HAE after treatment compared to healthy subjects. In some embodiments, the biomarker set includes one or more blood coagulation proteins as listed in Table 1. In some embodiments, the blood coagulation proteins of the biomarker set include alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin or plasma serine protease inhibitor (SERPINA5), or a combination thereof.

[0066] As described in Example 1, it was found that several proteins involved in cell adhesion were differentially present in samples from subjects with HAE before treatment compared to healthy subjects, and / or these proteins were normalized in samples from subjects with HAE after treatment (e.g., restored to levels comparable to those of healthy subjects) compared to healthy subjects. In some embodiments, the biomarker set includes one or more proteins involved in cell adhesion as listed in Table 1. In some embodiments, the 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), polyprotein-2 (MMRN2), olfactory protein-like protein 3 (OLFML3) or protocadherin γ-C3 (PCDHGC3) or a combination thereof.

[0067] In some embodiments, the biomarker set includes one or more proteins involved in cardiovascular function as listed in Table 1. In some embodiments, the protein involved in cardiovascular function of the biomarker set includes apolipoprotein B (APOB).

[0068] As described in Example 1, it was found that several proteins involved in proteolysis (including proteases and proteolytic inhibitors) were differentially present in samples from subjects with HAE before treatment compared to healthy subjects, and / or these proteins were normalized (e.g., restored to levels comparable to healthy subjects) in samples from subjects with HAE after treatment compared to healthy subjects. In some embodiments, the biomarker set includes one or more proteins involved in proteolysis as listed in Table 1. In some embodiments, the proteolysis-related proteins of the biomarker set include proteasome subunit β6 type (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 a combination thereof. In some embodiments, the proteolysis-related proteins of the biomarker set include thrombin (F2), tissue kallikrein 14 (KLK14), inter-alpha-trypsin inhibitor heavy chain H4 (ITIH4), or alpha-macroglobulin (A2M), or a combination thereof.

[0069] As described in Example 1, several proteins involved in complement activation were found to be differentially present in samples from subjects with HAE before treatment compared to healthy subjects, and / or to be normalized (e.g., restored to levels comparable to healthy subjects) in samples from subjects with HAE after treatment compared to healthy subjects. In some embodiments, the biomarker set includes one or more proteins involved in complement activation as listed in Table 1. In some embodiments, the complement activation proteins of the biomarker set are complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9).

[0070] In some embodiments, the biomarker set includes one or more proteins that are cytokines as 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 a combination thereof.

[0071] Table 1: Biomarkers of contact system activation

[0072]

[0073]

[0074]

[0075]

[0076]

[0077] Utility of protein biomarkers

[0078] Aspects of the present disclosure relate to methods for analyzing samples obtained from subjects (e.g., human patients) suffering from a disease or condition (such as a disease or condition associated with a contact activation system), suspected of suffering from a disease or condition, or at risk of a disease or condition by measuring the level of a biomarker set as described herein in the sample. In some aspects, the methods described herein relate to identifying subjects suffering from a condition associated with any of the proteins shown in Table 1 or at risk of suffering from the condition, and optionally, treating the subject with a plasma kallikrein inhibitor. Other aspects of the present disclosure relate to determining whether the condition is sensitive to treatment with a pKal inhibitor, and evaluating the efficacy of treatment of the condition in a subject. The results obtained from the methods described herein can be used for diagnostic and / or prognostic purposes, as well as for other non-clinical purposes, such as research purposes.

[0079] (i) Analysis of biological samples

[0080] The methods described herein relate to providing a biological sample obtained from a subject. As used herein, "biological sample" refers to a composition comprising a tissue (e.g., blood, plasma, or protein) from a subject. Samples include both the initial untreated sample taken from the subject and the form of subsequent treatment, such as partial purification or preservation. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a body fluid sample, such as a serum or plasma sample. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or more) biological samples can be collected from a subject over time or at specific time intervals, for example, to assess disease progression or to assess the efficacy of treatment. In some embodiments, multiple (e.g., at least 2, 3, 4, 5, or more) biological samples can be collected from a subject, for example, before treatment, during treatment, and / or after treatment, for example, to assess disease progression or to assess the efficacy of treatment. In some embodiments, the biological sample is plasma.

[0081] Any method known in the art can be used to obtain a biological sample from a subject. In some embodiments, a sample (e.g., a blood sample) is collected in a vacuumed collection tube (e.g., a vacuumed blood collection tube) to obtain a sample from a subject. In some embodiments, the vacuumed collection tube contains one or more protease inhibitors, for example, to reduce or prevent the in vitro activation of the contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitor includes at least one serine protease inhibitor and at least one cysteine ​​protease inhibitor. Such vacuumed collection tubes are known in the art. See, for example, PCT Application No. PCT / US2016 / 046681 (WO 2017 / 027771), which is incorporated herein by reference in its entirety. Optionally, the vacuumed blood collection tube may also include one or more anticoagulants.

[0082] The terms "patient", "subject" or "individual" can be used interchangeably and refer to a subject as described herein. In some embodiments, the subject is a human or non-human mammal. In some embodiments, the subject is suspected of having a disease or condition (such as a disease or condition mediated by any protein in Table 1) or is at risk of the disease or condition. In some embodiments, the subject is suspected of having a disease or condition associated with a contact activation system (e.g., HAE) or is at risk of the disease or condition. Such subjects may exhibit one or more symptoms associated with the disease. Alternatively or additionally, such subjects may carry one or more risk factors for the disease, for example, genetic factors associated with the disease (e.g., genetic defects in CI-INH).

[0083] Alternatively, the subject described herein can be a patient of the disease. Such subjects may currently be in the acute phase of the disease (e.g., experiencing an HAE attack), or may have suffered from the disease in the past (e.g., currently in the disease quiescent phase (baseline)). In some examples, the subject is a human patient who may be receiving disease treatment, for example, treatment involving agents targeting kallikrein-kinin system (KKS) (such as C1 esterase inhibitors (C1-INH), plasma kallikrein inhibitors or bradykinin inhibitors). In other cases, such human patients may not receive such treatment.

[0084] The methods described herein relate to determining whether a condition is susceptible to treatment with a pKal inhibitor. In such embodiments, the disease or condition may have been previously unrelated to pKal activity and / or contact activation systems. Such methods can be used to identify additional diseases or conditions for which treatment with a pKal inhibitor may be effective. In some embodiments, the disease or condition is associated with or mediated by abnormal expression or activity of any one or more of the proteins shown in Table 1. In some embodiments, the disease or condition is unrelated to a contact activation system. In some embodiments, the disease or condition is not hereditary angioedema.

[0085] In some embodiments, the disease or condition is associated with a contact activation system. Examples of diseases associated with a contact activation system include, but are not limited to, kallikrein-mediated conditions, such as bradykinin-mediated conditions, such as hereditary angioedema (HAE), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, 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 device or stent, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease and coronary heart disease with unstable angina, edema, eye disease, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spinal disease, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head Brain edema around trauma or tumors, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), vascular restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, nervous system diseases, diseases related to protein misfolding, diseases related to angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic diseases and respiratory diseases (e.g., anaphylactic shock, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, refractory rhinitis) and tissue damage (e.g., burns or chemical injuries).

[0086] In some embodiments, the disease or disorder associated with the contact activation system is hereditary angioedema (HAE). In some embodiments, the methods described herein can be used to identify whether a subject suffers from HAE or is at risk of suffering from HAE. Alternatively or additionally, the methods described herein can be used to evaluate the efficacy and / or disease progression of treatment. Alternatively or additionally, the methods described herein can be used to evaluate whether a subject suffers from an HAE attack or is at risk of suffering from an HAE attack. In some embodiments, the methods described herein can be used to determine whether to administer one or more additional doses of a therapeutic agent (e.g., a pKal inhibitor), adjust the dose of the therapeutic agent (e.g., increase or decrease the dose and / or frequency), or initiate or terminate the treatment of HAE. Hereditary angioedema (HAE) is also referred to as "Quincke edema", C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioedema (HANE). HAE is characterized by repeated attacks of severe swelling (angioedema), which can affect, for example, limbs, face, genitals, gastrointestinal tract, and airway. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction that may involve swelling of the throat and sudden hoarseness; recurring abdominal cramps for no apparent reason; and / or intestinal swelling that may be severe and may lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. About one-third of individuals with HAE develop a non-itchy rash called erythema marginale during an attack.

[0087] Swelling of the airways can be life-threatening and has resulted in death in some patients. The mortality rate is estimated to be 15%-33%. HAE results in approximately 15,000-30,000 emergency room visits each year.

[0088] Trauma or stress (e.g., dental surgery, illness (e.g., viral illnesses such as colds and flu), menstruation, and surgical procedures) may trigger an attack of angioedema. To prevent an acute attack of HAE, patients can try to avoid specific stimuli that previously caused an attack. However, in many cases, an attack occurs without a known trigger. Typically, HAE symptoms first appear in childhood and worsen during adolescence. On average, untreated individuals have an attack every 1 to 2 weeks, and most attacks last about 3 to 4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary greatly between individuals with hereditary angioedema, even between individuals in the same family.

[0089] There are three types of HAE, called types I, II, and III. It is estimated that HAE affects 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 females, but families with affected males have been identified.

[0090] HAE is inherited in an autosomal dominant pattern, so an affected person can inherit the mutation from an affected parent. New mutations can also occur in the gene, so HAE can occur in people with no family history of the condition. It is estimated that 20%-25% of cases are caused by new spontaneous mutations.

[0091] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene provides instructions for making the C1 inhibitor protein, which is important for controlling inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause hereditary angioedema type I result in low levels of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of dysfunctional C1 inhibitor. In the absence of appropriate levels of functional C1 inhibitor, excess bradykinin is produced. Bradykinin promotes inflammation by increasing the infiltration of fluid through blood vessel walls into body tissues. Excess fluid accumulation in body tissues results in episodes of swelling seen in individuals with hereditary angioedema types I and II.

[0092] Mutations in the F12 gene are associated with some cases of hereditary angioedema type III. The F12 gene provides instructions for making coagulation factor XII. In addition to playing a key role in blood clotting (coagulation), factor XII is an important stimulant of inflammation and is involved in the production of bradykinin. Certain mutations in the F12 gene lead to the production of factor XII with increased activity. As a result, more bradykinin is produced, and blood vessel walls become more leaky, leading to episodes of swelling. The cause of other cases of hereditary angioedema type III remains unknown. Mutations in one or more genes that have not yet been identified may be the cause of the condition in these cases.

[0093] The presentation of HAE may be similar to other forms of angioedema caused by allergies or other medical conditions, but it differs significantly in its etiology and treatment. When HAE is misdiagnosed as allergy, it is most often treated with antihistamines, steroids, and / or epinephrine, which are generally ineffective for HAE, although epinephrine can be used for life-threatening reactions. Misdiagnosis also leads to unnecessary exploratory surgery in patients with abdominal swelling, and in some patients with HAE, abdominal pain is incorrectly diagnosed as psychosomatic.

[0094] C1 inhibitor therapy and other treatments for HAE are described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0095] Acute treatment of HAE attacks is provided to stop the progression of edema as quickly as possible. Intravenous administration of C1 inhibitor concentrate from donor blood is an acute treatment; however, this treatment is not available in many countries. In emergency situations where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as a substitute because it also contains C1 inhibitor.

[0096] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are now available in the United States, and two C1 inhibitor products are now available in Canada. Pasteurized Berinert P (CSLBehring) was approved by the FDA in 2009 for acute attacks. Nanofiltered (Takeda) was approved by the FDA for prevention in 2008. Rhucin / Ruconest (Pharming) is a recombinant C1 inhibitor under development that does not pose the risk of infectious disease transmission due to pathogens carried in human blood.

[0097] Treatment of an acute HAE attack may also include medications and / or IV fluids for pain relief.

[0098] Other treatments can stimulate the synthesis of C1 inhibitor or reduce its consumption. Androgenic drugs such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0099] Helicobacter pylori can cause abdominal attacks. Antibiotics that treat Helicobacter pylori will reduce abdominal attacks.

[0100] Newer treatments attack the contact cascade. Ecallantide ( Takeda and lanariumab ( Takeda) inhibits plasma kallikrein and has been approved in the United States or the United States and Europe. Takeda) inhibits the bradykinin B2 receptor and has been approved in Europe and the United States.

[0101] The diagnosis of HAE can rely on, for example, family history and / or blood tests. Laboratory findings associated with type I, type II, and type III HAE are described, for example, in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5): 918-925. In type I HAE, C1 inhibitor levels are reduced, C4 levels are also reduced, but C1q levels are normal. In type II HAE, C1 inhibitor levels are normal or elevated; however, C1 inhibitor function is abnormal. C4 levels are reduced, and C1q levels are normal. In type III, C1 inhibitor, C4, and C1q levels can all be normal. The present disclosure is based, at least in part, on the identification of additional proteins, which have different levels in samples from HAE patients compared to healthy individuals (Table 1). Measuring the levels of biomarker sets 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 suffering from an HAE attack. In some embodiments, the method can be used to determine whether a treatment is or was effective in treating HAE.

[0102] Symptoms of a disease or condition such as HAE can be assessed, for example, using a questionnaire (e.g., a questionnaire completed 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, CV et al. Patient (2012) 5(2): 113-26.

[0103] The biological samples described herein can be analyzed by measuring the level of the biomarker set as described herein in the biological sample. The level (e.g., amount) of the biomarkers disclosed herein or the change of the biomarker level can be evaluated using the assay described herein and / or the assay known in the art. Conventional methods can be used to analyze one or more biomarkers described herein. In some embodiments, the level of the biomarker is evaluated or measured by directly detecting the protein in the biological sample. Alternatively or additionally, the level of the protein can be evaluated or measured indirectly in the biological sample, for example, by detecting the activity level of the protein (e.g., enzyme assay).

[0104] In some embodiments, biomarkers are measured using immunoassays. Examples of immunoassays include, but are not limited to, immunoblotting assays (western blots), enzyme-linked immunosorbent assays (ELISA) (e.g., sandwich ELISA), radioimmunoassays, electrochemiluminescence-based detection assays, magnetic immunoassays, lateral flow assays, and related techniques. It will be apparent to those skilled in the art that other suitable immunoassays for detecting biomarkers provided herein will be apparent to those skilled in the art. However, it will be apparent to those skilled in the art that the present disclosure is not limited to immunoassays, and detection assays not based on antibodies or antigen-binding antibody fragments (such as mass spectrometry) may also be used to detect and / or quantify biomarkers provided herein. Assays that rely on chromogenic substrates may also be used to detect and / or quantify biomarkers provided herein.

[0105] The type of detection assay used to detect and / or quantify biomarkers, such as those provided herein, will depend on the specific context in which the assay is used (e.g., clinical or research application), and on the type and number of biomarkers to be detected, and on the type and number of patient samples to be run in parallel, to name just a few parameters.

[0106] ELISA is known in the art (see, e.g., Crowther, John R (2009). "The ELISA Guidebook." 2nd Edition 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 also known in the art and commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0107] In some embodiments, immunoassays are used to measure the level of protein biomarkers. The immunoassays described herein can be in the form of a sandwich ELISA, in which a first binding agent that specifically binds to a protein of a biomarker set is fixed to a support member. The support member can then be incubated with a biological sample as described herein for a suitable period of time under conditions that allow a complex to form between the binding agent and the protein in the sample. Such complexes can then be detected using a detection agent that binds to a protein, a binding agent-protein complex, or a binding agent. The detection agent can be conjugated to a label that can release a signal directly or indirectly. The intensity of the signal represents the level of protein in the sample. In some embodiments, the detection agent is detected, and its level represents the level of protein in the sample.

[0108] Any binding agent that specifically binds to a desired protein can be used in the methods and kits described herein to measure the level of a protein in a biological sample. In some embodiments, the binding agent is an antibody that specifically binds to a desired protein. In some embodiments, the binding agent is an aptamer that specifically binds to a desired protein. In some embodiments, a sample can be contacted with more than one binding agent that binds to different proteins simultaneously or sequentially (e.g., multiplexed analysis, e.g., The biological sample is contacted with the binding agent under appropriate conditions. Generally, the term "contacting" refers to contacting the binding agent with the biological sample or reagent for a suitable period of time sufficient to form a complex between the binding agent and the protein (if any) in the sample. In some embodiments, the contacting is performed by capillary action, wherein the biological sample or agent moves across the surface of the carrier membrane.

[0109] In some embodiments, immunoassays can be performed on low-throughput platforms, including single immunoassay formats. For example, low-throughput platforms can be used to measure the presence and amount of proteins in biological samples (e.g., biological tissues, tissue extracts) for diagnostic methods, monitoring disease and / or treatment progress, and / or predicting whether a disease or condition may benefit from a particular treatment.

[0110] In some embodiments, it may be necessary to fix the binding agent to the support member. The method for fixing the binding agent will depend on factors such as the nature of the binding agent and the material of the support member, and may require a specific buffer. Such methods are obvious to those of ordinary skill in the art. For example, any kit and / or detection device as also described herein can be used to measure a biomarker set in a biological sample as described herein.

[0111] As used herein, the term "measuring" or "measurement" or alternatively "detecting" or "detection" means assessing the presence, absence, amount or quantity (which may be a significant amount) of a substance within a sample, including the derivation of a qualitative or quantitative concentration level of such a substance, or otherwise evaluating a value or classification of a subject.

[0112] The assay (e.g., a Western blot assay) may further involve the use of a quantitative imaging system, such as LICOR imaging technology, which is commercially available (see, e.g., LICOR Imaging from LI-COR Biosciences). CLx infrared imaging system). In some embodiments, an electrochemiluminescence detection assay or an assay relying on a combination of electrochemiluminescence and patterned array technology (e.g., ECL or MULTI-ARRAY technology assays from Meso Scale Discovery (MSD)) is used.

[0113] In any of the methods described herein, the protein levels of the biomarker set can be compared to the protein levels in a control sample or a reference sample. Alternatively or additionally, in any of the methods described herein, the protein levels of the biomarker set can be compared to a reference level (such as a predetermined reference level).

[0114] (ii) Clinical Application

[0115] The levels of the proteins presented in Table 1 detected in a sample from a subject can be used as biomarkers to identify a subject as having or at risk for a disease or condition, diagnose a disease, such as a disease associated with a contact activation system (e.g., HAE), monitor the progression of a disease, assess the efficacy of a disease treatment, and identify patients suitable for a specific treatment.

[0116] Thus, described herein are methods for diagnosing and prognosing diseases or conditions (such as diseases or conditions associated with expression of any of the proteins shown in Table 1) based on the levels of a set of biomarkers in a biological sample obtained from a subject. In some embodiments, a disease or condition associated with expression of any of the proteins shown in Table 1 can be treated with an agent (such as lanaritumumab). In some embodiments, these methods are methods for diagnosing and prognosing diseases associated with a contact activation system based on the levels of a set of biomarkers in a biological sample obtained from a subject.

[0117] In some embodiments, the level of a biomarker as measured using any of the methods described herein may be dependent on evaluating whether the subject (e.g., a human patient) from which the biological sample was obtained suffers from a disease or is at risk of a disease. In some embodiments, the level of a biomarker as measured using any of the methods described herein may be dependent on evaluating whether the subject (e.g., a human patient) from which the biological sample was obtained suffers from a disease associated with a contact activation system (such as a disease associated with plasma kallikrein, e.g., HAE or autoimmune diseases such as RA, UC, and Crohn's disease) or is at risk of the disease. In some embodiments, these methods are diagnostic and prognostic methods for diseases that may be mediated by or associated with the kallikrein-kinin system (contact activation system), and therefore may be treated with agents targeting the kallikrein-kinin system (e.g., lanarizumab).

[0118] In some embodiments, the level of the biomarker can then be compared with a reference sample or a control sample to determine the value indicating the amount of protein in the sample. In some embodiments, the value of the biomarker is obtained by comparing the level of the protein in the sample with the level of another protein (e.g., an internal control or an internal standard) in the sample. Such biomarker values ​​can be normalized values ​​relative to an internal control or an internal standard. The value of the biomarker can be compared with a reference value to determine whether the subject suffers from a disease or is at risk of a disease. The reference value can represent the level of the corresponding biomarker in a subject (e.g., a human subject) (e.g., a healthy subject) without a target disease. In some embodiments, if the level or value of the biomarker is higher than a reference level or value, the subject can be identified as suffering from a disease or being at risk of a disease. In some embodiments, if the level or value of the biomarker is lower than a reference level or value, the subject can be identified as suffering from a disease or being at risk of a disease.

[0119] In some embodiments, the level of the biomarker can be compared to a predetermined threshold value for the protein, and deviation from the threshold value may indicate that the subject has a disease. The predetermined threshold value can represent 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.

[0120] In some embodiments, the biomarker set includes more than one protein, for at least one of which an elevated level indicates that the subject has a disease or is at risk of having a disease, and for at least one protein, a decreased level indicates that the subject has a disease or is at risk of having a disease. In some embodiments, the biomarker set includes more than one protein, for each of which an elevated level indicates that the subject has a disease or is at risk of having a disease. In some embodiments, the biomarker set includes more than one protein, for each of which a decreased level indicates that the subject has a disease or is at risk of having a disease.

[0121] 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 control subject.

[0122] As used herein, a control subject can be a healthy individual, i.e., an individual who is apparently free of the target disease (e.g., a disease associated with the contact system) or a history of the disease when the level of the protein is measured. A control subject can also represent a population of healthy subjects, preferably having characteristics (e.g., age, sex, race) that match those of the subjects analyzed by the methods described herein.

[0123] The control level can be a predetermined level or threshold. Such a predetermined level can represent the protein level in a subject population that does not suffer from the target disease or is not at risk of the target disease (e.g., the average level in a healthy subject population). It can also represent the protein level in a subject population that suffers from the target disease.

[0124] The predetermined level can take many forms. For example, it can be a single critical value, such as a median or mean. In some embodiments, such a predetermined level can be established based on a comparison group, such as a group determined to be known to have a target disease, and another group determined to be not suffering from the target disease. Alternatively, the predetermined level can be a range, for example, representing a range of protein levels in a control population.

[0125] Control levels as described herein can be determined by conventional techniques. In some examples, control levels can be obtained by performing conventional methods (e.g., the same assay for obtaining protein levels of test samples as described herein) on control samples also as described herein. In other examples, protein levels can be obtained from members of a control population and can be analyzed, for example, by a computer program to obtain a control level (predetermined level) representing the protein level in the control population.

[0126] By comparing the level of the biomarker in the sample obtained from the candidate subject with the reference value as described herein, it can be determined whether the candidate subject suffers from the disease or is at risk of the disease. For example, if the level of the biomarker in the sample of the candidate subject deviates from the reference value (e.g., increases or decreases compared to the reference value), the candidate subject can be identified as suffering from the disease or at risk of the disease. When the reference value represents the value range of the biomarker level in the subject population suffering from the target disease, the value of the biomarker in the sample of the candidate falling within the range indicates that the candidate subject suffers from the target disease or is at risk of the target disease.

[0127] As used herein, "elevated levels" or "levels above a reference value" means that the level of a biomarker is above a reference value, such as a predetermined threshold value for the level of a biomarker in a control sample. Control levels are described in detail herein. Elevated levels of a biomarker include, for example, levels of a biomarker that are 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above a reference value. In some embodiments, the level of a biomarker in a test sample is at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 15 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2 times, 2.5 times, 3 times, 3.5 times, 4 times, 4.5 times, 5 times, 5.5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 50 times, 100 times, 150 times, 200 times, 300 times, 400 times, 500 times, 1000 times, 10000 times, or more than the level of the biomarker in a reference sample.

[0128] As used herein, "reduced levels" or "levels below a reference value" means that the level of a biomarker is below a reference value, such as a predetermined threshold value of a biomarker in a control sample. Control levels are described in detail herein. Reduced levels of a biomarker include, for example, levels of a biomarker that are 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more below a reference value. In some embodiments, the level of a biomarker in a test sample is at least 1 / 1.1, 1 / 1.2, 1 / 1.3, 1 / 1.4, 1 / 15, 1 / 1.6, 1 / 1.7, 1 / 1.8, 1 / 1.9, 1 / 2, 1 / 2.5, 1 / 3, 1 / 3.5, 1 / 4, 1 / 4.5, 1 / 5, 1 / 5.5, 1 / 6, 1 / 7, 1 / 8, 1 / 9, 1 / 10, 1 / 50, 1 / 100, 1 / 150, 1 / 200, 1 / 300, 1 / 400, 1 / 500, 1 / 1000, 1 / 10000, or less of the level of the biomarker in a reference sample.

[0129] In some embodiments, the methods described herein are used to identify a subject as a candidate for a specific treatment (e.g., treatment with a pKal inhibitor). In some embodiments, a candidate subject is a human patient with symptoms of a disease (such as a disease associated with a contact activation system, such as a pKal-mediated condition, e.g., HAE or an autoimmune disease, such as RA, UC, and Crohn's disease). For example, the subject suffers from edema, swelling, wherein the swelling is entirely or primarily peripheral; urticaria; redness, pain, and swelling without evidence of infection; recurrent episodes of non-histamine-mediated edema, swelling, or a combination thereof. In other embodiments, the subject has no symptoms of a pKal-mediated condition, no history of symptoms of a pKal-mediated condition, or no history of a pKal-mediated condition (such as HAE) at the time the sample is collected. In other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0130] A subject identified in the methods described herein can be treated with an appropriate therapy, such as treatment with a pKal inhibitor described herein.

[0131] Taking into account the correlation between the level of biomarkers and such diseases, the assay methods and kits described herein can also be used to evaluate the efficacy of the treatment of pKal inhibitors (such as those described herein). For example, multiple biological samples (e.g., blood or plasma samples) can be collected from the subject receiving treatment before and after treatment or during the treatment process. The level of the biomarker can be measured by any assay method as described herein, and the value (e.g., amount) of the biomarker can be determined accordingly. For example, if the level of the biomarker increases, it indicates that the subject suffers from the target disease and the level of the biomarker decreases after treatment or during the treatment process (the level of the biomarker in the sample collected later is compared with the level of the biomarker in the sample collected earlier), it indicates that the treatment is effective. As another example, if the level of the biomarker decreases, it indicates that the subject suffers from the target disease and the level of the biomarker increases after treatment or during the treatment process (the level of the biomarker in the sample collected later is compared with the level of the biomarker in the sample collected earlier), it indicates that the treatment is effective. If the level of a biomarker or set of biomarkers (e.g., a biomarker or set of biomarkers indicative of a disease) in a subject becomes normalized (e.g., does not deviate from the level of the same biomarker or set of biomarkers in a control subject (e.g., a healthy subject or a subject with a disease in a quiescent state) during or after the course of treatment, then the treatment can also be determined to be effective. In some embodiments, if there is no statistically significant difference between the level of the biomarker or set of biomarkers and the level of the control subject, the level of the biomarker or set of biomarkers does not substantially deviate from the level of the same biomarker or set of biomarkers in the control subject. As an example, an elevated biomarker indicating a disease before treatment can be reduced 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 another example, a reduced biomarker indicating a disease before treatment can be increased 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 one or more administrations of a plasma kallikrein inhibitor, such as lanarizumab.

[0132] If the subject is identified as unresponsive to treatment, or the treatment is determined to be ineffective, a higher dose and / or frequency of the therapeutic agent (e.g., a pKal inhibitor, such as lanarizumab) is administered to the identified subject. In some embodiments, the dose or frequency of administration of the therapeutic agent (e.g., a pKal inhibitor, such as lanarizumab) is maintained, reduced, or stopped in subjects identified as responsive to treatment or not requiring further treatment. Alternatively, a different treatment may be administered to subjects found to be unresponsive to the first treatment.

[0133] In other embodiments, the value of a biomarker or a set of biomarkers can also be used to identify a condition that is sensitive (treatable) to treatment with a pKal inhibitor. In order to practice the method, the level of a biomarker in a sample (e.g., a blood sample or a plasma sample) collected from a subject with a target disease can be measured by a suitable method (e.g., those methods described herein, such as Western blot or ELISA assay). If the level of the biomarker deviates from a reference value (e.g., increases or decreases), it indicates that the pKal inhibitor is effective in treating the disease. If the disease is identified as being sensitive to (can be treated by) a pKal inhibitor, the method may also include administering an effective amount of a pKal inhibitor to a subject with the disease, such as an anti-pKal antibody (e.g., lanarizumab) or an inhibitory peptide (e.g., icaraltide); a bradykinin 2 receptor inhibitor (e.g., icatibant); and / or C1-INH (e.g., human plasma-derived C1-INH).

[0134] (iii) Non-clinical applications

[0135] Further, the level of any biomarker set described herein can be used for research purposes. Although many diseases associated with the contact activation system have been identified, other diseases may be mediated by similar mechanisms or involve similar components, such as proteins whose levels are affected by lanarizumab treatment. In some embodiments, the methods described herein can be used to identify a disease as being associated with the contact activation system or with a component of the contact activation system (e.g., pKal activity). In some embodiments, the methods described herein can be used to study the mechanism of the disease (e.g., the discovery of novel biological pathways or processes involved in the development of the disease) or progress.

[0136] In some embodiments, the levels of biomarker sets as described herein can be used to develop new therapeutic agents for diseases. For example, the levels of biomarker sets can be measured in samples obtained from subjects to whom a new therapy (e.g., a clinical trial) has been administered. In some embodiments, the levels of biomarker sets can indicate the efficacy of a new therapeutic agent or the progression of a disease in a subject before, during, or after a new therapy.

[0137] Treatment

[0138] Also within the scope of the present disclosure are methods for identifying subjects with a disorder or at risk of a disorder, methods for identifying a subject as a candidate for treatment, and methods for treating a subject and a disorder of a subject. Subjects at risk of or suffering from a disorder (such as a disorder mediated by or associated with any biomarker in Table 1) can be identified using the methods described herein, and can be further treated with any appropriate therapeutic agent. In some embodiments, the methods provided include selecting treatment for a subject based on the output of the method (e.g., measuring the level of a biomarker set).

[0139] In some embodiments, the method comprises selecting or administering 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 the output of the assay (e.g., biomarker detection / level).

[0140] In some embodiments, one or more therapeutic agents are administered to a subject. In some embodiments, one or more plasma kallikrein inhibitors are administered to a subject. In some embodiments, pKal inhibitors are peptides, small molecule inhibitors, kallikrein antibodies or fragments thereof. In some embodiments, an antagonist of bradykinin B2 receptors is administered to a subject. In some embodiments, a C1 esterase inhibitor (C1-INH) is administered to a subject.

[0141] Therapeutic agents (e.g., pKal inhibitors, bradykinin B2 receptor inhibitors and / or C1-INH) can be administered together with another therapy as part of a combination therapy for treating a disease or illness involving a contact activation system. Combination therapy, such as a combination therapy with one or more of a pKal inhibitor, a bradykinin B2 receptor antagonist or a C1-INH substitute, such as a combination therapy with one or more of a pKal inhibitor, a bradykinin B2 receptor antagonist or a C1-INH substitute and another therapy, can be provided in a variety of different configurations. The first medicament can be administered before or after the administration of other therapies. In some cases, the first medicament and another therapy (e.g., therapeutic agent) are administered simultaneously, or in close proximity in time (e.g., a short time interval between injections, such as during the same course of treatment). The first medicament and other therapies can also be administered at larger time intervals.

[0142] Therapeutic agents

[0143] Plasma kallikrein binders (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binders, e.g., small molecules) are useful therapeutic agents for a variety of conditions and disorders (e.g., diseases and disorders associated with the expression of the biomarkers of Table 1). In some embodiments, the disease or disorder is associated with a contact activation system, e.g., involving pKal activity. In some embodiments, the disease or disorder involving plasma kallikrein activity is hereditary angioedema (HAE). In some embodiments, the disease or disorder is not hereditary angioedema. In some embodiments, a plasma kallikrein inhibitor is administered to a subject at risk of or suffering from a disease associated with a contact activation system.

[0144] Many useful protein inhibitors of kallikreins (tissue and / or plasma kallikreins) include Kunitz domains. As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and containing at least two, and preferably three, disulfides. The domain is folded so that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form a disulfide bond (e.g., in a Kunitz domain having 58 amino acids, cysteines may be present at positions corresponding to amino acids 5, 14, 30, 38, 51, and 55, and disulfides may be formed between cysteines at positions 5 and 55, 14 and 38, and 30 and 51, according to the number of BPTI homologous sequences provided below), or, if two disulfides are present, they may be formed between their corresponding subsets of cysteines. According to the numbering of the BPTI sequence provided below, the spacing between each cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino acids corresponding to the following spacing between positions 5 to 55, 14 to 38, and 30 to 51. The BPTI sequence can be used as a reference to refer to a specific position in any general Kunitz domain. Comparison of the Kunitz domain of interest to BPTI can be performed by identifying the best fit alignment in which the number of aligned cysteines is maximized.

[0145] The 3D structure (high resolution) of the Kunitz domain of BPTI is known. One of the X-ray structures is deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of some BPTI homologs (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are known. At least eighty-one Kunitz domain sequences are known. Known human homologs include the three Kunitz 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 Kunitz domains of the inter-α-trypsin inhibitor APP-I (Kido et al., J. Biol. Chem. (1988) 263(34):18104-18107), one Kunitz domain from collagen, and the three Kunitz domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91: 3353-3357), Kunitz domain of hepatocyte growth factor activator inhibitor type 1, Kunitz domain of hepatocyte growth factor activator inhibitor type 2, Kunitz domain described in U.S. Patent Publication No.: 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa (amino acid sequence in Table 2), which contains three Kunitz domains.

[0146] Table 2: Exemplary native Kunitz domains

[0147]

[0148] The above-mentioned Kunitz domains are called LACI-K1 (residues 50 to 107), LACI-K2 (residues 121 to 178) and LACI-K3 (213 to 270). Wun et al. (J. Biol. Chem. (1988) 263 (13): 6001-6004) reported the cDNA sequence of LACI. Girard et al. (Nature (1989) 338: 518-20) reported mutation studies in which the P1 residue of each of the three Kunitz domains was changed. When F.VIIa is complexed with tissue factor, LACI-K1 inhibits factor VIIa (F.VIIa) and LACI-K2 inhibits factor Xa.

[0149] Exemplary Kunitz domain-containing proteins include the following, where the SWISS-PROT accession number is in parentheses:

[0150] A4_HUMAN(P05067), A4_MACFA(P53601), A4_MACMU(P29216), A4_MOUSE(P12023), A4_RAT(P08592 ), A4_SAISC(Q95241), AMBP_PLEPL(P36992), APP2_HUMAN(Q06481), APP2_RAT(P15943), AXP1_ANT AF(P81547), AXP2_ANTAF(P81548), BPT1_BOVIN(P00974), BPT2_BOVIN(P04815), CA17_HUMAN(Q0 2388), CA36_CHICK(P15989), CA36_HUMAN(P12111), CRPT_BOOMI(P81162), ELAC_MACEU(Q62845),

[0151] ELAC_TRIVU(Q29143), EPPI_HUMAN(O95925), EPPI_MOUSE(Q9DA01), HTIB_MANSE(P26227), IBP_CARCR(P00993), IBPC_BOVIN(P00976), IBPI_TACTR(P). 16044)、IBPS_BOVIN(P00975)、ICS3_BOMMO(P07481)、IMAP_DROFU(P11424). )、IP52_ANESU(P10280)、ISC1_BOMMO(P10831)、ISC2_BOMMO(P10832)、ISH1 _STOHE(P31713)、ISH2_STOHE(P81129)、ISIK_HELP(P00994)、ISP2_GALM E(PS1906)、IVB1_BUNFA(P25660)、IVB1_BUNMU(P00987)、IVB1_VIPAA(P009). 91) IVB2_BUNMU(P00989) IVB2_DABRU(P00990) IVB2_HEMMA(P00985) I VB2_NAJNI(P00986)、IVB3_VIPAA(P00992)、IVBB_DENPO(P00983)、IVBC_NA JNA(P19859) 、IVBC_OPHHA(P82966) 、IVBE_DENPO(P00984) 、IVBI_DENAN(P00980) 、IVBI_DENPO(P00979) 、IVBK_DENAN(P00982) IVBK_DENPO(P00981) 、IVBT_ERIMA(P24541)、IVBT_NAJNA(P20229)、MCPI_MELCP(P82968)、SBPI _SARBU(P26228)、SPT3_HUMAN(P49223)、TKD1_BOVIN(Q28201)、TKD1_SHEEP (Q29428), TXCA_DENAN (P81658), UPTI_PIG (Q29100), AMBP_BOVIN (P00978), AMBP_HUMAN (P02760), AMBP_MERUN (Q62577), AMBP_MESAU (Q60559), AMBP _MOUSE(Q07456)、AMBP_PIG(P04366)、AMBP_RAT(Q64240)、IATR_HORSE(P0 4365) IATR_SHEEP(P13371) SPT1_HUMAN(O43278) SPT1_MOUSE(Q9R097)SPT2_HUMAN(O43291)、SPT2_MOUSE(Q9WU03)、TFP2_HUMAN(P48307)、TFP2_MOUSE(O35536)、TFPI_HUMAN(P10646)、TFPI_MACMU(Q28864)、TFPI_MOUSE(O54819)、TFPI_RABIT(P19761)、TFPI_RAT(Q02445)、YN81_CAEEL(Q03610)、

[0152] A variety of methods can be used to identify Kunitz domains from sequence databases. For example, known amino acid sequences, consensus sequences or motifs (e.g., ProSite Motifs) of Kunitz domains can be searched against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda MD), for example, using BLAST searches; against the Pfam database for HMMs (Hidden Markov Models) (e.g., using default parameters for Pfam searches; against the SMART database; or against the ProDom database. For example, Pfam accession number PF00014 of Pfam version 9 provides many Kunitz domains and HMMs for identifying Kunitz domains. A description of the Pfam database can be found in Sonhammer et al. Prot eins (1997) 28(3): 405-420, and a detailed description of the HMM can be found in, for example, Gribskov et al. Meth. Enzymol. (1990) 183: 146-159; Gribskov et al. Proc. Natl. Acad. Sci. USA (1987) 84: 4355-4358; Krogh et al. J. Mol. Biol. (1994) 235: 1501-1531; and Stultz et al. Protein Sci. (1993) 2: 305-314. The SMART database of HMM (Simple Modular Architecture Research Tool, EMBL, Heidelbem, DE) is described in Schultz et al. Proc. Natl. Acad. Sci. USA (1998) 95: 5857 and Schultz et al. Nucl. Acids Res (2000) 28: 231. The SMART database contains domains identified by analysis of hidden Markov models using the HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database has also been annotated and monitored. The ProDom protein domain database consists of an automated compilation of homologous domains (Corpet et al. Nucl. Acids Res. (1999) 27: 263-267).The current version of ProDom was constructed using SWISS-PROT 38 and a recursive PSI-BLAST search of the TREMBL protein database (Altschul et al. Nucleic Acids Res. (1997) 25:3389-3402; Gouzy et al. Computers and Chemistry (1999) 23:333-340). This database automatically generates a consensus sequence for each domain. Prosite lists Kunitz domains as motifs and identifies proteins that include Kunitz domains. See, e.g., Falquet et al. Nucleic Acids Res. (2002) 30:235-238.

[0153] Kunitz domains primarily use amino acids in two loop regions ("binding loops") to interact with target proteases. The first loop region is between approximately residues corresponding to amino acids 13-20 of BPTI. The second loop region is between approximately residues corresponding to amino acids 31-39 of BPTI. An exemplary Kunitz domain library will vary one or more amino acid positions in the first and / or second loop regions. When screening for Kunitz domains that interact with kallikrein or when selecting improved affinity variants, particularly useful positions to vary include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 relative to the BPTI sequence. At least some of these positions are expected to be in close contact with the target protease. It is also useful to vary other positions, for example, positions adjacent to the above positions in the three-dimensional structure.

[0154] The "framework region" of a Kunitz domain is defined as those residues that are part of the Kunitz domain, but specifically excludes residues in the first and second binding loop regions, i.e., approximately residues corresponding to amino acids 13-20 of BPTI and amino acids 31-39 of BPTI. In contrast, residues not in the binding loops may tolerate a wider range of amino acid substitutions (e.g., conservative and / or non-conservative substitutions).

[0155] In one embodiment, these Kunitz domains are variant forms of the ring structure of Kunitz domain 1 of human lipoprotein-associated coagulation inhibitor (LACI) protein. LACI contains three internal, well-defined peptide ring structures, which are paradigmatic Kunitz domains (Girard, T. et al., Nature (1989) 338: 518-520). Variants of Kunitz domain 1 of LACI described herein have been screened, isolated, and bind to kallikrein with enhanced affinity and specificity (see, e.g., U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to other Kunitz domain frameworks to obtain other Kunitz domains that interact with kallikrein (e.g., plasma kallikrein). As determined using kallikrein binding and inhibition assays, useful kallikrein function modulators generally bind and / or inhibit kallikrein.

[0156] In some aspects, plasma kallikrein inhibitors are combined with the active form of plasma kallikrein. In some embodiments, plasma kallikrein inhibitors are combined with and inhibit plasma kallikrein, such as human plasma kallikrein and / or mouse kallikrein. Exemplary polypeptide plasma kallikrein inhibitors are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983 and U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991 and U.S. Publication No. 20110086801, the entire contents of each of these patents are incorporated herein by reference. In some embodiments, plasma kallikrein inhibitors are inhibitory polypeptides or peptides. In some embodiments, the inhibitory peptide is ecallantide (also known as DX-88 or SEQ ID NO:3). In some embodiments, the plasma kallikrein inhibitor comprises or consists of about 58 amino acid sequences of amino acids 3-60 of SEQ ID NO:3 or a DX-88 polypeptide having a 60 amino acid sequence of SEQ ID NO:3:

[0157] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Gly Pro Cys ArgAlaHis Pro Arg Trp Phe Asn Ile Phe Thr Arg Gln Cys Glu Phe Ile Tyr GlyCys GluGly Asn Gln Asn Arg Phe Glu Ser Leu Glu Cys Lys Met Cys ThrArg Asp (SEQ ID NO: 3).

[0158] The plasma kallikrein inhibitor can be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE)) or can include only an antigen binding fragment (e.g., a Fab, F(ab')2, or scFv fragment). The plasma kallikrein binding antibody can include two heavy chains and two light chains, or can be a single-chain antibody. The plasma kallikrein inhibitor can be a recombinant protein, such as a humanized antibody, a CDR-grafted antibody, a chimeric antibody, a deimmunized antibody, or an antibody produced in vitro, and can optionally include a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0159] Exemplary plasma kallikrein binding proteins are disclosed in U.S. 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 known 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 lanaritumomab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein competes for or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein 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 lanarumab. See U.S. Publication No. 2011 / 0200611 and U.S. Publication No. 2012 / 0201756, which are incorporated herein by reference.

[0160] An example of a plasma kallikrein inhibitory antibody is lanariumab ( It may also be referred to as TAK-743, SHP943 or DX-2930). The amino acid sequences of the heavy and light chain variable regions of lanaritumab are provided below, with the CDR regions identified in bold and underlined.

[0161] Lanariumab heavy chain variable region sequence (SEQ ID NO: 4)

[0162]

[0163] Lanariumab light chain variable region sequence (SEQ ID NO: 5)

[0164]

[0165] The heavy and light chain constant and variable sequences of lanariumab are provided below, with the signal sequence in italics. The CDRs are in bold and underlined.

[0166] Lanariumab heavy chain amino acid sequence (451 amino acids, 49439.02 Da)

[0167]

[0168] Lanariumab light chain amino acid sequence (213 amino acids, 23419.08 Da)

[0169]

[0170] Table 4. CDRs of lanariumab

[0171] CDR Amino acid sequence Heavy chain CDR1 HYIMM (SEQ ID NO: 8) Heavy chain CDR2 GIYSSGGITVYADSVKG(SEQ ID NO:9) Heavy chain CDR3 RRIGVPRRDEFDI (SEQ ID NO: 10) Light chain CDR1 RASQSISSWLA (SEQ ID NO: 11) Light chain CDR2 KASTLES (SEQ ID NO: 12) Light chain CDR3 QQYNTYWT (SEQ ID NO: 13)

[0172] In some embodiments, the plasma kallikrein inhibitor may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a plasma kallikrein inhibitor described herein. In some embodiments, the plasma kallikrein inhibitor may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a plasma kallikrein inhibitor described herein in the HC and / or LC framework regions (e.g., HC and / or LC FR 1, 2, 3 and / or 4). In some embodiments, the plasma kallikrein inhibitor may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein in the HC and / or LC CDRs (e.g., HC and / or LC CDR1, 2, and / or 3). In some embodiments, the plasma kallikrein inhibitor may have at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein in the constant region (e.g., CH1, CH2, CH3, and / or CL1).

[0173] In some aspects, the small molecule binds to and inhibits the active form of plasma kallikrein.

[0174] Bradykinin B2 receptor inhibitors

[0175] In some embodiments, a bradykinin B2 receptor inhibitor (eg, antagonist) is administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant It is a peptide mimetic drug containing 10 amino acids that blocks the binding of native bradykinin to the bradykinin B2 receptor.

[0176] C1-INH replacement

[0177] In some embodiments, a C1 esterase inhibitor (C1-INH), such as an alternative C1-INH agent, is administered to a subject. Exemplary C1-INH alternative agents are publicly available and include, for example, human plasma-derived C1-INH, such as and

[0178] Kits and assays for measuring protein biomarker panels

[0179] The present disclosure also provides kits and detection devices for measuring the levels of biomarker sets as described herein. Such kits or detection devices may include binding agents that specifically bind to protein biomarkers (such as those listed in Table 1). For example, such kits or detection devices 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 binding agents that are specific for all members of the protein biomarker set described herein.

[0180] In some embodiments, one or more of these binding agents is an antibody that specifically binds to a protein of a biomarker set. In some embodiments, one or more binding agents is an aptamer, such as a peptide aptamer or an oligonucleotide aptamer, that specifically binds to a protein of a biomarker set.

[0181] In some embodiments, the kit further comprises a detector (e.g., an antibody bound to a binding agent) for detecting the binding of an agent to a protein of a biomarker set. The detector may be conjugated to a label. In some embodiments, the detector is an antibody that specifically binds to at least one of the binding agents. In some embodiments, the binding agent comprises a label that can be identified and directly or indirectly bound by the detector.

[0182] In some embodiments, the kit may further include a support member, for example, for performing any of the methods described herein. 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 a Western blot assay format or a lateral flow assay format.

[0183] 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 (e.g., 24-, 48-, 96-, 384- or more well plates) can be used for high-throughput immunoassays. Individual immunoassays can be performed in parallel in each well. Therefore, it is generally desirable to use a plate reader to measure multiple wells in parallel to increase the assay throughput. In some embodiments, a plate reader capable of imaging multiple wells (e.g., 4, 16, 24, 48, 96, 384 or more wells) in parallel can be used for the platform. For example, a commercially available plate reader (e.g., the plate::vision system of Perkin Elmer, Waltham, MA) can be used. This plate reader is capable of performing kinetic-based fluorescence analysis. The plate::vision system has high collection efficiency optics and has special optics designed for parallel analysis of 96 wells. Other suitable parallel plate readers include, but are not limited to, SAFIRE (Tecan, San Jose, CA), (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ) and CellLux (Perkin Elmer, Waltham, MA).

[0184] In the kit or detection device, one or more of the binding agents can be immobilized on a support member (e.g., a membrane, a bead, a slide, or a multiwell plate). The selection of a suitable support member for use in an immunoassay will depend on factors such as the number of samples and the method of detecting the signal released from the label conjugated to the second agent.

[0185] The kit may also include one or more buffers as described herein, but not limited to a coating buffer, a blocking buffer, a wash buffer, and / or a stop buffer.

[0186] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of how to use the components contained in the kit to measure protein levels of a biomarker set in a biological sample collected from a subject (such as a human patient).

[0187] The instructions associated with the use of the kit typically include information about the amount of each component and suitable conditions for carrying out the assay methods described herein. The components in the kit can be unit doses, bulk packages (e.g., multi-dose packages), or subunit doses. The instructions provided in the kit of the present disclosure are typically written instructions on a label or package insert (e.g., paper included in the kit), but machine-readable instructions (e.g., instructions carried on a magnetic or optical storage disk) are also acceptable.

[0188] The label or package insert indicates that the kit is used to assess protein levels of a biomarker panel.Instructions for practicing any of the methods described herein may be provided.

[0189] The kit of the present disclosure is in a suitable package. Suitable packages include, but are not limited to, vials, bottles, cans, flexible packages (e.g., sealed Mylar or plastic bags), etc. Packages used in combination with specific devices such as inhalers, nasal application devices (e.g., nebulizers), or infusion devices (such as miniature pumps) are also contemplated. The kit can have a sterile inlet port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). The container can also have a sterile inlet port (e.g., the container can be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle).

[0190] The kit may optionally provide additional components, such as explanatory information, such as controls and / or standards or reference samples. Typically, the kit includes a container and a label or package insert on or connected to the container. In some embodiments, the disclosure provides an article comprising the contents of the above-mentioned kit.

[0191] Without further elaboration, it is believed that, based on the above description, those skilled in the art can make the most of the present disclosure. Therefore, the following specific embodiments should be interpreted as being merely illustrative and not limiting the remainder of the present disclosure in any way. For the purpose of the subject matter mentioned herein, all publications cited herein are incorporated by reference.

[0192] Example

[0193] Example 1: Identification of proteins differentially present in samples from HAE patients compared to healthy individuals

[0194] Despite the clinical validation of the role of the kallikrein-kinin system (KKS) in the pathophysiology of hereditary angioedema (HAE) C1 esterase inhibitor (C1-INH) (HAE-C1-INH), novel disease state biomarkers for the diagnosis and evaluation of HAE patients remain to be identified. Such biomarkers can be used to further elucidate the disease biology of HAE and other plasma kallikrein (pKal)-mediated diseases, for the development of improved diagnostic assays, and for the identification of additional indications other than HAE that may be sensitive to treatment with established therapeutic agents (such as lanariumab). In order to identify previously unidentified HAE biomarkers, the plasma proteome of HAE subjects was analyzed before (baseline) and after 6 months (26 weeks) of lanariumab treatment and compared with the levels of analytes present in plasma samples from healthy control subjects (subjects without HAE).

[0195] Positive determination of HAE in patients was performed by collecting plasma samples from subjects and measuring the plasma levels of complement component 4 (C4) and functional C1-INH by ELISA and chromogenic assays known in the art, respectively. Plasma proteomics analysis was performed using a multiplex approach that was able to assess the relative levels of 7,113 proteins using DNA aptamers specific for each protein assessed (SOMALogic, Denver, CO, USA), and comparing plasma samples obtained from healthy control subjects (n = 30), plasma samples obtained from HAE patients (HAE C1-INH) before lanariumab treatment (baseline, n = 125), and plasma samples obtained from HAE patients after lanariumab treatment of 300 mg lanariumab every two weeks (week 26, post-treatment, n = 114). Among the 114 HAE-C1-INH subjects for whom baseline / pre-dose plasma was available, 112 subjects had matched post-dose samples available.

[0196] Plasma collection

[0197] Citrated plasma samples from healthy controls were matched for age and sex (n = 30; BioIVT, Westbury, NY, USA). Citrated plasma samples from HAE-C1-INH subjects before lanarumab treatment (n = 125) were collected as part of a Phase III study (HELP study) and as non-rollover samples from an open-label extension study (open-label extension of the HELP study) (National Institutes of Health identifiers NCT02586805 and NCT02741596, respectively). Citrated plasma samples from HAE-C1-INH subjects were collected after 26 weeks of lanarumab treatment (300 mg lanarumab administered every 2 weeks; n = 114), of which 112 subjects had patient-matched pre- and post-treatment samples available. Plasma (sodium citrate) was collected from age- and sex-matched healthy controls (n = 30) by BioIVT (Westbury, NY). In order to minimize the ex vivo activation of the contact pathway system during blood collection, plasma was collected from subjects with HAE-C1-INH and healthy controls by clean venipuncture with a butterfly needle / catheter kit (BD Biosciences) and removing the tourniquet while the blood was flowing to reduce stagnation. The first tube of blood was discarded and the blood was collected into polypropylene vacuum tubes containing 3.8% sodium citrate (BD Biosciences, San Jose, CA, USA). The blood samples were centrifuged within 1 hour, and the plasma was aliquoted and stored at -80°C until processing.

[0198] ELISA

[0199] To verify the differentially expressed HAE biomarkers, protein levels were assessed by ELISA. A2M was measured in plasma samples using a commercially available ELISA kit (Raybiotech, catalog number ELH-A2M). APOB was measured in plasma samples using a commercially available ELISA kit (Raybiotech, catalog number ELH-ApoB). IL21 was measured in plasma samples using a commercially available ELISA kit (Raybiotech, catalog number ELH-IL21).

[0200] result

[0201] Of the 7,113 proteins evaluated, 1,041 were identified as having statistically significant differential levels in samples obtained from HAE patients at baseline compared with healthy controls ( Figure 1As validation of this approach, various biomarkers previously known to be differentially present during HAE were identified in this panel. For example, C1-INH and C4 are each biomarkers used in the laboratory diagnosis of HAE-C1-INH (Grumach et al. (2021) Front Immunol, 12:785736), and were found to be decreased in HAE baseline samples (see Figure 2 and Figure 3 Also observed in the analysis was cleaved high molecular weight kininogen (cHMWK), which is known to be elevated during HAE-C1-INH and decreased after lanariumab treatment ( Figure 4A and 4B ). See, e.g., 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) NEngl J Med., 376: 717-728. Several additional proteins previously reported to be differentially present during the HAE-C1-INH disease state were also observed in the analysis, including thrombin, ITH4, IL-36A, and IL-21 (see Figure 5-8 ). See, e.g., 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.

[0202] Statistical analysis was performed to identify proteins that differed between samples from HAE-C1-INH patients compared to healthy subjects, as well as to identify proteins that did not differ after lanarumab treatment (i.e., returned to control levels). As shown in Table 1, it was further found that the levels of 120 proteins were significantly different in HAE-C1-INH plasma compared to healthy controls at baseline, but were not significantly different after 26 weeks of treatment with lanarumab (normalized). These proteins included several biomarkers previously known to differ significantly during HAE-C1-INH, namely cleaved HMWK (cHMWK; Figure 4A ), thrombin (F2; Figure 5 ), inter-α-trypsin inhibitor heavy chain H4 (ITH4; Figure 6 )、IL-21( Figure 8), as well as a number of proteins not previously identified as differentially present during HAE-C1-INH and / or after treatment with plasma kallikrein inhibitors such as lanariumab. Additional proteins identified included alpha-macroglobulin (A2M; Fig. 10A ), kininogen (KNG1; Fig. 9B , 10D and 13A-13H), complement component C3 (C3; Figures 14A-14D ) and plasminogen (PLG; data not shown).

[0203] As an example of how these 120 proteins were identified, DNA aptamers that specifically bind to cHMWK (ID 15343-337 and ID 19631-13; Table 1 ; Figures 13A-13D ) showed that after 26 weeks of treatment of HAE-C1-INH subjects with lanarumab, cHMWK levels were elevated in HAE-C1-INH baseline plasma but no longer different from healthy control plasma levels. DNA aptamers generated against intact HMWK (without additional specificity information provided by SOMALogic) showed similar relative expression levels in plasma samples from healthy controls, HAE-C1-INH patients at baseline, and HAE-C1-INH patients after 26 weeks of lanarumab treatment (ID 4918-21; Table 1; Figures 13E-13F A second DNA aptamer selective for intact HMWK (which was observed to bind cHMWK and low molecular weight kininogens with up to 1 / 10 lower affinity than intact HMWK) showed reduced expression levels in HAE-C1-INH baseline plasma relative to healthy controls and increased expression levels in HAE-C1-INH subjects treated with lanariumab for 26 weeks (ID 7784-1; Table 1; Figures 13G-13H ).

[0204] Complement C3 (C3) is another example from the list of 120 proteins identified as potential biomarkers by proteomics using HELP plasma samples. Increased expression levels of C3 and C3 fragments were observed in baseline plasma samples of HAE-C1-INH subjects compared with healthy controls ( Figures 14A-14D ). C3b expression level (ID 4480-59; Table 1; Fig. 14B ) was elevated in baseline plasma samples of HAE-C1-INH subjects and decreased after 26 weeks of receiving 300 mg of lanariumab every 4 weeks.

[0205] These proteins also include a number of biomarkers that have not previously been identified as being significantly different during HAE-C1-INH or normalized to levels comparable to healthy subjects following treatment with plasma kallikrein inhibitors such as lanariumab. Examples of such proteins include apolipoprotein B (APOB; Fig.9A ), G antigen 2 (GAGE2B; Fig. 9C ), mortality factor 4-like protein 2 (MORF4L2; Fig.9D ), complement C1q, and tumor necrosis factor-related protein 9A (C1QTNF9; Fig.9E ), plasma serine protease inhibitor (SERPINA5; Fig.9F ) and cadherin-15: cytoplasmic domain (CDH15; Figure 9G ), α-2-macroglobulin (A2M; Fig. 10A ), interleukin-12 (IL12A-IL12B; Fig. 10B ) and liver-expressed antimicrobial peptide 2 (LEAP2; Fig. 10C )( Figures 9A-9G , 10A-10C; Table 1). Table 1 also provides the gene name, UniProt identifier, and the protein expression level used to analyze each protein. Identifier of the aptamer.

[0206] To determine whether these 120 proteins represent components of pathways that are functionally relevant to the pathophysiology of HAE, pathway enrichment analysis was performed using the Metabase process network (bio.tools / metabase; Bolser et al., (2012) Nucleic Acids Res, 40: D1250-D1254) and a known knowledge network method called CASnet (consistently active subnetworks of known protein-protein interactions). Proteins functionally involved in the kallikrein-kinin system were the most significantly enriched. However, proteins involved in coagulation, cell adhesion, cytokines, cardiovascular proteins, and proteolysis (including proteases and proteolysis inhibitors) pathways were also found to be significantly enriched, indicating that these pathways are also dysregulated during HAE-C1-INH and become normalized after lanariumab treatment. Tables 3 and 5 and Fig.11 and 12 Summary results of the Metabase pathway enrichment analysis are provided in .

[0207] CASnet incorporates the directionality of changes in protein levels, with proteins in red being elevated and proteins in blue being decreased when HAE baseline is compared to healthy control plasma ( Fig.12). The designated proteins were added to the active subnetwork by known pathway association. Proteins identified as potential disease state biomarkers affected by 26 weeks of lanaritumab treatment by CASnet analysis included proteases and protease inhibitors (e.g., thrombin, tissue kallikrein 14, inter-alpha trypsin inhibitor heavy chain 4, alpha 2-macroglobulin), apolipoproteins, and complement system proteins.

[0208] Table 3: Metabase pathway analysis of 120 proteins differentially expressed in plasma samples from HAE patients before but not after lanaritumab treatment

[0209]

[0210] Pathway enrichment analysis was performed using the hypergeometric test, where “r” represents the number of proteins belonging to a given pathway, “R” represents the total number of proteins tested, “n” represents the number of proteins in a given pathway, and “N” represents the total number of proteins in all pathways.

[0211] Table 5: Pathway analysis comparing HAE-C1-INH baseline to healthy control plasma

[0212]

[0213] *Number of proteins identified out of 1041 proteins identified in each pathway. Values ​​in parentheses are percentages of total proteins in the pathway.

[0214] Proteomic analysis identified 120 proteins whose presence levels differed between samples from patients with HAE and healthy individuals (controls) but normalized after 6 months (26 weeks) of lanariumab treatment.

[0215] For the three proteins identified herein (A2M, APOB, and IL21), 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) and HAE-C1-INH subjects from the HELP study (NCT02586805) at baseline (before receiving lanarumab) and after receiving 150 mg or 300 mg lanarumab once monthly up to day 98 after the first dose ( Figure 15A-1 5D).

[0216] The results of the analysis are as follows Fig.15A (A2M), Fig. 15B (APOB) and Fig. 15C(IL21). Plasma concentrations of A2M in HAE-C1-INH subjects were significantly different from those in healthy controls (Kruskal-Wallis test, p<0.05), but in contrast to SomaScan measurements of A2M, there were no differences between Day 0 and Day 98 following lanarumab treatment. Also in contrast to SomaScan measurements, concentrations of APOB and IL21 measured by ELISA were not significantly different from healthy controls or between Day 0 and Day 98 for each dose of lanarumab.

[0217] Further studies could elucidate whether somamers targeting A2M also bind to the A2M-PKa covalent complex to determine whether the observed increase in A2M is due to an increase in free A2M protein or in complex with proteases. Plasma A2M concentrations in plasma before HAE-C1-INH administration were also observed to be significantly higher than those in healthy controls. However, A2M concentrations in plasma before HAE-C1-INH administration did not decrease with lanariumab treatment by ELISA. The lack of reduction in A2M plasma levels observed by ELISA due to lanarumab treatment can be attributed to several considerations: 1) Results from different immunoassays can be inconsistent with each other, so the discordance between ELISA and SomaScan results does not clearly imply that one is superior; 2) Differences in assay characteristics (e.g., target immobilization and washing conditions) can affect comparisons 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 a higher dose of lanarumab (300 mgq2wk) for a longer period of time (6 months) compared to the plasma samples used in the ELISA analysis (300 mg or 150 mg q4wk for 98 days), suggesting that higher and longer exposure to lanarumab is required to reduce A2M. The same plasma samples used to measure A2M by ELISA were also used to measure IL21 and APOB. However, no increase in plasma levels of HAE-C1-INH was observed in subjects by ELISA, which could be attributed to the above-mentioned A2M.

[0218] Any of the proteins identified herein can be used as a biomarker (alone or combined as a biomarker set) for identifying a disease associated with a contact activation system, for example, in a method for identifying patients at risk for a disease associated with a contact activation system (e.g., HAE), selecting candidates for treatment, monitoring disease progression or disease state, evaluating the efficacy of a treatment for a disease, determining a course of treatment, identifying whether a disease or disorder is associated with a contact activation system, and / or for research purposes (including, for example, studying the mechanisms of a disease, which may be relied upon for developing new therapies).

[0219] Other Implementations

[0220] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0221] From the above description, those skilled in the art can easily determine the basic characteristics of the present disclosure, and without departing from its spirit and scope, various changes and modifications can be made to the present disclosure to adapt it to various uses and conditions. Therefore, other embodiments are also within the scope of the claims.

[0222] Equivalent solutions and scope

[0223] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation numerous equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0224] In the claims, articles such as "a", "an", and "the" may refer to one or more than one, unless indicated to the contrary or otherwise apparent from the context. Claims or descriptions containing "or" between one or more members of a group are deemed to be satisfied if one, more than one, or all of the members of the group are present in, used in, or otherwise related to a specified product or process, unless indicated to the contrary or otherwise apparent from the context. The present disclosure includes embodiments in which exactly one member of the group is present in, used in, or otherwise associated with a given product or process. The present disclosure includes embodiments in which 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.

[0225] In addition, the present disclosure encompasses all variations, combinations and arrangements, wherein one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim subordinate to another claim can be modified to be included in one or more limitations found in any other claim subordinate to the same basic claim. In the case where the element is presented in a list form such as a Markush group format, each subgroup of the element is also disclosed, and any one or more elements can be removed from the group. It should be understood that, in general, in the case where the present disclosure or aspects of the present disclosure are referred to as including specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are composed of such elements and / or features, or are substantially composed of such elements and / or features. For simplicity, those embodiments are not specifically described in the same words herein. It should also be noted that the terms "including" and "containing" are intended to be open and allow the inclusion of additional elements or steps. When a range is given, the end value is included. In addition, unless otherwise indicated or otherwise obvious from the context and understanding of one of ordinary skill in the art, values ​​expressed as ranges can be assumed in different embodiments of the present disclosure to any specific value or sub-range within the stated range, to the tenth of the unit of the lower limit of the stated range, unless the context clearly dictates otherwise.

[0226] This application relates to various granted patents, published patent applications, journal articles and other publications, which are all incorporated herein by reference. If there is a conflict between any incorporated reference and this specification, the specification shall prevail. In addition, any specific embodiment of the present disclosure that falls within the prior art can be explicitly excluded from any one or more claims. Because such embodiments are considered to be known to those of ordinary skill in the art, even if such exclusion is not explicitly proposed herein, it can also be excluded. For any reason, whether or not it is related to the existence of the prior art, any specific embodiment of the present disclosure can be excluded from any claim.

[0227] Those skilled in the art will recognize or be able to determine many equivalents to the specific embodiments described herein using no more than routine experimentation. The scope of the present embodiments described herein is not intended to be limited to the above description, but as described in the appended claims. Those of ordinary skill in the art will appreciate that various modifications and changes may be made to this description without departing from the spirit or scope of the present disclosure as defined in the appended claims.

Claims

1. A method for determining whether a condition is susceptible to treatment with a plasma kallikrein (pKal) inhibitor, comprising: measuring the level of a biomarker set comprising at least one protein selected from Table 1 in a biological sample of a subject suffering from the condition, and If the levels of the biomarker set deviate from the reference values, the condition is identified as being sensitive to treatment with a pKal inhibitor.

2. The method of claim 1, further comprising administering the pKal inhibitor to the subject if the condition is identified as being susceptible to treatment with the pKal inhibitor.

3. A method for identifying a subject as a candidate for treatment with a plasma kallikrein (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 if the level of the biomarker set in the biological sample deviates from a reference value, then the subject is identified as a candidate for treatment with the pKal inhibitor.

4. The method of claim 3, further comprising administering the pKal inhibitor to a subject identified as a candidate for treatment.

5. A method for identifying a subject as having a disorder or being 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 if the level of the biomarker set in the biological sample deviates from the level of the biomarker set in a control sample, the subject is identified as having or being at risk of having the disorder.

6. The method of claim 5, further comprising administering to the subject an effective amount of a plasma kallikrein (pKal) inhibitor if the subject is identified as having or being at risk for the condition.

7. A method for treating a condition in a subject, comprising administering to the subject an effective amount of a plasma kallikrein (pKal) inhibitor, wherein the level of the biomarker set in the subject deviates from the level of the biomarker set in a control sample, Wherein the biomarker set comprises at least one protein selected from Table 1.

8. The method of claim 7, wherein the disorder is not hereditary angioedema (HAE).

9. The method of any one of claims 1 to 8, wherein the pKal inhibitor is lanaritumomab.

10. The method of any one of claims 1 to 9, wherein the biomarker panel consists of 2-10 proteins selected from Table 1.

11. The method of any one of claims 1 to 10, wherein the biological sample is a serum sample or a plasma sample.

12. The method of any one of claims 1 to 11, wherein the condition is a disease associated with a contact activation system.

13. The method of any one of claims 1 to 7 or 9 to 12, wherein the disorder is hereditary angioedema (HAE).

14. The method of any one of claims 1 to 7 or 9 to 13, wherein the disorder is HAE Type I or HAE Type II.

15. The method of any one of claims 1 to 14, wherein the at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1) and kininogen.

16. The method of any one of claims 1 to 15, wherein the 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).

17. The method of any one of claims 1 to 16, wherein 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), multimeric protein-2 (MMRN2), olfactory stromal protein-like 3 (OLFML3) and protocadherin gamma-C3 (PCDHGC3).

18. The method of any one of claims 1 to 17, wherein the at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3).

19. The method of any one of claims 1 to 18, wherein 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).

20. The method of any one of claims 1 to 19, wherein 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), 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).

21. The method of any one of claims 1 to 20, wherein the biological sample is provided in an evacuated blood collection tube comprising one or more protease inhibitors.

22. The method of any one of claims 1 to 21, wherein the level of the biomarker panel is measured by enzyme-linked immunosorbent assay (ELISA), immunoblot assay, or lateral flow assay.

23. The method of any one of claims 1 to 22, wherein the subject is a human patient.

24. A composition for treating a subject suffering from a disorder, the composition comprising a plasma kallikrein (pKal) inhibitor, wherein the level of the biomarker set in the subject deviates from the level of the biomarker set in a control sample, Wherein the biomarker set comprises at least one protein selected from Table 1.

25. The composition of claim 24, wherein the pKal inhibitor is lanaritumomab.

26. The composition of claim 24 or 25, wherein the biomarker panel consists of 2-10 proteins selected from Table 1.

27. A composition as described in any one of claims 24 to 26, wherein the condition is a disease associated with a contact activation system.

28. The composition of any one of claims 24 to 27, wherein the disorder is hereditary angioedema (HAE).

29. The composition of claim 28, wherein the disorder is HAE Type I or HAE Type II.

30. The composition of any one of claims 24 to 29, wherein the at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1), and kallikrein.

31. The composition of any one of claims 24 to 30, wherein the 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).

32. The composition of any one of claims 24 to 31, wherein 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), multimeric protein-2 (MMRN2), olfactory stromal protein-like protein 3 (OLFML3) and protocadherin gamma-C3 (PCDHGC3).

33. The composition of any one of claims 24 to 32, wherein the at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3).

34. The composition of any one of claims 24 to 33, wherein 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).

35. The composition of any one of claims 24 to 34, wherein 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), 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).

36. The composition of any one of claims 24 to 35, wherein the level of the biomarker panel is measured by enzyme-linked immunosorbent assay (ELISA), immunoblot assay, or lateral flow assay.

37. The composition of any one of claims 24 to 36, wherein the subject is a human patient.

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

39. The method of claim 38, wherein the treatment comprises administering to the subject a plasma kallikrein (pKal) inhibitor.

40. The method of claim 39, wherein the pKal inhibitor is lanaritumomab.

41. The method of any one of claims 38 to 40, wherein the biomarker panel consists of 2-10 proteins selected from Table 1.

42. The method of any one of claims 38 to 41, wherein the biological sample is a serum sample or a plasma sample.

43. The method of any one of claims 38 to 42, wherein the at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1), and kallikrein.

44. The method of any one of claims 38 to 43, wherein the 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).

45. The method of any one of claims 38 to 44, wherein 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), multimeric protein-2 (MMRN2), olfactory stromal protein-like 3 (OLFML3) and protocadherin gamma-C3 (PCDHGC3).

46. ​​The method of any one of claims 38 to 45, wherein the at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3).

47. The method of any one of claims 38 to 46, wherein the at least one protein is a complement activating protein selected from the group consisting of complement CIq and tumor necrosis factor-related protein 9A (C1QTNF9).

48. The method of any one of claims 38 to 47, wherein 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), 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).

49. The method of any one of claims 38 to 48, wherein the biological sample is obtained in an evacuated blood collection tube comprising one or more protease inhibitors.

50. The method of any one of claims 38 to 49, wherein the level of the biomarker panel is measured by enzyme-linked immunosorbent assay (ELISA), immunoblot assay, or lateral flow assay.

51. The method of any one of claims 38 to 50, wherein the subject is a human patient.

52. The method of any one of claims 38 to 51, wherein the subject has, is suspected of having, or is at risk of a disease associated with a contact activation system.

53. The method of claim 52, wherein the disease associated with a contact activation system is hereditary angioedema (HAE).

54. The method of claim 53, wherein the HAE is type I HAE or type II HAE.

55. The method of claim 54, wherein the disease associated with exposure to an activated system is not HAE.

56. A method for analyzing a sample, the method comprising: (i) providing a collection of biological samples obtained from subjects having, suspected of having, or at risk of having a disease associated with an activated system; as well as (ii) measuring the level of a biomarker set comprising at least one protein selected from Table 1, wherein if the biomarker set consists of one protein, the protein is not high molecular weight kininogen (KNG1), alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin or interleukin-21 (IL-21).

57. The method of claim 56, wherein the biomarker panel consists of 2-10 proteins selected from Table 1.

58. The method of claim 56 or 57, wherein the biological sample is a serum sample or a plasma sample.

59. The method of any one of claims 56 to 58, wherein the disease associated with a contact activation system is hereditary angioedema (HAE).

60. The method of claim 59, wherein the HAE is type I HAE or type II HAE.

61. The method of any one of claims 56 to 60, wherein the at least one protein is a kallikrein-kinin system protein selected from the group consisting of kallikrein-13 (KLK13), kallikrein-14 (KLK14), 2-chain high molecular weight kininogen (KNG1), and kallikrein.

62. The method of any one of claims 56 to 61, wherein the 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).

63. The method of any one of claims 56 to 62, wherein 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), multimeric protein-2 (MMRN2), olfactory stromal protein-like protein 3 (OLFML3) and protocadherin gamma-C3 (PCDHGC3).

64. The method of any one of claims 56 to 63, wherein the at least one protein is a proteolysis-related protein selected from the group consisting of proteasome subunit beta 6 type (PSMB6), ubiquitin conjugating enzyme E2 R2 (UBE2R2), ubiquitin-protein ligase E3A (UBE3A), ubiquitin conjugating factor E4 A (UBE4A) and E3 ubiquitin-protein ligase ZNRF3 (ZNRF3).

65. The method of any one of claims 56 to 64, wherein the at least one protein is a complement activating protein selected from the group consisting of complement CIq and tumor necrosis factor-related protein 9A (C1QTNF9).

66. The method of any one of claims 56 to 65, wherein 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), 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).

67. The method of any one of claims 56 to 66, wherein step (i) comprises collecting the biological sample into an evacuated blood collection tube comprising one or more protease inhibitors.

68. The method of any one of claims 56 to 67, wherein step (ii) is performed using an enzyme-linked immunosorbent assay (ELISA), an immunoblot assay, or a lateral flow assay.

69. The method of any one of claims 56 to 68, wherein the subject is a human patient.

70. The method of any one of claims 56 to 69, further comprising identifying the subject as a patient suffering from the disease associated with a contact activation system if the level of the biomarker set in the subject deviates from the level of the same biomarker set in a control subject.

71. The method of claim 70, further comprising administering to the subject an effective amount of a therapeutic agent if the subject is identified as having the disease.

72. The method of any one of claims 56 to 71, wherein the subject is a human patient to whom a treatment for the disease has been administered, and the method further comprises 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 course of treatment with the levels of the same set of biomarkers measured in a biological sample obtained from the subject before treatment; Wherein the treatment is determined to be effective if the level of the set of biomarkers in the sample obtained after or during the course of treatment deviates from the level of the set of biomarkers in the sample obtained before treatment.

73. The method of any one of claims 56 to 72, wherein the subject is a human patient to whom a treatment for the disease has been administered, and the method further comprises 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 course of treatment with the levels of the same set of biomarkers measured in a control sample obtained from a healthy subject; Wherein the treatment is determined to be effective if the level of the biomarker set in the sample obtained after or during the course of the treatment does not substantially deviate from the level of the biomarker set in the control sample.

74. The method of claim 72 or 73, further comprising administering to the subject an effective amount of a therapeutic agent or an increasing dose of the therapeutic agent if the treatment is determined to be ineffective.

75. The method of claim 74, wherein the therapeutic agent is a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R) inhibitor, and / or a C1 esterase inhibitor.

76. The method of claim 75, wherein the pKal inhibitor is an anti-pKal antibody or an inhibitory peptide.

77. The method of claim 76, wherein the pKal inhibitor is lanaritumomab or ecallantide.

78. The method of claim 75, wherein the B2R inhibitor is an inhibitory peptide.

79. The method of claim 76, wherein the inhibitory peptide is icatibant.

80. The method of claim 75, wherein the C1 esterase inhibitor is a human plasma-derived C1 esterase inhibitor.

81. 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 comprising: (i) a first binding agent specific for a first protein biomarker selected from Table 1; and (ii) a second binding agent specific for a second protein biomarker selected from Table 1; wherein the first protein biomarker and the second protein biomarker are different and are not both selected from the group consisting of 2-chain high molecular weight kininogen (KNG1), alpha-2-macroglobulin (A2M), complement C1q and tumor necrosis factor-related protein 9A (C1QTNF9), thrombin and interleukin-21 (IL-21).

82. The kit of claim 81, further comprising a first detection agent that binds to the first binding agent and a second detection agent that binds to the second binding agent.

83. The kit of claim 81 or 82, wherein the first binding agent is an antibody specific for the first protein biomarker, and / or the second binding agent is an antibody specific for the second protein biomarker.

84. The kit of claim 81 or 82, wherein the first binding agent is an aptamer specific for the first protein biomarker; and / or the second binding agent is an aptamer specific for the second protein biomarker.

85. The kit of any one of claims 81 to 84, wherein the first binding agent and the second binding agent are immobilized on a support member.

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