Immunoassay for detecting cleaved high-molecular-weight kininogen

Abstract

To develop an assay for detecting a level of cleaved HMWK in a biological sample, which has high sensitivity and is reliable.SOLUTION: Provided is an immunoassay for detecting cleaved high-molecular-weight kininogen (HMWK) with high sensitivity and specificity. A method includes: (i) preparing a support element to which a first agent (for example, an antibody such as 559B-M004-B04) specifically binding to cleaved HMWK is bound; (ii) contacting the support element of (i) with a biological sample suspected of containing cleaved HMWK; (iii) contacting the support element obtained in (ii) with a second agent which is conjugated with a label and binds to HMWK; and (iv) detecting a signal released from the label of the second agent which is bound directly or indirectly to the support element, in order to determine a level of cleaved HMWK in the biological sample.SELECTED DRAWING: Figure 9

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Patent Application No. 62 / 243,505, filed October 19, 2015, and U.S. Provisional Patent Application No. 62 / 335,311, filed May 12, 2016, each of which is incorporated herein by reference in its entirety. [Background technology]

[0002] Kininogens are precursors of kinins such as bradykinin and kallidin. There are two splicing variants of kininogen in humans: high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK). HMWK primarily functions as a cofactor in coagulation and inflammation and is the preferred substrate for plasma kallikrein (pKal)-mediated bradykinin production.

[0003] Plasma kallikrein (pKal) is the major bradykinin-generating enzyme in the circulation. Activation of pKal occurs via the contact pathway, which has been implicated in the pathology of hereditary angioedema (HAE). pKal cleaves HMWK (a single-chain polypeptide) to produce bradykinin and a truncated form of HMWK containing two polypeptide chains linked by disulfide bonds. Cugno et al., Blood (1997) 89:3213-3218.

[0004] During attacks of hereditary angioedema (HAE), cleaved HMWK increases to approximately 47% of total kininogen (Cugno et al., Blood (1997) 89:3213-3218), making it a biomarker for monitoring HAE attacks. Therefore, there is interest in developing a sensitive and reliable assay to detect the level of cleaved HMWK in biological samples. Summary of the Invention

[0005] Some embodiments of the present disclosure provide an immunoassay for detecting cleaved high molecular weight kininogen (HMWK) with high sensitivity and specificity. The method includes: (i) preparing a support element to which a first agent (e.g., an antibody such as 559B-M004-B04) that specifically binds to cleaved HMWK is bound; (ii) contacting the support element of (i) with a biological sample suspected of containing cleaved HMWK; (iii) contacting the support element obtained in (ii) with a second agent that is conjugated to a label and binds to HMWK; and (iv) detecting a signal emitted from the label of the second agent directly or indirectly bound to the support element to determine the level of cleaved HMWK in the biological sample. In some cases, step (ii) may be performed in the presence of ZnCl.

[0006] In some embodiments, the support element of (i) is incubated with a blocking buffer prior to step (ii).

[0007] In some embodiments, the second agent is a polyclonal antibody, a monoclonal antibody, or a mixture of two or more monoclonal antibodies that bind to HMWK. Two or more monoclonal antibodies in the mixture may bind to different epitopes of HMWK. In some embodiments, the label is a signal-emitting substance. In some embodiments, the label is a member of a receptor-ligand pair. In this case, the immunoassay further includes, prior to step (iv), contacting the second agent (iii) immobilized on a support element with the other member of the receptor-ligand pair, wherein the other member is conjugated with a signal-emitting substance. In one example, the receptor-ligand pair is biotin and streptavidin.

[0008] Another aspect of the present disclosure provides a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample, the method comprising: (i) contacting a sample suspected of containing cleaved HMWK with any of the antibodies described herein (e.g., 559B-M004-B04); (ii) measuring the cleaved HMWK-antibody complex formed in step (i); and (iii) determining the level of cleaved HMWK in the sample based on the results of step (ii). In some embodiments, step (i) is performed in the presence of ZnCl. In some embodiments, step (i) is performed using enzyme-linked immunosorbent assay (ELISA) or immunoblotting.

[0009] In any of the methods described herein, the sample can be a biological sample collected from a subject (e.g., a human patient), such as a serum sample of a plasma sample, etc. In some embodiments, the method can further include collecting the sample in an evacuated blood collection tube containing one or more protease inhibitors.

[0010] Any of the assay methods (eg, immunoassays) described herein can be an ELISA assay, a Western blot assay, or a lateral flow assay.

[0011] In some embodiments, a biological sample is collected from a subject (e.g., a human patient) with a disease. The assay method can further include determining whether the disease is mediated by plasma kallikrein based on the level of cleaved HMWK, where a deviation in the level of cleaved HMWK in the sample from that in a control sample indicates that the disease is mediated by plasma kallikrein.

[0012] Any of the assay methods described herein may further include identifying a patient with a plasma kallikrein-mediated disease or disorder or evaluating the effectiveness of a treatment for the disease or disorder based on the level of cleaved HMWK. In some embodiments, if the subject is identified as having a disorder, the method may further include administering to the subject an effective amount of a therapeutic agent, such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R) inhibitor, and / or a C1 esterase inhibitor, to treat the disorder. In some embodiments, the pKal inhibitor is an anti-pKal antibody. In some embodiments, the therapeutic agent is lanadelumab, ecallantide, icatibant, or a human plasma-derived C1 esterase inhibitor.

[0013] In some embodiments, the subject is a human patient undergoing treatment for a disorder, and the method further comprises evaluating the effectiveness of the treatment based on the level of cleaved HMWK in step (iii), wherein a deviation of the level of cleaved HMWK in the sample from the subject from the level in the control sample indicates that the treatment is effective. In some embodiments, the method further comprises identifying an appropriate treatment for the subject based on the level of cleaved HMWK. In some embodiments, the method may further comprise identifying the subject as a candidate for treatment for the disease based on the level of cleaved HMWK.

[0014] In some embodiments, the human patient has a history of disease (e.g., HAE). In some embodiments, the method further comprises assessing the subject's risk of developing the disease based on the level of cleaved HMWK, and if the level of cleaved HMWK in the sample from the subject deviates from that of the control sample, this indicates a risk of developing the disease. In some embodiments, the method further comprises administering a therapeutic agent to the subject if the subject is at risk of developing the disease.

[0015] In another aspect, a kit for detecting cleaved high molecular weight kininogen (HMWK) is provided, the kit comprising a first agent (e.g., an antibody described herein) that specifically binds to cleaved HMWK. In some embodiments, the kit further comprises a second agent that binds to HMWK, a support element, or both, and, optionally, instructions for detecting cleaved HMWK. In some examples, the support element is a 96-well plate.

[0016] In another aspect of the present disclosure, an isolated antibody that specifically binds to cleaved high molecular weight kininogen (HMWK) is provided. In some embodiments, the antibody binds to the same epitope as 559B-M004-B04 or competes with 559B-M004-B04 for binding to cleaved HMWK. In some embodiments, the antibody comprises the same heavy chain complementarity determining regions and light chain complementarity determining regions as 559B-M004-B04, e.g., the same heavy chain variable regions and light chain variable regions as 559B-M004-B04. In one example, the antibody is 559B-M004-B04.

[0017] Any antibody specific for cleaved HMWK described herein can be used in a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample. Such a method can include (i) contacting a sample suspected of containing cleaved HMWK with an antibody; (ii) measuring the cleaved HMWK-antibody complex formed in step (i); and determining the level of cleaved HMWK in the sample based on the results of step (ii). In some embodiments, the sample is a biological sample, such as a serum sample or plasma sample, collected from a human subject. The results obtained from this method can be used to determine the risk of the subject from whom the sample was obtained developing a plasma kallikrein-mediated disorder, such as HAE. In some cases, step (i) can be performed in the presence of ZnCl.

[0018] Any of the immunoassay methods described herein can be performed in a Western blot or ELISA format.

[0019] In yet another embodiment, an isolated antibody that binds to both intact high molecular weight kininogen (HMWK) and cleaved HMWK is provided.

[0020] In some embodiments, antibodies that bind to both intact and cleaved HMWK do not bind to low molecular weight kininogen (LMWK). In some embodiments, the antibody binds to the same epitope as 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08. In some embodiments, the antibody competes with 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08 for binding to intact and / or cleaved HMWK.

[0021] In some embodiments, the antibody comprises the same heavy chain and light chain CDRs as 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08. In some examples, the antibody is selected from the group consisting of 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, and 559B-M0004-E08.

[0022] In other embodiments, antibodies that bind to both intact and cleaved HMWK also bind to LMWK. In some embodiments, the antibodies are selected from the group consisting of 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03 , 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 or 559B-M0065-B10. In some embodiments, the antibody is selected from the group consisting of 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M00 Compatible with 053-G01, 559B-M0038-H03, 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 or 559B-M0065-B10.

[0023] In some embodiments, the antibody is selected from the group consisting of 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 55 and 559B-M0064-B10. In some examples, the antibody is selected from the group consisting of 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 and 559B-M0065-B10.

[0024] The following description sets forth the details of one or more embodiments of the present disclosure. Other features or advantages of the present disclosure will become apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims.

[0025] The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present disclosure, and such aspects may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments described herein. [Brief explanation of the drawings]

[0026] [Figure 1]FIG. 1 is a graph showing the binding of 559B-M0004-B04 to intact HMWK (dark grey bars) or cleaved HMWK (light grey bars) under the indicated ELISA conditions. [Figure 2] Figure 2 is a graph showing the binding of various Fab clones to intact single-chain (intact) HMWK, double-chain (truncated) HMWK, or LMWK. A: Fab clones identified using the phage display screening method described herein. Intact HWMK is shown as a dark gray bar, truncated HMWK as a light gray bar, and LMWK as a medium gray bar. B: Binding of several example Fab clones. LWMK is shown as a dark gray bar, intact HMWK as a light gray bar, and truncated HWMK as a medium gray bar. [Figure 3] Figure 3 is a graph showing the specificity of 559B-M0004-B04 for intact HMWK, truncated HMWK, or LMWK. Purified truncated HMWK was added to SBT assay buffer (circles) or HMWK-deficient plasma (squares). Purified intact HMWK was added to SBT assay buffer (triangles). Purified LMWK was added to SBT assay buffer (diamonds). The y-axis represents the ELISA signal in absorbance units, and the x-axis represents the kininogen concentration in μg / mL. [Figure 4] Figure 4 is a graph showing the detection of double-chain HMWK (truncated HMWK) in plasma or assay buffer. Purified truncated HMWK was added to SBT assay buffer (open circles) and analyzed in the presence of 10% plasma (squares) or HMWK-deficient plasma, and analyzed in the presence of 10% plasma (triangles). Purified truncated HMWK was similarly added to assay buffer and analyzed in the presence of 2.5% plasma (diamonds) or HMWK-deficient plasma, and analyzed in the presence of 2.5% plasma (filled circles). The y-axis represents the ELISA signal in absorbance units, and the x-axis represents the kininogen concentration in μg / mL. [Figure 5]Figure 5 is a graph showing the levels of cleaved HMWK in the indicated human plasma samples before and after contact system activation. A: Before and after contact system activation with FXIIa or ellagic acid. B: Before and after contact system activation with FXIIa, pKal, or ellagic acid. [Figure 6] FIG. 6 is a graph showing the levels of cleaved HMWK in plasma samples from 12 healthy human donors before and after contact system activation with ellagic acid. [Figure 7] Figure 7 shows graphs showing levels of cleaved HMWK after inhibition with pKal inhibitors. A: Inhibition by lanadelumab / DX-2930 or C1-INH before contact system activation by ellagic acid. B: Inhibition of pooled sodium citrate plasma samples by lanadelumab / DX-2930 before contact system activation by 10 nM FXIIa. [Figure 8] FIG. 8 is a graph showing cleaved HMWK production at the indicated time points after activation of the contact system by FXIIa or ellagic acid. [Figure 9] FIG. 9 is a graph showing the levels of double-chain HMWK in plasma samples from normal subjects and subjects with HAE. [Figure 10] Figure 10 is a photograph showing the results obtained from Western blot analysis of HMWK, which are consistent with the results obtained from the ELISA assay of two-chain HMWK described herein. Human citrated plasma samples (normal plasma, FXII-deficient plasma, and prekallikrein-deficient plasma) were probed with a mouse monoclonal anti-HMWK light chain antibody followed by a goat anti-mouse detection antibody. Plasma samples analyzed were either untreated or activated with 100 nM pKal, 10 nM FXIIa, or 10% ellagic acid. [Figure 11] Figure 11 is a graph showing that adding ZnCl to either citrated or EDTA plasma samples increased the double-chain HMWK signal in an ELISA assay. The x-axis shows the concentration of ZnCl in the assay wells after a 40-fold dilution. [Figure 12]Figure 12 is a schematic representation of the discovery and development of assays using the antibodies described herein. A: Schematic representation of the phage display method used to discover double-chain HMWK-binding antibodies. B: An example of a sandwich ELISA assay in which double-chain HMWK-specific antibodies / Fabs (e.g., 559B-M0004-B04) are immobilized on 96-well plates to capture double-chain HMWK in citrated plasma, followed by washing and detection with a label-conjugated anti-HMWK antibody (anti-HMWK-HRP). [Figure 13] FIG. 13 is a graph showing the results obtained from a standard curve of a double-chain HMWK sandwich ELISA in which double-chain HMWK was added to citrated plasma samples (final dilution 10%). [Figure 14] Figure 14 shows the identification of double-chain HMWK-specific antibodies by phage display selection and screening. A: For each antibody (Fab) tested, the ratio of the double-chain HMWK binding assay results to the LMWK binding assay results on the y-axis is plotted against the ratio of the double-chain HMWK binding assay results to the single-chain HMWK binding assay results on the x-axis. Recombinant Fab fragments were passively immobilized on a 384-well plate, followed by the addition of biotinylated double-chain HMWK, single-chain HMWK, or LMWK, followed by streptavidin-HRP. B: Binding of the indicated isolated Fab fragments to single-chain HMWK, double-chain HMWK, or LMWK is shown. [Figure 15] FIG. 15 is a graph showing competition of two-chain HMWK and kininogen peptides (HKH20 and GCP28) for binding to 559B-M0004-B04. [Figure 16] FIG. 16 is a graph showing the standard curve of an optimized sandwich ELISA for the detection of two-chain HMWK in human plasma samples. [Figure 17]Figure 17 is a graph of a Western blot analysis comparing the levels of double-chain HMWK in citrated plasma samples from healthy subjects and HAE patients. A: Scatter plot comparing the percent double-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Basal") and during an HAE attack ("Attack"). B: Receiver operating characteristic (ROC) analysis comparing the sensitivity and specificity of detection between samples from HAE basal and healthy subjects (AUC=0.977). C: ROC analysis comparing the sensitivity and specificity of detection between samples from HAE attacks and healthy subjects (AUC=1). D: ROC analysis comparing the sensitivity and specificity of detection between samples from HAE attacks and HAE basal (AUC=0.625). [Figure 18] Figure 18 is a graph of a Western blot analysis comparing the levels of double-chain HMWK in SCAT169 plasma samples from healthy subjects and HAE patients. A: Scatter plot comparing the percentage of double-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Basal") and during an HAE attack ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection between HAE basal samples and healthy samples (AUC=0.915). C: ROC analysis comparing the sensitivity and specificity of detection between HAE attack samples and healthy subjects (AUC=0.967). D: ROC analysis comparing the sensitivity and specificity of detection between HAE attack samples and HAE basal samples (AUC=0.597). [Figure 19]Figure 19 is a graph of an ELISA analysis comparing the levels of double-chain HMWK in citrated plasma samples from healthy subjects and HAE patients. A: Scatter plot comparing the percent double-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Basal") and during an HAE attack ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection between samples from HAE basal and healthy subjects (AUC=0.795). C: ROC analysis comparing the sensitivity and specificity of detection between samples from HAE attacks and healthy subjects (AUC=0.866). D: ROC analysis comparing the sensitivity and specificity of detection between samples from HAE attacks and HAE basal (AUC=0.709). [Figure 20] Figure 20 is a graph of an ELISA analysis comparing the levels of double-chain HMWK in SCAT169 samples from healthy subjects and HAE patients. A: Scatter plot comparing the percentage of double-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Basal") and during an HAE attack ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection between basal state samples and healthy subject samples (AUC=0.999). C: ROC analysis comparing the sensitivity and specificity of detection between HAE attack samples and healthy subject samples (AUC=1). D: ROC analysis comparing the sensitivity and specificity of detection between HAE attack samples and HAE basal state samples (AUC=0.8176). DETAILED DESCRIPTION OF THE INVENTION

[0027] DETAILED DESCRIPTION OF THE DISCLOSURE Plasma kallikrein (PKal) is a serine protease component of the contact system and the major bradykinin-generating enzyme in circulating blood. The contact system is activated by factor XIIa (activated factor XII or factor FXII) upon contact with foreign or negatively charged surfaces, or by prolylcarboxypeptidase on endothelial cell surfaces (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Plasma kallikrein activation amplifies intrinsic coagulation through feedback activation of factor XII and proteolytically cleaves the kininogen precursor, high-molecular-weight kininogen (HMWK), releasing the proinflammatory nonapeptide bradykinin and the cleaved HMWK (also known as two-chain HMWK), which contains two polypeptide chains linked by disulfide bonds.

[0028] Plasma kallikrein, the major kininogenase in the circulation, is primarily responsible for the production of bradykinin in the vasculature. Genetic defects in the C1-inhibitory protein (C1-INH) cause hereditary angioedema (HAE). Patients with HAE often suffer from acute, painful attacks precipitated by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). The plasma kallikrein-kinin system (plasma KKS) has been implicated in a variety of diseases through the use of pharmacological agents in animal models and genetic studies.

[0029] It has been found that the level of truncated HMWK is elevated in HAE attacks and other pKal-related disorders. Therefore, truncated HMWK can serve as a biomarker for monitoring the onset of disease and / or the effectiveness of treatment. However, the art lacks suitable agents and / or assays that can effectively distinguish between intact HMWK and truncated HMWK.

[0030] The present disclosure is based, at least in part, on the development of a specific immunoassay capable of detecting cleaved HMWK with high specificity and sensitivity. It was observed that a sandwich ELISA in which an agent that specifically binds to cleaved HMWK is immobilized on a support element (e.g., a multi-well plate) unexpectedly increases detection efficiency compared to an ELISA in which the antigen (in this case, cleaved HMWK) is immobilized on a support element. Furthermore, it was unexpectedly observed that the use of a low-cross blocking buffer (containing casein) rather than a blocking buffer containing bovine serum albumin (BSA) increases detection specificity and sensitivity during initial screening to discover antibodies specific to cleaved HMWK. Furthermore, the use of a 96-well plate further increases detection specificity and sensitivity compared to a 384-well plate. The present disclosure is also based, at least in part, on the isolation of an antibody that specifically binds to cleaved HMWK.

[0031] Thus, provided herein is an immunoassay that detects the presence or measures the level of cleaved HMWK in a biological sample suspected of containing HMWK species using an agent (e.g., an antibody) that specifically binds to cleaved HMWK (e.g., cleaved HMWK with a molecular weight of 46 kDa). Given the correlation between the level of cleaved HMWK and pKal-related or pKal-mediated disorders (e.g., HAE), the immunoassay described herein can be applied to identifying patients at risk for such diseases, monitoring disease progression, and / or monitoring the effectiveness of treatments for such disorders.

[0032] I. Immunoassay specifically detecting cleaved HMWK One aspect of the present disclosure relates to an immunoassay that detects cleaved HMWK with high sensitivity and specificity.Such immunoassay can include a sandwich ELISA method, in which an agent that specifically binds to cleaved HMWK is immobilized on a support element, which can be a 96-well plate.The immunoassay described herein can selectively detect cleaved HMWK in biological samples that may contain both intact HMWK and cleaved HMWK, as well as LMWK, such as serum or plasma samples.

[0033] (i) High molecular weight kininogen High molecular weight kininogen (HMWK) exists in plasma as a single polypeptide (single chain) multidomain (domains 1-6) protein with a molecular weight of approximately 110 kDa, and is referred to herein as intact HWMK. The human gene encoding HMWK is kininogen 1 (KNG1). KNG1 is transcribed and alternatively spliced ​​to form mRNA encoding either HMWK or low molecular weight kininogen (LMWK). Exemplary protein sequences for HMWK are listed below: >gi|156231037|ref|NP_001095886.1|Kininogen-1 isoform 1 precursor [Human (Homo sapiens)] MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTAQY DCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEE TLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS (SEQ ID NO: 1)

[0034] Intact HMWK, also referred to herein as "intact kininogen," can be assayed, for example, using clotting or immunological methods, such as radioimmunoassays (see, e.g., Kerbiriou-Nabias, DM, Br J Haematol, 1984, 56(2):2734-86). Monoclonal antibodies against the light chain of human HMWK are known. See, e.g., Reddigari, SR and Kaplan, AP, Blood, 1999, 74:695-702. Assays for HMWK that rely on chromogenic substrates can also be used. See, e.g., Scott, CF et al., Thromb Res, 1987, 48(6):685-700; Gallimore, MJ et al., Thromb Res, 2004, 114(2):91-96.

[0035] HMWK is cleaved by pKal within domain 4 to release the nine-amino acid proinflammatory peptide bradykinin and a two-chain form of HMWK, referred to herein as cleaved HMWK. HMWK consists of two chains, a heavy chain comprising domains 1-3 and a light chain comprising domains 5 and 6, which are linked by disulfide bonds. When intact HMWK is first cleaved, the heavy and light chains have molecular weights of approximately 65 kDa and 56 kDa, respectively. Further proteolytic processing yields a 46 kDa light chain.

[0036] Exemplary sequences of the heavy and light chains of truncated kininogen are set forth below.

[0037] >Cleaved kininogen-1 heavy chain QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTA QYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQ PPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISL MK (SEQ ID NO: 2) >Cleaved kininogen-1 light chain SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNK GKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTT QMKESYYFDLTDGLS (SEQ ID NO: 3)

[0038] (ii) Antibodies specific to cleaved HMWK The immunoassays described herein can use any agent capable of specifically binding to cleaved HMWK, for example, an agent that recognizes a neoepitope on cleaved HMWK that is not present on intact HMWK. In some embodiments, the cleaved HMWK-binding agent is an antibody.

[0039] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to targets such as carbohydrates, polynucleotides, lipids, and polypeptides through at least one antigen recognition site present in the immunoglobulin variable region. As used herein, the term "antibody" encompasses not only intact (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (Fab, Fab', F(ab')2, Fv), single-chain (scFv), variants thereof, fusion proteins containing antibody portions, humanized antibodies, chimeric antibodies, diabodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other immunoglobulin molecule that contains an antigen recognition site with the required specificity and has a modified conformation, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and an antibody need not be of any particular class. Immunoglobulins can be divided into various classes depending on the antibody amino acid sequence of the constant domain of their heavy chains. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and some of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the various classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional configurations of the various classes of immunoglobulins are well known.

[0040] Any antibody described herein can be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, while a "polyclonal antibody" refers to a heterogeneous antibody population. The two terms do not limit the source of the antibody or the method by which it is produced.

[0041] An antibody that "specifically binds" to cleaved HMWK or an epitope thereof is a term well understood in the art, and methods for determining such specific binding are also well known in the art. A molecule is said to exhibit "specific binding" if it reacts or associates with a particular target antigen (here, cleaved HMWK) with greater frequency, rapidity, longer duration, and / or greater affinity than it binds to alternative targets (e.g., intact HMWK and / or LMWK). An antibody "specifically binds" to a target antigen if it binds with greater affinity, greater avidity, more rapidity, and / or longer duration than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to cleaved HMWK or an epitope thereof is an antibody that binds to this target antigen with greater affinity, greater avidity, more readily, and / or longer duration than it binds to other antigens (e.g., intact HMWK or LMWK) or other epitopes of the same antigen. By reading this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Generally, when referring to binding, it can mean, but does not necessarily mean, preferential binding.

[0042] In some embodiments, antibodies that specifically bind to cleaved HMWK described herein (as well as other antibodies that bind to both cleaved and intact HMWK, and optionally LMWK) have a suitable binding affinity for cleaved HMWK (or another target antigen described herein). As used herein, "binding affinity" refers to the apparent binding constant or K A K A is the dissociation constant (K D ) is the reciprocal of the antibody. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10-10 Binding affinity (K D The increased binding affinity can be expressed as K D An antibody that binds to a first target with higher affinity than to a second target corresponds to a decrease in the K for binding to the first target. A is the K for binding to the second target A (or K D (or K D In such cases, the antibody has specificity for a first target (e.g., a protein or mimetic thereof in a first conformation) compared to a second target (e.g., the same protein or mimetic thereof in a second conformation; or a second protein). The difference in binding affinity (e.g., when comparing specificity or otherwise) may be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50-fold, 70-fold, 80-fold, 91-fold, 100-fold, 500-fold, 1000-fold, 10,000-fold, or 10 5 For example, the binding affinity of an antibody that specifically binds to truncated HMWK described herein can be 10 times, 100 times, 10,000 times, or 10 times greater than the binding affinity of that antibody to intact HMWK and / or LMWK. 5 It could be double.

[0043] Binding affinity can be determined by various methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance, or spectroscopy (e.g., using a fluorescence assay). An exemplary condition for assessing binding affinity is in HBS-P buffer (10 mM HEPES (pH 7.4), 150 mM NaCl, 0.005% (v / v) surfactant P20). Using these techniques, the concentration of bound binding protein can be measured as a function of target protein concentration. The concentration of bound binding protein ([bound]) is calculated using the following equation: [Bound]=[N][Free] / (Kd+[Free]) It relates the free target protein concentration ([free]) and the concentration of binding sites for the binding protein on the target by (N), where (N) is the number of binding sites per target molecule.

[0044] Not necessarily K A It is not necessary to precisely determine K, as this can be determined using methods such as ELISA or FACS analysis, A This is because it is sufficient to obtain a quantitative measure of affinity that is proportional to and can thus be used for comparison, e.g., to determine whether higher affinity is, e.g., 2-fold higher affinity, to obtain a qualitative measure of affinity, or to obtain an inference of affinity, e.g., by activity in a functional assay, e.g., an in vitro assay or an in vivo assay.

[0045] In some embodiments, an antibody that specifically binds to cleaved HMWK (also referred to as an anti-cleaved HMWK antibody) binds to the same epitope as 559B-M004-B04 of cleaved HMWK. "Epitope" refers to the site on a target antigen to which a binding protein (e.g., an antibody such as a Fab or full-length antibody) binds. This site may consist exclusively of amino acid elements, exclusively of chemically modified amino acids of the protein (e.g., glycosyl moieties), or a combination thereof. Overlapping epitopes contain at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular structure. Epitopes may be linear; in other examples, epitopes are conformational epitopes.

[0046] A first antibody "binds to the same epitope" as a second antibody if it binds to the same site on the target antigen as the second antibody, or if it binds to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90% or 100% overlaps), e.g., in amino acid sequence or other molecular structure (e.g., glycosyl groups, phosphate groups or sulfate groups) with the site bound by the second antigen.

[0047] In some embodiments, an antibody that specifically binds to cleaved HMWK competes with 559B-M004-B04 for binding to HMWK. A first antibody "competes for binding" with a second antibody if binding of the first antibody to its epitope reduces (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the amount of the second antibody that binds to that epitope. Competition can be direct (e.g., the first antibody binds to an epitope that is the same as or overlaps with the epitope bound by the second antibody) or indirect (e.g., binding of the first antibody to its epitope causes a conformational change in the target antigen that reduces the ability of the second antibody to bind to that epitope).

[0048] In some instances, an antibody that specifically binds to cleaved HMWK is identified as having the corresponding V H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the CDR H CDR1, V H CDR2 and / or V H including CDR3, V H Alternatively or additionally, antibodies that specifically bind to truncated HMWK include the corresponding V chain of 559B-M004-B04. L V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the CDR L CDR1, V L CDR2 and / or V L In some embodiments, antibodies that specifically bind to cleaved HMWK have the same heavy and / or light chain complementarity determining regions (CDRs) as 559B-M004-B04.

[0049] "Complementarity-determining regions" or "CDRs" are known in the art as noncontiguous amino acid sequences within antibody variable regions that confer antigen specificity and binding affinity. Generally, there are three CDRs in each heavy chain variable region and three CDRs in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al. (1991) (Public Health Service, 5th ed., National Institutes of Health, Bethesda, MD) (Kabat numbering scheme), Al-Lazikani et al. (1997) (JMB 273,927-948) (Chothia numbering scheme), MacCallum et al. (J. Mol. Biol. 262:732-745 (1996)) (Contact numbering scheme), Lefranc MP et al. (Dev Comp Immunol, 2003 January;27(1):55-77) (IMGT numbering scheme), and Honegger A and Pluckthun A (J Mol Biol, 2001 Jun. 8;309(3):657-70) (AHo numbering scheme).

[0050] The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignment, while the Chothia scheme is based on structural information. The Contact scheme is based on the analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Therefore, unless otherwise specified, the terms "complementarity determining region" or "CDR" of a given antibody should be understood to encompass the complementarity determining regions determined by any of the above known schemes.

[0051] It is possible that an antibody has the same V as 559B-M004-B04 (as well as other exemplary antibodies disclosed herein) as determined by the same numbering scheme. HCDR and / or V L If the antibody has the same CDRs as clone 559B-M004-B04 (or other exemplary antibodies disclosed herein), then such antibody is considered to have the same CDRs as clone 559B-M004-B04 (or other exemplary antibodies disclosed herein) and is within the scope of the present disclosure. For example, such an antibody may have the same V H CDR and / or V L In another example, an anti-cleaved HMWK antibody within the scope of the present disclosure may have the same V CDRs as clone 559B-M004-B04 as determined by the Kabat numbering scheme. H CDR and / or V L It may have CDRs.

[0052] Alternatively or additionally, the anti-cleaved HMWK antibody may be V of 559B-M004-B04. H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to a V strand. H Chain and / or V of 559B-M004-B04 L V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to a V strand. L In some embodiments, the antibody is 559B-M004-B04.

[0053] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-68, 1990), modified as in Karlin and Altschul, Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm has been incorporated into the NBLAST program of Altschul et al. (J. Mol. Biol. 215:403-10, 1990) and the XBLAST program (version 2.0). The XBLAST program performs BLAST protein searches with a score of 50 and word length of 3 to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be used as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0054] The sequences of the heavy and light chain variable regions of 559B-M004-B04 are shown below, with the sequences of CDR1, CDR2, and CDR3 of the heavy chain and CDR1, CDR2, and CDR3 of the light chain underlined and in bold (identified by one scheme as an example).

[0055] [ka]

[0056] In some cases, an antibody that specifically binds to truncated HMWK may contain one or more (e.g., up to five, up to three, or up to one) conservative mutations in one or more heavy chain CDRs or one or more light chain CDRs in 559B-M0004-B04, for example, at positions where the residues are not likely to be involved in the interaction with truncated HMWK. As used herein, a "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants can be prepared according to methods for altering polypeptide sequences known to those skilled in the art, such as those described in references that summarize such methods, such as Molecular Cloning: A Laboratory Manual, edited by J. Sambrook et al., 2nd Edition (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989) or Current Protocols in Molecular Biology, edited by FMA Usubel et al. (John Wiley & Sons, Inc., New York). Conservative substitutions of amino acids include substitutions made between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0057] Antibodies capable of binding to the cleaved HMWK described herein (as well as antibodies capable of binding to intact HMWK and / or LMWK) can be produced by any method known in the art. See, for example, Harlow and Lane (1988) Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory, New York).

[0058] In some embodiments, antibodies specific to a target antigen (truncated HMWK, intact HMWK, and / or LMWK) can be produced by conventional hybridoma technology. A host animal can be immunized with a full-length target antigen or a fragment thereof, optionally conjugated to a carrier protein such as KLH, to produce antibodies that bind to the antigen. The route and schedule for immunizing the host animal generally conform to established conventional techniques for stimulating and producing antibodies, as further described herein. General techniques for producing murine, humanized, and human antibodies are known in the art and are described herein. It is contemplated that any mammalian subject, including humans, or antibody-producing cells derived therefrom can be engineered to serve as a basis for producing mammalian, including human, hybridoma cell lines. Typically, a host animal is inoculated intraperitoneally, intramuscularly, intraorally, subcutaneously, intraplantarly, and / or intradermally with an amount of an immunogen, including those described herein.

[0059] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975) (Nature 256:495-497) or a modification thereof by Buck, DW et al. (In Vitro, 18:377-381 (1982)). Available myeloma cell lines, including but not limited to X63-Ag8.653 and those from the Salk Institute for Cell Distribution Center (San Diego, CA, USA), can be used for hybridization. This technique generally involves fusing myeloma cells with lymphoid cells using a fusing agent such as polyethylene glycol or by electrical means well known to those skilled in the art. After fusion, the cells are separated from the fusion medium and grown in a selective growth medium, such as hypoxanthine-aminopterin-thymidine (HAT) medium, to remove unhybridized parent cells. Hybridomas secreting monoclonal antibodies can be cultured in any of the media described herein, with or without the addition of serum. As an alternative to cell fusion techniques, EBV-immortalized B cells can be used to produce the anti-PKal monoclonal antibodies described herein. Hybridomas can be expanded and subcloned as needed, and supernatants assayed for anti-immunogen activity by conventional immunoassay methods (e.g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).

[0060] Hybridomas that can be used as antibody sources include any derivatives or progeny of the parent hybridoma that produce monoclonal antibodies capable of blocking PKal activity. Hybridomas producing such antibodies can be grown in vitro or in vivo using known methods. If necessary, the monoclonal antibodies can be isolated from culture media or body fluids by conventional immunoglobulin purification techniques, such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration. Undesirable activity, if present, can be removed, for example, by passing the preparation over an adsorbent made with the immunogen bound to a solid phase, thereby eluting or dissociating the desired antibody from the immunogen. A population of antibodies (e.g., monoclonal antibodies) can be obtained by immunizing a host animal with the target antigen or with a fragment containing the target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional agent or derivatizing agent, such as maleimidobenzoyl sulfosuccinimide ester (conjugation via cysteine ​​residues), N-hydroxysuccinimide (conjugation via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or RN=C=NR, where R and R are different alkyl groups.

[0061] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) can be sequenced, and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector within a host cell, which can then be expanded and frozen for later use. Alternatively, the polynucleotide sequence can be used in genetic engineering to improve the affinity (affinity maturation) or other properties of the antibody. It may be desirable to genetically engineer the antibody sequence to obtain greater affinity and / or specificity for the target antigen. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity for its target antigen.

[0062] In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Similarly, transgenic animals designed to generate a more desirable (e.g., fully human) or robust immune response can also be used to generate humanized or human antibodies. Examples of such technology are Xenomouse® from Amgen, Inc. (Fremont, CA), and HuMAb-Mouse® and TC Mouse™ from Medarex, Inc. (Princeton, NJ). In another alternative, antibodies can be produced recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al. (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, phage display technology (McCafferty et al., (1990) Nature 348:552-553) can be used to produce human antibodies and antibody fragments in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0063] Antigen-binding fragments of intact (full-length) antibodies can be prepared by routine methods, for example, by pepsin digestion of the antibody molecule to produce F(ab')2 fragments and by reduction of the disulfide bridges of the F(ab')2 fragment to produce Fab fragments.

[0064] Single-chain antibodies can be prepared by recombinantly linking a nucleotide sequence encoding a heavy-chain variable region with a nucleotide sequence encoding a light-chain variable region. It is preferable to incorporate a flexible linker between the two variable regions. Alternatively, techniques described for the production of single-chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be applied to generate phage or yeast scFv libraries, and PKa1-specific scFv clones can be identified from the libraries using routine methods. Positive clones can be further screened to identify clones that specifically bind to target antigens, such as cleaved HMWK.

[0065] In some embodiments, antibodies specific to cleaved HMWK (or intact HMWK or LMWK) may be isolated from an antibody library, which may be a synthetic or natural library. A natural antibody library refers to a library obtained from a natural source (e.g., a human donor) according to routine procedures. A synthetic antibody library refers to a library designed according to predetermined rules (e.g., having completely randomized CDR regions, such as CDRs, or semi-randomized CDR regions, such as CDR1 or CDR2, of the heavy chain, light chain, or both).

[0066] In some cases, the antibody library is a display library (e.g., a phage display library or a yeast display library). A display library is a collection of entities, each of which contains available polypeptide elements and recoverable elements that encode or identify the polypeptide elements. The polypeptide elements are diverse and therefore represented by a variety of amino acid sequences. The polypeptide elements can be of any length, e.g., from 3 amino acids to over 300 amino acids. A display library entity can contain more than one polypeptide element, e.g., the two polypeptide chains of an sFab. In an exemplary embodiment, a display library can be used to identify proteins that bind to truncated HMWK (and other target antigens described herein). For selection, the polypeptide element of each library member is probed with truncated HMWK (or a fragment thereof), and if the polypeptide element binds to truncated HMWK, the display library member is typically identified by retention on a support. An example of using a phage display antibody library to identify antibodies specific to truncated HMWK is shown in Figure 12.

[0067] The retained display library members are recovered from the support and analyzed. Analysis can include amplification followed by selection under the same or different conditions. For example, alternating positive and negative selections can be performed. Analysis can also include determining the amino acid sequence of the polypeptide elements and purifying the polypeptide elements for detailed characterization.

[0068] Antibodies obtained by methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which an antigen binds, i.e., "epitope mapping." There are many methods known in the art for mapping and characterizing the location of an epitope on a protein, including, for example, analysis of the crystal structure of an antibody-antigen complex, as described in Chapter 11 of Harlow and Lane, "Using Antibodies," a Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999), competitive assays, gene fragment expression assays, and synthetic peptide-based assays. In another example, epitope mapping can be used to determine the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., an epitope contained in the form of a single stretch of amino acids, or a conformational epitope formed by the three-dimensional interaction of amino acids that do not necessarily contain a single stretch of amino acids (linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids in length) can be isolated or synthesized (e.g., recombinantly) and used in antibody binding assays. In another example, the epitope to which an antibody binds can be determined by systematically screening overlapping peptides derived from the target antigen sequence to determine antibody binding. In gene fragment expression assays, the open reading frame encoding the target antigen is fragmented either randomly or by specific gene constructs, and the reactivity of the expressed antigen fragments with the test antibody is determined. Gene fragments can be generated, for example, by PCR, and then transcribed in vitro in the presence of radioactive amino acids and translated into protein. Binding of the antibody to the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0069] Specific epitopes can also be identified using large libraries of random peptide sequences (phage libraries) displayed on the surface of phage particles. Alternatively, libraries of defined overlapping peptide fragments can be tested for binding to a test antibody in a simple binding assay. In another example, mutagenesis of the antigen-binding domain, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues necessary, sufficient, and / or essential for epitope binding. For example, domain swapping experiments can be performed using mutants of labeled antigen in which various fragments of the HMWK polypeptide are replaced (swapped) with sequences from closely related but antigenically distinct proteins. By assessing the binding of the antibody to the mutant HMWK, the importance of specific antigen fragments for antibody binding can be assessed.

[0070] Alternatively, competition assays can be performed with other antibodies known to bind to the same antigen to determine whether one antibody binds to the same epitope as another antibody. Competition assays are well known to those skilled in the art.

[0071] Any anti-cleaved HMWK antibody is within the scope of this disclosure.

[0072] (iii) Immunoassay Provided herein are immunoassays for detecting cleaved HMWK. As used herein, the term "immunoassay" may be interchangeably referred to as an immune-based assay or an immuno-based assay. Immunoassays generally use agents such as antibodies that bind to a molecule (e.g., HMWK) to detect the presence and / or concentration (level) of that molecule in a sample. Examples of immunoassays include Western blotting, enzyme-linked immunosorbent assays (ELISAs), lateral flow assays, radioimmunoassays, electrochemiluminescence-based detection assays, magnetic immunoassays, and related techniques. In some embodiments, the immunoassay is an ELISA assay. In some embodiments, the immunoassay is a sandwich ELISA assay. In some embodiments, the immunoassay is a lateral flow assay.

[0073] ELISAs are known in the art (see, e.g., Crowther, John R (2009). "The ELISA Guidebook." 2nd ed. Humana Press and Lequin R (2005). "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)." Clin. Chem. 51(12):2415-8), and exemplary ELISAs are described herein. Kits for performing ELISAs are also known in the art and commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0074] In performing the immunoassays described herein, a sample may be obtained from a subject. As used herein, a "sample" refers to a composition containing tissue, e.g., blood, plasma, or protein, from a subject. Samples include both the initial, unprocessed sample collected from a subject and samples that are subsequently processed, e.g., partially purified or preserved. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a bodily fluid sample, such as a serum sample or plasma sample. The sample analyzed by the immunoassays described herein may be the initial, unprocessed sample collected from a subject or samples that are subsequently processed, e.g., partially purified or preserved. In some embodiments, multiple samples (e.g., at least two, three, four, five, or more) may be collected from a subject over time or at specific time intervals, e.g., to assess the progression of a disease or disorder or the effectiveness of a treatment. Multiple samples may be collected before and after treatment or during the course of treatment.

[0075] A sample can be collected from a subject using any means known in the art. In some embodiments, the sample is obtained from the subject by collecting a sample (e.g., a blood sample) in an evacuated collection tube (e.g., a vacuum blood collection tube). In some embodiments, the evacuated collection tube contains one or more protease inhibitors, e.g., to reduce or prevent ex vivo activation of the contact system during sample collection. Such protease inhibitors may be included in the form of a liquid formulation. In some embodiments, the protease inhibitors include at least one serine protease inhibitor and at least one cysteine ​​protease inhibitor. Such collection tubes are known in the art. See, e.g., International Application No. PCT / US2016 / 046681. Optionally, the evacuated collection tube may further contain one or more anticoagulants.

[0076] A "patient," "subject," or "host" (these terms are used interchangeably) treated by the methods of the invention can refer to either a human or a non-human animal. In some embodiments, the subject is a patient suffering from a kallikrein-mediated disorder, e.g., a bradykinin-mediated disorder, e.g., hereditary angioedema (HAE), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with a ventricular assist device or stent, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye disease, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis-degenerative spine disease, postoperative ileus, aortic aneurysm. , osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., post-angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis), and tissue injury (e.g., burn or chemical injury).

[0077] Alternatively or additionally, the subject who needs the analysis described herein can be a patient with disease or disorder.This subject can be a subject who is currently experiencing an attack of disease (for example, HAE) or a subject who has previously suffered from disease (for example, currently in the dormant phase of disease).In some examples, the subject is a human patient who can be undergoing treatment for disease, for example, treatment with C1 esterase inhibitor (C1-INH), plasma kallikrein inhibitor or bradykinin inhibitor.In other cases, this human patient can be a patient who has not undergone such treatment.

[0078] The sample described herein can be analyzed using an agent that specifically binds to cleaved HMWK to determine the level of cleaved HMWK in the sample. In some embodiments, the immunoassay described herein can be in the form of a sandwich ELISA, in which a first agent that specifically binds to cleaved HMWK (e.g., an antibody described herein) is immobilized on a support element. The support element can then be incubated with the sample described herein for an appropriate time under conditions that allow the formation of a cleaved HMWK / first agent (e.g., antibody) complex. A second agent that binds to HMWK can then be used to detect such complexes. The second agent can be conjugated with a label that can directly or indirectly emit a signal. The intensity of the signal represents the level of cleaved HMWK in the sample.

[0079] This method can use any support element known in the art, including, but not limited to, membranes, beads, slides, or multi-well plates. The selection of an appropriate support element for an immunoassay depends on various factors, such as the number of samples and the method for detecting the signal emitted from the label conjugated to the second agent.

[0080] In some embodiments, the support element is a membrane, such as a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, or a cellulose acetate membrane. In some examples, the immunoassay can be in a Western blot or lateral flow assay format.

[0081] In some embodiments, the support element is a multiwell plate, such as an ELISA plate. In some embodiments, the immunoassays described herein can be performed on a high-throughput platform. In some embodiments, high-throughput immunoassays can use multiwell plates, e.g., 24-well, 48-well, 96-well, 384-well, or more well plates. Individual immunoassays can be performed simultaneously in each well. For this reason, it is generally desirable to increase assay throughput using a plate reader that simultaneously measures multiple wells. In some embodiments, plate readers capable of simultaneously imaging multiple wells (e.g., 4-well, 16-well, 24-well, 48-well, 96-well, 384-well, or more wells) can be used with this platform. For example, commercially available plate readers (e.g., the plate::vision system available from Perkin Elmer, Waltham, Massachusetts) can be used. These plate readers are capable of kinetic-based fluorescence analysis. The plate::vision system has highly efficient collection optics and specialized optics designed to simultaneously analyze 96 wells. Other suitable parallel plate readers include, but are not limited to, SAFIRE (Tecan, San Jose, CA), FLIPRTETRA® (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ), and CellLux (Perkin Elmer, Waltham, MA).

[0082] It has been surprisingly discovered that the surface area and / or volume of the wells of a multi-well plate can affect the results of an immunoassay, as described in Example 1. In some embodiments, the immunoassays described are performed in 96-well plates, such as 96-well ELISA plates.

[0083] In other embodiments, the high-throughput screening immunoassays of the present disclosure can be automated (eg, adapted for robotic assays).

[0084] In some embodiments, immunoassays can be performed in low-throughput platforms, including single immunoassay formats. For example, low-throughput platforms can be used to measure the presence and amount of cleaved HMWK in biological samples (e.g., biological tissues, tissue extracts) for diagnostic methods, monitoring disease and / or treatment progression, and / or predicting whether a disease or disorder may benefit from a particular treatment.

[0085] Any method known in the art can be used to immobilize an agent that specifically binds to cleaved HMWK, such as an antibody described herein, on a support element also described herein. In some embodiments, immobilization involves binding the agent (e.g., an antibody) to the support element. In other embodiments, immobilization involves adsorbing the antibody to the support element. Such adsorption methods can be performed, for example, by incubating an antibody in a buffer solution with the wells of a multiwell plate. In some embodiments, an agent, such as an antibody, is placed in a coating buffer and incubated with the wells of a multiwell plate. Coating buffers will be apparent to those skilled in the art and may be prepared or obtained from commercial sources. Non-limiting examples of coating buffers include 50 mM sodium bicarbonate, pH 9.6; 0.2 M sodium bicarbonate, pH 9.4; phosphate buffer solution (50 mM phosphate, pH 8.0, 0.15 M NaCl); carbonate-bicarbonate solution; and TBS (50 mM Tris, pH 8.0, 0.15 M NaCl).

[0086] In some embodiments, the first agent is immobilized on the support element by hydrophobic interactions between the first agent and the support element, hi some embodiments, the first agent is immobilized on the support element using electrophoretic transfer.

[0087] The support element may be incubated with a blocking buffer before or after immobilization. A blocking buffer is typically used to block any exposed surface of the support membrane (e.g., the site on the support membrane occupied by the first agent). The use of a blocking buffer can reduce the baseline signal detected (i.e., "background interference"), improve the sensitivity of the immunoassay, and / or reduce nonspecific binding of sample components to the support membrane. As described in Example 1, the choice of blocking buffer influenced the results of the immunoassay. In some embodiments, the blocking buffer contains serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the blocking buffer is a BSA buffer (e.g., 2% BSA in PBS buffer). In some embodiments, the blocking buffer does not contain serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the blocking buffer contains casein fragments, and optionally NaCl and Tween, and may have a pH of 7.0-7.4. In some embodiments, the casein fragments are highly purified casein fragments. Such blocking buffers may be prepared or obtained from commercial sources (e.g., Blocking Solution LowCross from CANDOR Bioscience).

[0088] A support element having an agent specific for cleaved HMWK bound thereto can be contacted (incubated) with a sample described herein suspected of containing cleaved HMWK. The term "contacting" generally refers to exposing the support element to the biological sample or agent for a suitable period of time sufficient to form a complex between the agent, such as an antibody, and the cleaved HMWK (if present) in the sample. The sample can then be removed from the support element, and the support element can then be washed several times to remove unbound cleaved HMWK. In some embodiments, contacting is achieved by capillary action, which migrates the biological sample or agent across the surface of the support membrane.

[0089] The support element can then be incubated with a second agent that binds to the HMWK for a period of time suitable to allow binding of the second agent to the HMWK bound to the support element.

[0090] The second agent can be any agent capable of binding to HMWK, such as an antibody capable of binding to HMWK (specific for cleaved HMWK or capable of cross-reacting with both cleaved and intact HMWK). In some embodiments, the second agent comprises one or more antibodies that bind to HWMK (cleaved HWMK and / or intact HMWK). In some embodiments, the antibody is a mouse monoclonal antibody or a monoclonal sheep antibody. This is conjugated to a label, which is a compound capable of directly or indirectly (e.g., through interaction with one or more additional compounds) emitting a signal.

[0091] In some embodiments, the label is a signal-emitting agent that is either an agent that emits a signal directly (e.g., a dye or fluorophore) or an agent that emits a signal upon interaction with a substrate (e.g., an enzyme such as HRP or β-galactosidase that can convert a colorless substrate to a colored product). As used herein, the term "fluorophore" (also called "fluorescent label" or "fluorochrome") refers to a moiety that absorbs light energy at a particular excitation wavelength and emits light energy at a different wavelength.

[0092] In other embodiments, the label may be a member of a receptor-ligand pair. As used herein, "ligand-receptor pair" refers to a pair of molecules (e.g., biomolecules) that have specific affinity for each other, such as biotin-streptavidin. In this case, the support element carrying the first agent-cleaved HMWK-second agent may be further incubated with the other member of the ligand-receptor pair for a suitable time period to allow the two members of the receptor-ligand pair to interact. The other member of the receptor-ligand pair is conjugated to a signal-emitting substance as described herein. In one example, the second agent is conjugated to biotin, and HRP-conjugated streptavidin is used for detection.

[0093] After washing away any unbound conjugate, a substrate solution may be added to aid detection. For example, after a period of time, the reaction can be stopped (e.g., by adding 1N NaOH), and the concentration of the colored product produced can be measured spectrophotometrically. The intensity of the color is proportional to the concentration of bound antigen.

[0094] The signal emitted from the label described herein can then be detected / measured by routine methodologies depending on the particular immunoassay format and the signal-emitting substance used therein. As used herein, the terms "measuring" or "measurement" or "detecting" or "detection" refer to assessing the presence, absence, quantity, or amount (which may be an effective amount) of a substance in a sample, including inducing a qualitative or quantitative concentration level of such substance, or otherwise assessing a value or classification of interest.

[0095] Assays, e.g., Western blot assays, may further involve the use of quantitative imaging systems, such as commercially available LICOR imaging technology (see, e.g., LI-COR Biosciences' Odyssey® CLx Infrared Imaging System). In some embodiments, electrochemiluminescence detection assays or assays relying on a combination of electrochemiluminescence and patterned array technology are used (e.g., Meso Scale Discovery (MSD)'s ECL or MULTI-ARRAY technology).

[0096] Any immunoassay described herein, for example, one or more steps of an immunoassay, may be performed in a suitable assay buffer that will be apparent to one skilled in the art. In some embodiments, the assay buffer contains ZnCl or is supplemented with ZnCl. In some embodiments, the assay buffer contains at least about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM, or more of ZnCl. In some embodiments, such a ZnCl-containing assay buffer is used in the step in which a cleaved HMWK-specific agent (e.g., an antibody specific to cleaved HMWK) binds to cleaved HMWK. ZnCl increases the binding activity of the agent (e.g., an antibody) to cleaved HMWK.

[0097] In some embodiments, the assay buffer contains serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the assay buffer contains at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.12%, 0.014%, 0.16%, 0.18%, 0.2%, 0.25%, 0.3%, 0.4%, or more BSA. In some embodiments, the assay buffer contains a detergent, such as Tween-20. In some embodiments, the assay buffer contains at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, or more detergent. In one example, the assay buffer contains 0.1% BSA and 0.05% Tween-20 in PBS.

[0098] (iv) Diagnostic and prognostic applications Given the correlation between the level of cleaved HMWK and diseases or disorders associated with plasma kallikrein, the assays and kits described herein can be applied (e.g., as biomarkers) to assess such diseases or disorders, such as those described herein (e.g., HAE). Alternatively, or in addition, the assays and kits described herein can be used to monitor the progression of such diseases, evaluate the effectiveness of treatments for the disease, identify patients suitable for particular treatments, and / or predict the disease state (e.g., attack vs. quiescence) of a subject.

[0099] In some embodiments, the level of cleaved HMWK determined by the immunoassay described herein can be used to assess whether a subject (e.g., a human patient) from whom a biological sample was obtained has or is at risk for a plasma kallikrein-related disease or disorder, such as HAE or an autoimmune disease (such as RA, UC, and Crohn's disease). The level of cleaved kininogen can then be compared with either the amount of intact kininogen or total kininogen in the sample to determine the level (e.g., percentage) of cleaved kininogen, the level of intact kininogen, or both in the sample. The level of cleaved kininogen and / or intact kininogen can be compared with a reference value to determine whether a subject has or is at risk for a PKal-mediated disorder, such as HAE or an autoimmune disease such as RA, UC, and Crohn's disease. For example, if the percentage of cleaved kininogen is equal to or greater than the reference value, the subject can be identified as having or at risk for a PKal-mediated disorder, such as HAE, RA, UC, and Crohn's disease. Alternatively, if the percent intact kininogen is below the reference value, the subject can be considered to have or be at risk for a pKal-mediated disorder, such as HAE, RA, UC, and Crohn's disease.

[0100] In some embodiments, the samples analyzed by the methods described herein are derived from human subjects with or at risk of hereditary angioedema (HAE). HAE is also known as "Quincke's edema," C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent episodes of severe swelling (angioedema), which can occur, for example, in the extremities, face, genitals, gastrointestinal tract, and respiratory tract. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or pharynx; airway obstruction, which may be accompanied by pharyngeal swelling and sudden hoarseness; recurrent episodes of abdominal pain with unknown cause; and / or intestinal swelling, which can become severe and cause abdominal pain, vomiting, dehydration, diarrhea, pain, and / or shock. Approximately one-third of patients with HAE develop a non-itchy rash called erythema marginatum during attacks.

[0101] Airway swelling can be life-threatening and, in some patients, fatal. Mortality rates are estimated at 15-33%. HAE results in approximately 15,000-30,000 emergency room visits each year.

[0102] Trauma or stress, such as dental procedures, illness (e.g., viral illnesses such as colds and influenza), menstruation, and surgery can trigger attacks of angioedema. To prevent acute attacks of HAE, patients can try to avoid specific stimuli that have previously triggered attacks. However, attacks often occur without a known trigger. HAE symptoms usually first appear in childhood and worsen during adolescence. Untreated patients experience attacks on average every 1–2 weeks, with most episodes lasting approximately 3–4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary widely among patients with hereditary angioedema, even within the same family.

[0103] There are three types of HAE, known as types I, II, and III. It is estimated that 1 in 50,000 people suffer from HAE, with type I accounting for approximately 85 percent of cases, type II accounting for approximately 15 percent of cases, and type III being extremely rare. Type III is the most recently described type and was initially thought to occur only in women, although families with affected men have been identified.

[0104] HAE is inherited in an autosomal dominant manner, meaning that affected individuals may inherit the mutation from one affected parent. Because de novo gene mutations can occur, HAE can occur in individuals with no family history of the condition. It is estimated that 20-25% of cases are due to de novo spontaneous mutations.

[0105] Mutations in the SERPING1 gene cause types I and II hereditary angioedema. The SERPING1 gene directs the production of the C1 inhibitor protein, which is important in controlling inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema result in low levels of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of abnormally functional C1 inhibitor. Inadequate levels of functional C1 inhibitor result in the production of excessive amounts of bradykinin. Bradykinin promotes inflammation by increasing fluid leakage from blood vessel walls through body tissues. Excessive fluid accumulation in body tissues leads to the swelling episodes seen in patients with types I and II hereditary angioedema.

[0106] Mutations in the F12 gene are associated with some cases of type III hereditary angioedema. The F12 gene directs the production of clotting factor XII. Factor XII plays a vital role in blood clotting (clotting), as well as being an important stimulator of inflammation and is involved in the production of bradykinin. Mutations in the F12 gene result in the production of highly active factor XII. This results in increased bradykinin production and increased leakiness of blood vessel walls, which leads to swelling episodes. The cause of other cases of type III hereditary angioedema remains unknown. In these cases, mutations in one or more unidentified genes are likely responsible for the disorder.

[0107] Although HAE may share similarities with other forms of angioedema caused by allergies or other medical conditions, the causes and treatment are significantly different. When misdiagnosed as an allergy, HAE is most commonly treated with antihistamines, steroids, and / or epinephrine; however, these medications are generally ineffective against HAE, although epinephrine can be used for life-threatening reactions. Misdiagnosis has also led to unnecessary exploratory surgery in patients with abdominal swelling, and some patients with HAE have had their abdominal pain mistakenly diagnosed as psychogenic.

[0108] Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925 describes C1 inhibitor therapy as well as other treatments for HAE.

[0109] In an HAE attack, acute treatment is administered to halt the progression of edema as quickly as possible. One acute treatment is intravenous injection of C1 inhibitor concentrate derived from donor blood, but this therapy is not available in many countries. In emergency situations where C1 inhibitor concentrate is unavailable, an alternative is fresh frozen plasma (FFP), which also contains C1 inhibitor.

[0110] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are currently available in the United States, and two C1 inhibitor products are currently available in Canada. For acute attacks, sterile Berinert P (CSL Behring) was approved by the FDA in 2009. For prophylaxis, nanofiltered CINRYZE® was approved by the FDA in 2008. Rhucin / Ruconest (Pharming) is an investigational recombinant C1 inhibitor that does not pose a risk of transmission of infections caused by human bloodborne pathogens.

[0111] Treatment for acute HAE attacks also includes the administration of pain relievers and / or IV fluids.

[0112] Other treatment modalities can stimulate C1 inhibitor synthesis or reduce C1 inhibitor consumption. Administration of androgens, such as danazol, can reduce the frequency and severity of attacks by stimulating C1 inhibitor production.

[0113] Helicobacter pylori can trigger abdominal attacks. Treating H. pylori with antibiotics reduces abdominal attacks.

[0114] Novel treatments target the contact cascade. Ecallantide (KALBITOR®) inhibits plasma kallikrein and is approved in the United States. Icatibant (FIRAZYR®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0115] Diagnosis of HAE can be based on, for example, family history and / or blood tests. Laboratory findings associated with types I, II, and 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 decreased, as are C4 levels, but C1q levels are normal. In type II HAE, C1 inhibitor levels are normal or increased, but C1 inhibitor function is abnormal. C4 levels are decreased and C1q levels are normal. In type III, C1 inhibitor, C4, and C1q levels are all normal. The present disclosure is based, at least in part, on the identification of additional proteins whose levels differ in samples from HAE patients compared to those from healthy individuals (Table 1). Measuring the level or presence of 2-HMWK can be used to determine whether a subject has a disease such as HAE. In some embodiments, this method may be used to determine whether a patient has had or is currently experiencing an HAE attack.

[0116] Symptoms of HAE can be evaluated using questionnaires, such as questionnaires that are completed by patients, clinicians or family members.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.

[0117] The level of cleaved kininogen and / or intact kininogen detected in a sample from a subject can be compared with a reference value to determine whether the subject has or is at risk for a PKa1-mediated disorder (e.g., HAE). Alternatively, or in addition, the level of cleaved kininogen and / or intact kininogen detected in a sample from a subject can be compared with a reference value to assess the effectiveness of treatment of the disorder, the prognosis or severity of the disorder, and / or identify the patient as a candidate for treatment.

[0118] The reference value can be the percentage of cleaved kininogen of the control level. In some embodiments, the control level is the percentage of cleaved kininogen in a control sample, for example, a sample (e.g., a blood sample or a plasma sample) taken from a healthy subject or a population of healthy subjects, and the healthy subject preferably belongs to the same species as the candidate subject. As used herein, a healthy subject is a subject who does not have or has no apparent history of a target disease (e.g., HAE or an autoimmune disease, for example, a PKal-mediated disorder such as RA, US, and Crohn's disease) at the time the level of cleaved kininogen and / or intact kininogen is measured.

[0119] The control level can also be a predetermined level or threshold. Such a predetermined level can represent the percentage of cleaved kininogen in a population of subjects who do not have or are not at risk for the target disease. The predetermined level can also represent the percentage of cleaved kininogen in a population of subjects who have the target disease.

[0120] The predetermined level can take a variety of forms. For example, the predetermined level can be a single cutoff value, such as a median or mean. In some embodiments, such a predetermined level can be established based on comparison groups, for example, one particular group known to have the target disease and another particular group known to not have the target disease. Alternatively, the predetermined level can be a range, for example, a range representing the percent of cleaved kininogen in a subject population within a predetermined percentile.

[0121] The control levels described herein can be determined by routine techniques. In some examples, the control level can be determined by performing conventional methods (e.g., the same assays described herein for obtaining the levels of cleaved and / or intact kininogen in a test sample) on a control sample as also described herein. In another example, the levels of cleaved and / or intact kininogen in members of a control group are obtained, and the results are analyzed, for example, by a calculation program, to obtain a control level (a predetermined level) representing the levels of cleaved and / or intact kininogen in the control population.

[0122] By comparing the percentage of cleaved kininogen in a sample obtained from a candidate subject with the reference value described herein, it is possible to determine whether the candidate subject has or is at risk for a PKal-mediated disease (e.g., HAE or an autoimmune disease such as RA, UC, and Crohn's disease). For example, if the percentage of cleaved kininogen in a candidate subject's sample deviates from the reference value (e.g., is increased compared to the reference value or is decreased compared to the reference value), the candidate subject can be identified as having or at risk for the disease. If the reference value represents a range of the percentage of cleaved kininogen in a population of subjects with a target disease, if the percentage of cleaved kininogen in a candidate sample falls within that range, it indicates that the candidate subject has or is at risk for the target disease. In some cases, the reference value may represent a background level indicating the absence of cleaved kininogen. The presence of cleaved kininogen would be considered to deviate from such a background reference value. As used herein, "deviation from" a control sample or reference value encompasses the level of cleaved HMWK as well as the presence or absence of cleaved HMWK in a sample.

[0123] As used herein, "high level, or level above reference value" means that the level / percentage of cleaved kininogen is higher than a reference value, e.g., a pre-defined threshold value for the level / percentage of cleaved kininogen in a control sample. Control levels are described in detail herein.

[0124] An elevated percentage of cleaved kininogen includes, for example, a percentage of cleaved kininogen that is 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more above the reference value. An elevated percentage of cleaved kininogen also includes increasing an event from a zero state (e.g., no or undetectable cleaved and / or intact kininogen that binds to the capture reagent in the sample) to a non-zero state (e.g., some presence or detectable presence of cleaved and / or intact kininogen).

[0125] As used herein, a "low percentage / level, or a percentage / level below a reference value" means that the percentage / level of cleaved kininogen is lower than a reference value, e.g., a pre-defined threshold value for cleaved kininogen in a control sample. Control levels are described in detail herein.

[0126] A low level of cleaved kininogen includes, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more lower cleaved kininogen than the reference level. A low level of cleaved kininogen binding to the capture reagent also includes a reduction of an event from a non-zero state (e.g., some or detectable cleaved kininogen in the sample) to a zero state (e.g., no or no detectable cleaved kininogen in the sample).

[0127] In some embodiments, the candidate subject is a human patient with symptoms of pKal-mediated disorders, such as HAE or autoimmune diseases such as RA, UC and Crohn's disease. For example, the subject has edema, swelling that is mostly or entirely peripheral; hives; redness, pain and swelling without evidence of infection; non-histamine-mediated edema, recurrent bouts of swelling, or a combination thereof. In other embodiments, the subject does not have symptoms of pKal-mediated disorders at the time of sample collection, or has no history of symptoms of pKal-mediated disorders, or has no history of pKal-mediated disorders such as HAE. In yet another embodiment, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0128] Subjects identified by the methods described herein may be administered an appropriate treatment.

[0129] Given the correlation between the level of cleaved HMWK and plasma kallikrein-related diseases, such as those described herein, the assay methods and kits described herein can be applied to assess the effectiveness of the treatment of such diseases.For example, a plurality of biological samples (e.g., blood samples or plasma samples) can be collected from a subject undergoing treatment before and after treatment or during the course of treatment.The level of cleaved kininogen and / or intact kininogen can be measured by any of the assay methods described herein, and the value (e.g., percentage) of cleaved kininogen and / or intact kininogen can be determined accordingly.A decrease in the percentage of cleaved kininogen after treatment or during the course of treatment (comparing the percentage of cleaved kininogen in the sample collected later with that in the sample collected earlier) or an increase in the percentage of intact kininogen after treatment or during the course of treatment indicates that the treatment is effective.In some examples, the treatment involves a therapeutic agent such as a kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH supplement. Examples of therapeutic agents include, but are not limited to, lanadelumab (DX-2930), ecallantide (DX-88), icantibant, and human plasma-derived C1-INH.

[0130] If a subject is identified as not responding to treatment, the identified subject is administered with a high dose and / or frequent dose of the therapeutic agent.In some embodiments, for a subject who is identified as responding to treatment or who does not need further treatment, the dosage or frequency of administration of the therapeutic agent is maintained, reduced, or discontinued.Alternatively, for a subject who is found not to respond to the first treatment, a different treatment can be applied.

[0131] In other embodiments, the level of cleaved kininogen, alone or in combination with the level of intact kininogen, can be used to identify disorders that may be treatable with pKal inhibitors. To carry out this method, the level of cleaved kininogen and / or the level of intact kininogen can be measured in a sample (e.g., a blood sample or plasma sample) collected from a subject with the target disease using an appropriate assay, such as a Western blot or ELISA assay described herein. Values ​​such as the percentage of cleaved kininogen and / or intact kininogen can be determined as described herein. The level of cleaved kininogen and / or intact kininogen can be compared with a reference value described herein. A deviation (e.g., higher or lower) of the level of cleaved kininogen and / or intact kininogen from the reference value indicates that a pKal inhibitor may be effective in treating the disease. For example, a decrease in the percentage of cleaved kininogen after or during treatment can identify the treatment as effective. Alternatively, a treatment is identified as effective if the percent of intact kininogen increases after or during the course of treatment.

[0132] If the disease is identified as being sensitive to (treatable with) a pKal inhibitor, the method may further include administering to a subject having the disease an effective amount of a pKal inhibitor, such as ecallantide (DX-88), EPIKAL-2, or lanadelumab (DX-2930).

[0133] Methods for assessing the severity or condition of a disease or disorder associated with plasma kallikrein are also within the scope of the present disclosure. For example, as described herein, HAE may be in a dormant state (basal state) in which a subject exhibits no disease symptoms. Attacks of HAE are typically recurrent episodes, and subjects may experience pain and swelling, for example, in the hands, feet, face, gastrointestinal tract, genitals, and larynx (pharynx), which may last for 2 to 5 days. In some embodiments, the level of 2-HMWK is an indicator of whether a subject will experience, is experiencing, or will soon experience an HAE attack. In some embodiments, the method involves comparing the level of 2-HMWK in a sample obtained from a subject with HAE with the level of 2-HMWK in a sample from the same subject, e.g., a sample obtained from the same subject in a basal state or during an HAE attack.

[0134] (v) Non-clinical applications Furthermore, assays for detecting the level of cleaved 2-HMWK described herein may be used for research purposes. Although numerous diseases and disorders associated with or mediated by plasma kallikrein have been identified, it is possible that other diseases may be mediated by similar mechanisms or have similar components. In some embodiments, the methods described herein can be used to identify a disease as being associated with plasma kallikrein, mediated by plasma kallikrein, or having a component of the contact activation system. In some embodiments, the methods described herein can be used to investigate the mechanism (e.g., discovery of new biological pathways or biological processes involved in disease development) or progression of a disease.

[0135] In some embodiments, the level of cleaved 2-HMWK measured using the assays described herein can be used to develop new therapeutic agents for diseases associated with the contact activation system. For example, the level of cleaved 2-HMWK can be measured in samples obtained from subjects receiving a new therapeutic agent (e.g., in a clinical trial). In some embodiments, the level of cleaved 2-HMWK before, during, or after a new treatment can indicate the effectiveness of the new therapeutic agent or the progression of the disease in the subject.

[0136] II. Treatment of plasma kallikrein-related diseases Subjects at risk for or suffering from a plasma kallikrein-associated disorder identified using the methods and assays described herein can be treated with any appropriate therapeutic agent. In some embodiments, the methods provided include selecting a treatment for the subject based on the results of the described methods, e.g., measuring the level of cleaved 2-HMWK.

[0137] In some embodiments, the method includes one or both of selecting or administering a therapeutic agent to the subject, e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1 esterase inhibitor, based on the results of the assay, e.g., 2-HMWK detection.

[0138] In some embodiments, the subject is administered one or more doses of a therapeutic agent. In some embodiments, the subject is administered a plasma kallikrein inhibitor. In some embodiments, the kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein antibody or a fragment thereof. In some embodiments, the subject is administered an antagonist of the bradykinin B2 receptor. In some embodiments, the subject is administered C1-INH.

[0139] As part of a combination therapy for treating a disease or condition involving the contact activation system, a therapeutic agent, such as a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or C1-INH, can be administered with another therapy. Combination therapy, such as a combination with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH supplement, for example, a combination with one or more of a kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a C1-INH supplement, and a combination therapy with another therapy, can be provided in several different configurations. A first agent can be administered before or after the other therapy. In some situations, the first agent and the other therapy (e.g., administration of a therapeutic agent) are administered simultaneously or within close proximity (e.g., with a short time interval between injections, such as in the same treatment session). The first agent and the other therapy can also be administered at a further time interval.

[0140] Plasma kallikrein binding agents (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding agents, e.g., small molecules) are useful therapeutic agents for various diseases and conditions, e.g., diseases and conditions associated with plasma kallikrein activity. For example, in some embodiments, the disease or condition associated with plasma kallikrein activity is hereditary angioedema (HAE). In some embodiments, a plasma kallikrein binding agent, such as a plasma kallikrein inhibitor, is administered to a subject at risk for or suffering from a disease associated with the contact activation system.

[0141] Many useful protein inhibitors of tissue kallikrein and / or plasma kallikrein contain Kunitz domains. As used herein, a "Kunitz domain" refers to a polypeptide domain having at least 51 amino acids and containing at least two, preferably three, disulfides. This domain folds so that disulfide bonds can be formed between the first and sixth cysteines, the second and fourth cysteines, and the third and fifth cysteines (e.g., in a 58-amino acid Kunitz domain, disulfides can be formed between the 5th and 55th, 14th and 38th, and 30th and 51st cysteines according to the numbering of the BPTI homologous sequence described below), or, if two disulfides are present, disulfides can be formed between the corresponding subsets of cysteines. The spacing between each cysteine ​​can be within 7, 5, 4, 3, 2, 1, or 0 amino acids of the spacing between positions corresponding to positions 5-55, 14-38, and 30-51 in the BPTI sequence numbering described below. The BPTI sequence can be used as a reference to identify specific positions of any general Kunitz domain. Comparison of a Kunitz domain of interest with BPTI can be performed by identifying the optimal alignment that maximizes the number of aligned cysteines.

[0142] The three-dimensional structure (at high resolution) of the Kunitz domain of BPTI is known. One of the X-ray structures has been deposited in the Brookhaven Protein Data Bank as "6PTI." The three-dimensional structures of several BPTI homologs are known (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022). The sequences of at least 81 Kunitz domains are known. Known human homologues 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), inter-alpha trypsin inhibitor, two Kunitz domains of APP-I (Kido et al., J. Biol. Chem. (1988) 263(34):18104-18107), a Kunitz domain from collagen, and three Kunitz domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91:3353-3357), the Kunitz domain of hepatocyte growth factor activator inhibitor type 1, the Kunitz domain of hepatocyte growth factor activator inhibitor type 2, and the 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 shown in Table 1) that contains three Kunitz domains.

[0143] [Table 1]

[0144] The Kunitz domains are designated LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (residues 213-270). The cDNA sequence of LACI was reported by Wun et al. (J. Biol. Chem. (1988) 263(13):6001-6004). Girard et al. (Nature (1989) 338:518-20) reported mutation studies in which the P1 residues of each of the three Kunitz domains were altered. LACI-K1 inhibits factor VIIa (F.VIIa) when it forms a complex with tissue factor, and LACI-K2 inhibits factor Xa.

[0145] Various methods can be used to identify Kunitz domains from sequence databases. For example, the amino acid sequences, consensus sequences, or motifs (e.g., ProSite motifs) of known Kunitz domains can be searched against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, Maryland), for example, using BLAST; against the Pfam database of HMMs (hidden Markov models), against the SMART database; or against the ProDom database (e.g., using default parameters for the Pfam search). For example, Pfam Accession Number PF00014 for Pfam Release 9 provides numerous Kunitz domains and HMMs for identifying Kunitz domains. A description of the Pfam database can be found in Sonhammer et al., Proteins (1997) 28(3):405-420, and a detailed description of HMMs can be found, for example, in Gribskov et al., Meth. Enzymol. (1990) 183:146-159; Gribskov et al., Proc. Natl. Acad. Sci. USA (1987) 84:4355-4358; Krogh et al., J. Mol. Biol. (1994) 235:1501-1531; and Stultz et al., Protein Sci. (1993) 2:305-314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, Germany) 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 profiling using hidden Markov models in the HMMer2 search program (RR. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). This database is annotated and monitored.The ProDom protein domain database consists of an automated compilation of homologous domains (Corpet et al., Nucleic Acids Res. (1999) 27:263-267). The latest version of ProDom was constructed using recursive PSI-BLAST searches of the SWISS-PROT 38 protein database and the TREMBL protein database (Altschul et al., Nucleic Acids Res. (1997) 25:3389-3402; Gouzy et al., Computers and Chemistry (1999) 23:333-340). The database automatically generates consensus sequences for each domain. Prosite lists Kunitz domains as motifs and identifies proteins containing Kunitz domains. See, e.g., Falquet et al., Nucleic Acids Res. (2002) 30:235-238.

[0146] Kunitz domains interact with target proteases primarily using amino acids in two loop regions ("binding loops"). The first loop region lies between residues corresponding approximately to amino acids 13-20 of BPTI. The second loop region lies between residues corresponding approximately to amino acids 31-39 of BPTI. Exemplary libraries of Kunitz domains vary at one or more amino acid positions in the first and / or second loop regions. Particularly useful positions to vary when screening for Kunitz domains that interact with kallikrein or selecting variants with improved affinity include positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 of the BPTI sequence. At least some of these positions are expected to make close contact with the target protease. Varying other positions, such as those adjacent to these positions in the three-dimensional structure, is also useful.

[0147] The "framework regions" of a Kunitz domain are defined as residues that are part of the Kunitz domain, but specifically exclude residues in the first and second binding loop regions, i.e., residues corresponding approximately to amino acids 13-20 of BPTI and amino acids 31-39 of BPTI. Conversely, residues not present within the binding loops may tolerate a broader range of amino acid substitutions (e.g., conservative and / or non-conservative substitutions).

[0148] In one embodiment, these Kunitz domains are variants of the loop structure comprising Kunitz domain 1 of the human lipoprotein-associated coagulation inhibitor (LACI) protein. LACI contains three distinct internal peptide loop structures that are paradigm Kunitz domains (Girard, T. et al., Nature (1989) 338:518-520). Variants of Kunitz domain 1 of LACI described herein have been screened and isolated and bind to kallikrein with high 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, such as plasma kallikrein. Useful modulators of kallikrein function typically bind to and / or inhibit kallikrein, as determined using kallikrein binding assays and kallikrein inhibition assays.

[0149] In some aspects, the plasma kallikrein inhibitor binds to the active form of plasma kallikrein. In some embodiments, the plasma kallikrein inhibitor binds to and inhibits plasma kallikrein, for example, human plasma kallikrein and / or mouse kallikrein. Exemplary polypeptide plasma kallikrein agents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983, U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991, and U.S. Patent Application Publication No. 20110086801 (each of which is incorporated herein by reference in its entirety). In some embodiments, the plasma kallikrein inhibitor is an inhibitory polypeptide or inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also known as DX-88 or KALBITOR®; SEQ ID NO: 80). In some embodiments, the kallikrein inhibitor comprises or consists of a DX-88 polypeptide having a sequence of about 58 amino acids, amino acids 3-60 of SEQ ID NO: 80, or a sequence of about 60 amino acids of SEQ ID NO: 80.

[0150] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO: 80).

[0151] The plasma kallikrein inhibitor may be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) or may comprise only an antigen-binding fragment (e.g., a Fab fragment, a F(ab')2 fragment, or an scFv fragment). The binding protein may comprise two heavy chain immunoglobulins and two light chain immunoglobulins, or may be a single-chain antibody. The plasma kallikrein inhibitor may be a recombinant protein, such as a humanized antibody, a CDR-grafted antibody, a chimeric antibody, a deimmunized antibody, or an in vitro-generated antibody, and may optionally comprise a constant region derived from a human germline immunoglobulin sequence. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0152] Exemplary plasma kallikrein binding proteins are disclosed in U.S. Patent Application Publication No. 20120201756, the entire contents of which are incorporated herein by reference. In some embodiments, the kallikrein binding protein is 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-E10, X115-E11, X115-E12, X115-E13, X115-E14, X115-E15, X115-E16, X115-E17, X115-E18, X115-E19, X115-E20, X115-E21, X115-E22, X115-E23, X115-E24, X115-E25, X115-E26, X115-E27, X115-E28, X115-E29, X115-E30, X115-E31, X115-E32, X115-E33, X115-E34, X115-E35, X115-E36, X115-E37, X115-E38, X115-E39, X115-E40, X115-E41, X115-E42, X115-E43, X115-E4 and 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 115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or lanadelumab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to 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 lanadelumab. See U.S. Patent Application Publication Nos. 20110200611 and 20120201756, which are incorporated herein by reference.

[0153] An example of a plasma kallikrein inhibitor antibody is lanadelumab. The amino acid sequences of the heavy and light chain variable regions of lanadelumab are set forth below, with the CDR regions shown in bold and underlined:

[0154] Lanadelumab heavy chain variable region sequence (SEQ ID NO: 81) EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY ADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SS Lanadelumab light chain variable region sequence (SEQ ID NO: 82) DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPKLLIY KASTLESGVP SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI

[0155] In some embodiments, a plasma kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the framework regions of the HC and / or LC (e.g., FR1, FR2, FR3 and / or FR4 of the HC and / or LC) to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a plasma kallikrein inhibitor described herein in its HC and / or LC CDRs (e.g., CDR1, CDR2, and / or CDR3 of the HC and / or LC). In some embodiments, a plasma kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with a plasma kallikrein inhibitor described herein in its constant region (e.g., CH1, CH2, CH3, and / or CL1).

[0156] In some embodiments, the small molecule binds to and inhibits the active form of plasma kallikrein.

[0157] Bradykinin B2 receptor inhibitor In some embodiments, the subject is administered a bradykinin B2 receptor inhibitor (e.g., antagonist). Exemplary bradykinin B2 receptor antagonists include icatibant (Firazyr®), a 10-amino acid peptidomimetic that blocks native bradykinin from binding to the bradykinin B2 receptor.

[0158] C1-INH supplements In some embodiments, the subject is administered a C1 esterase inhibitor (C1-INH), such as a C1-INH supplement. Exemplary C1-INH supplements have been published, including, for example, human plasma-derived C1-INH, such as Berinert® and CINRYZE®.

[0159] III. Kit for detecting cleaved HMWK The present disclosure also provides a kit for use in evaluating cleaved HMWK in a sample suspected of containing cleaved HMWK, e.g., a biological sample from a human patient. Such a kit may include a first agent that specifically binds to cleaved HMWK compared to intact HMWK or LMWK. In some embodiments, the first agent is an antibody, e.g., any antibody described herein that specifically binds to cleaved HMWK (e.g., 559B-M004 or a functional variant thereof described herein). In some embodiments, the kit further includes a second agent (e.g., an antibody that binds to HMWK) that detects binding of the first agent to cleaved HMWK. The second agent can be conjugated to a label. In some embodiments, the second agent is an antibody that specifically binds to cleaved HMWK. In other embodiments, the second agent is an antibody that cross-reacts with both cleaved HMWK and intact HMWK.

[0160] The kit may further comprise a support element for carrying out immunoassay and immobilizing the first agent. In some embodiments, the support element is a 96-well plate, such as a 96-well ELISA plate. The kit may also comprise one or more buffers described herein, including but not limited to, a coating buffer; an assay buffer, such as an assay buffer containing ZnCl; a blocking buffer; a washing buffer; and / or a stop buffer.

[0161] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for using the components included in the kit to measure the level of cleaved HMWK and / or intact HMWK in a sample, and the sample may be a biological sample collected from a human patient. Alternatively, or in addition, the kit may include instructions for using the components included therein to measure the level of LMWK.

[0162] The instructions for using the kit generally include information on the amount of each component and the appropriate conditions for carrying out the assay methods described herein. The components of the kit may be present in unit doses, bulk packaging (e.g., multi-dose packaging), or sub-unit doses. The instructions provided with the kits of the present disclosure are typically written instructions on a label or package insert (e.g., paper included in the kit), although machine-readable instructions (e.g., instructions written on a magnetic or optical storage disk) are also acceptable.

[0163] The label or package insert indicates that the kit is for use in assessing levels of cleaved HMWK and / or intact HMWK. In some embodiments, the kit is for use in assessing levels of LWMK. Instructions can be provided for practicing any of the methods described herein.

[0164] The kits of the present disclosure are packaged in suitable packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, and flexible packaging (e.g., sealable Mylar bags or plastic pouches). Packaging for use in combination with specific devices, such as inhalers, nasal spray devices (e.g., nebulizers), or injection devices such as minipumps, is also contemplated. The kits may have a sterile connection port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle). The container may also have a sterile connection port (e.g., the container may be an intravenous solution bag or vial with a stopper pierceable by a hypodermic injection needle).

[0165] The kit may optionally provide additional components, such as information for use in interpretation, such as a control sample and / or a standard or reference sample. The kit typically includes a container and a label or package insert(s) on or attached to the container. In some embodiments, the present disclosure provides an article of manufacture containing the contents of the above-described kit.

[0166] IV. Other Antibodies that Bind to Cleaved HMWK Provided herein are isolated antibodies that bind to both cleaved HMWK and intact HMWK. In some embodiments, such antibodies do not bind to LMWK or bind to LMWK with low affinity. In other embodiments, such antibodies also bind to LMWK.

[0167] In some embodiments, the antibodies described herein that specifically bind to cleaved and intact HMWK (or even LMWK) have suitable binding affinity for one or more target antigens. -5 M, 10 -6 M, 10 -7 M, 10 -8 M, 10 -9 M, 10 -10 Binding affinity (K D ).

[0168] Examples of the above antibodies and their binding specificities are provided in Table 2 in Example 2 below. The amino acid sequences of the heavy and light chain variable regions are set forth below, with the CDR regions in bold and underlined (determined by one scheme as an example): [ka] TIFF2025081553000005.tif223162TIFF2025081553000006.tif228162TIFF2025081553000007.tif22716 2TIFF2025081553000008.tif221162TIFF2025081553000009.tif230162TIFF2025081553000010.tif23162

[0169] Functional equivalents of any of the exemplary antibodies listed above are also included within the scope of the present disclosure. Such functional equivalents may bind to the same epitope of cleaved HMWK and / or intact HMWK or sample epitope of LMWK as one of the exemplary antibodies listed above. In some embodiments, functional equivalents compete with one of the exemplary antibodies listed above for binding to the target antigen.

[0170] In some embodiments, the functional equivalent is the corresponding V of one of the exemplary antibodies above. H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the CDR H CDR1, V H CDR2 and / or V H V containing CDR3 H Alternatively or additionally, functional equivalents include V chains that are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the exemplary antibodies described above. L CDR1, V L CDR2 and / or V LIn some embodiments, the functional equivalent comprises the same heavy chain complementarity determining regions (CDRs) and / or light chain CDRs as one of the above exemplary antibodies.

[0171] Alternatively or additionally, functional equivalents may be the V of an exemplary antibody. H V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to a V strand. H Chains and / or V of Exemplary Antibodies L V that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to a V strand. L Includes chains.

[0172] In some cases, a functional equivalent may include one or more (e.g., up to five, up to three, or up to one) conservative mutations in one or more heavy chain CDRs or one or more light chain CDRs of an exemplary antibody, for example, at positions where the residues are unlikely to be involved in interactions with the target antigen.

[0173] Without further elaboration, it is believed that one skilled in the art can utilize the present disclosure to its fullest extent in light of the above description. The following specific embodiments should, therefore, be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. Any publications cited herein are incorporated by reference for the purposes or subject matter discussed herein.

[0174] (Example) Example 1 : Development of an immunoassay for the specific detection of cleaved HMWK First, we developed an ELISA-based immunoassay screen to identify Fab fragments that bind to truncated or intact HMWK in a phage display library. The assay conditions generally involved immobilizing biotinylated intact or truncated HMWK on streptavidin-coated 384-well assay plates, blocking with bovine serum albumin (BSA) blocking buffer, and contacting the immobilized HMWK with Fab displayed on phage from an overnight culture of Escherichia coli (E. coli) (detected with anti-M13-HRP antibody).

[0175] As shown in panel A of Figure 12, first, approximately 1 × 10 antibody was added to biotinylated single-chain HMWK immobilized on streptavidin-coated magnetic beads (Dynabeads M280, Thermo Fisher Scientific). 12Selection aimed at obtaining double-chain HMWK-specific antibodies was performed by negative selection of a library containing 100 phage. The depleted library was then contacted with biotinylated double-chain HMWK immobilized on streptavidin-coated magnetic beads. The beads were washed extensively with PBS buffer and used to infect Escherichia coli (E. coli) to amplify phage output and complete the selection round. After three rounds of selection, individual phage colonies were screened by ELISA using biotinylated single-chain HMWK and double-chain HMWK immobilized on streptavidin-coated plates, followed by detection with horseradish peroxidase (HRP) conjugated to anti-M13 antibody and absorbance detection via substrate hydrolysis of 3,3',5,5'-tetramethylbenzidine (TMB). The recombinant Fab fragments were expressed in Escherichia coli (E. coli) and purified by protein A-Sepharose chromatography (Wassaf et al., Anal. Biochem. (2006) 351:241-253). The specificity of each purified Fab was determined by measuring binding to biotinylated single-chain HMWK, biotinylated double-chain HMWK, or biotinylated LMWK in coated 384-well plates using HRP-conjugated streptavidin and TMB detection. These assay conditions identified the 559B-M004-B04 isolate, which specifically bound cleaved HMWK over intact HMWK (Figure 1).

[0176] Immobilized HMWK was also contacted with crude (unpurified) 559B-M004-B04 Fab preparation obtained from an overnight culture of E. coli. Fab bound to HMWK was detected using an anti-human Fab-HRP antibody, but specific binding to cleaved HMWK was not observed (Figure 1).

[0177] The immunoassay configuration was reversed by passively immobilizing the purified Fab fragment of 559B-M004-B04 on a polystyrene 384-well assay plate. The Fab was contacted with biotinylated HMWK, and bound HMWK was detected with streptavidin-HRP (Figure 1).

[0178] Unexpectedly, substituting the BSA blocking buffer with a commercially available blocking buffer, Candor Biosciences LowCross Blocking Solution, during the initial screening assay unexpectedly increased the specificity of 559B-M004-B04 Fab for cleaved HMWK (Figure 1). Furthermore, performing the immunoassay in a 96-well assay plate rather than a 384-well plate further enhanced the observed specificity of 559B-M004-B04 for cleaved HMWK (Figure 1).

[0179] Based on the results obtained with the 559B-M004-B04 isolate, we developed an immunoassay (ELISA) to detect 2-HMWK in samples (Figure 12, panel B). This assay can also be used to further evaluate the binding properties of other Fab fragments and antibodies. Briefly, multiwell plates are coated with Fab overnight. The next day, the plates are washed and then blocked with BSA buffer. After washing, samples, standards, and QCs diluted in LowCross buffer are added to the plate, followed by incubation and washing. Bound double-chain HMWK is detected by the addition of an HRP-conjugated sheep anti-HMWK polyclonal detection antibody. After incubation with the detection antibody, the plate is washed and TMB substrate is added to the plate. After a short incubation, the reaction is stopped with phosphoric acid. The absorbance is then measured between 450 nm and 630 nm.

[0180] Example 2 : Evaluation of the binding specificity of Fab clones using the immunoassay described herein The immunoassay described in Example 1 was used to evaluate the binding of 36 purified Fab clones (Table 2 below) to cleaved HMWK, intact HMWK, and LWMK. Specifically, each purified Fab clone was immobilized in 100 μL of PBS at a concentration of 1 μg / L on a 96-well assay plate and incubated overnight at 2–8°C. The assay plate was blocked with LowCross blocking buffer. Biotinylated intact HMWK, biotinylated cleaved HMWK, or biotinylated LMWK (1 μg / L each) was added to each well in a total volume of 100 μL, incubated for 2 hours, and then washed with wash buffer. HRP-labeled streptavidin was added to each well at a concentration of 100 ng / mL, and signal was generated using Ultra TMB Substrate. The signal observed when biotinylated protein was added to uncoated wells was used to calculate the signal-to-noise ratio (Figure 2, panels A and B). Based on the ELISA results, the antibodies can be classified into five types (Table 2).

[0181] [Table 2]

[0182] Several antibodies, such as 559B-M0064-H02, were obtained that bind to both single-chain HMWK, two-chain HMWK, and LMWK. These antibodies likely bind to epitopes in domains 1–4 common to HMWK and LMWK. M070-H10 is an example of an antibody that is predicted to bind to an epitope common to both single-chain and two-chain HMWK but not LMWK. LMWK is a splice variant of kininogen that produces a truncated protein consisting of domains 1–4 and part of domain 5 (Colman et al., Blood (1997) 90:3819–3843). Therefore, antibodies such as M070-H10 likely bind to domains 5 or 6.

[0183] As shown in Figure 14, panel A, 559B-M0004-B04 showed selectivity for double-chain HMWK over both single-chain HMWK and LMWK and was selected for further assay optimization. To detect cleaved HMWK in human plasma samples, a sandwich ELISA was developed in which 559B-M0004-B04 (100 μL at 2 μg / mL) was passively immobilized on a 96-well plate (Nunc Maxisorp plate) (Figure 12, panel B). The next day, the plate was washed and then blocked with 2% BSA in PBS buffer (protease / IgG-free). After washing, samples containing cleaved HMWK were placed in 0.1% BSA buffer in PBS with 0.05% Tween-20 (double-chain HMWK assay buffer). Purified protein standards (e.g., double-chain HMWK, intact HMWK, or LMWK) were spiked into HNKW HMWK-deficient plasma and diluted 1:320 in double-chain HMWK assay buffer. After washing the plate with PBST, a mixture of two mouse monoclonal antibodies (11H05 and 13B12) at 1 μg / mL in double-chain HMWK assay buffer was added for 1 h at room temperature. Unbound detection antibody was washed away, and a 1:2000 dilution of a goat anti-mouse secondary antibody conjugated with horseradish peroxidase (HRP) was added. The assay containing the secondary antibody was incubated for 1 h at room temperature, and unbound secondary antibody was removed by washing with double-chain HMWK assay buffer. Signal was detected by the addition of the HRP substrate, 3,3',5,5'-tetramethylbenzidine (TMB). The reaction was stopped with phosphoric acid. Hydrolysis of the TMB substrate was detected at 450 nm to 630 nm using a microplate reader (Figure 3). Furthermore, ELISA assays were performed using samples containing truncated HMWK in either the two-chain HMWK assay buffer or HMWK-deficient plasma, and comparable binding was observed when analyzed in the presence of either 2.5% or 10% plasma (Figure 4). These immunoassay conditions were used to detect specific binding to truncated HMWK. Comparable performance was observed in this assay whether HMWK was provided in the two-chain HMWK assay buffer or in HMWK-deficient plasma (Figures 3 and 4). Furthermore, no binding of 559B-M0004-B04 to LMWK was observed.

[0184] An ELISA assay was evaluated for the detection of truncated HMWK generated by contact activation in human plasma (Fig. 5A and 5B). The amount of truncated HMWK in normal human plasma was measured in the absence or presence of catalytic amounts of FXIIa, pKal, or ellagic acid, which trigger the autoactivation of FXII to FXIIa and thereby generate truncated HMWK (Fig. 5, panels A and B). Consistent with the role of plasma kallikrein as the primary plasma enzyme required for the generation of two-chain HMWK, the addition of ellagic acid or FXIIa did not generate truncated HMWK in prekallikrein-deficient plasma. The contact system in FXI-deficient plasma was activated to the same extent by FXIIa, pKal, or ellagic acid, a result consistent with the notion that FXIa is generated by FXIIa and does not generate two-chain HMWK.

[0185] The results obtained with the two-chain HMWK ELISA were confirmed by Western blot analysis using the mouse monoclonal antibody 11H05, which detects cleaved HMWK generated by contact activation in human plasma (Fig. 10). The 11H05 antibody specifically binds to the light chain of HMWK and reveals both the 56-kDa light chain and the further proteolytically cleaved 46-kDa light chain generated by the proteolytic activity of plasma kallikrein at a site near the N-terminus of the HMWK light chain (Colman et al., Blood (1997) 90:3819-3843).

[0186] We also evaluated whether the ELISA assay could detect cleaved HMWK produced in the plasma of 12 healthy donors (Figure 6). In each of the 12 samples, cleaved HMWK was detected after ellagic acid activation of the contact activation system. Similarly, we measured the amount of cleaved HMWK after inhibiting the contact activation system in normal plasma using various concentrations of lanadelumab (DX-2930; a specific inhibitor of plasma kallikrein) or an inhibitor of serpin C1-INH, followed by activation with ellagic acid (Panels A and B of Figure 7). Lanadelumab (DX-2930) potently inhibits plasma kallikrein (K). iLanadelumab is a fully human antibody that specifically inhibits FXIIa-induced double-chain HMWK formation (IC = 0.12 nM), discovered using phage and currently in clinical development for the preventive treatment of HAE-C1INH attacks (Chyung et al., Ann. Allergy Asthma Immunol. (2014) 113:460-466; Kenniston et al., J. Biol. Chem. (1994) 289:23596-23608). Lanadelumab, when added to citrated plasma at various concentrations, effectively inhibited FXIIa-induced double-chain HMWK formation, as shown by Western blot and sandwich ELISA (Figure 7B). The IC of lanadelumab-induced double-chain HMWK formation inhibition was 0.12 nM. 50 The activity of M004-B04 was 212±28 nM, which is consistent with the expected value (approximately 500 nM) for the activation of all prekallikrein in undiluted plasma. The signal was completely inhibited by lanadelumab in plasma treated with a contact activator, confirming that M004-B04 is specific for the two-chain HMWK generated by plasma kallikrein.

[0187] In this preliminary assay, using M004-B04 as the capture antibody and HRP-conjugated sheep polyclonal anti-kininogen as the detection antibody, activation of the contact system with kininogen-deficient plasma did not result in an increase in the ELISA signal (data not shown).

[0188] Similarly, Figure 10 shows that plasma collected from healthy subjects using EDTA as an anticoagulant was activated to the same extent as citrated plasma, supporting the finding that metal ions are not required for contact system activation (Colman et al., Blood (1997) 90:3819-3843). However, double-chain HMWK was not detected by ELISA in EDTA-treated plasma (Figure 5B), suggesting that the binding of the M004-B04 antibody to double-chain HMWK is metal ion dependent. The zinc-binding site of HMWK (amino acids 479–498) in domain 5 of the light chain has been previously identified and shown to mediate the interaction of kininogen with the endothelial cell surface receptor gC1qR, cytokeratin 1, and urokinase plasminogen activator receptor, thereby enhancing contact system activation (Kaplan et al., Adv. Immunol. (2014) 121:41–89; Bjorkqvist et al., Biol. Chem. (2013) 394:1195–1204). Addition of ZnCl2 to the assay buffer at various concentrations was tested and found to increase antibody binding to cleaved HMWK (Figure 11). The effect of increasing concentrations of ZnCl2 on the ELISA signal observed in ellagic acid-activated citrated plasma and EDTA-added plasma was examined. The ELISA signal for EDTA-added plasma increased to an apparent maximum at ZnCl2 concentrations above 400 μM (concentration in the well).

[0189] Electron microscopy has previously demonstrated that zinc binding to single-chain HMWK promotes a more compact, globular quaternary structure (Herwald et al., Eur J. Biochem. (2001) 268:396-404). Similarly, electron microscopy has also demonstrated that double-chain HMWK adopts a more elongated, less globular quaternary structure than single-chain HMWK in EDTA-containing buffers (Herwald et al., Eur J. Biochem. (2001) 268:396-404). Although the effect of zinc on the structure of double-chain HMWK has not yet been reported, the apparent zinc-dependent binding of M004-B04 described herein suggests that double-chain HMWK exists in a unique conformation in the presence of zinc.

[0190] The EDTA concentration in plasma collected in commercially available spray-coated K2EDTA tubes was approximately 4 mM, which, when diluted 1:20, resulted in a well concentration of approximately 200 μM, consistent with the restoration of zinc-dependent binding upon the addition of sufficient ZnCl2 to overcome the chelating capacity of EDTA. In contrast, the ELISA signal from citrated plasma activated with ellagic acid did not increase in the presence of 25 μM or 50 μM (well concentrations), but at ZnCl2 concentrations above 100 μM, the ELISA signal increased to a maximum when ZnCl2 exceeded approximately 200 μM (Figure 11). Normal zinc concentrations in plasma from healthy subjects are 10–17 μM (Wessells et al., J. Nutr. (2014) 144:1204–1210). The increase in ELISA signal observed when ZnCl2 concentrations in the wells exceeded 50 μM was limited to activated citrated plasma, which corresponds to a plasma concentration of >1 mM ZnCl2, suggesting that the ELISA is not affected by physiological variations in plasma zinc concentration. Therefore, ZnCl2 was not added to the assay buffer in subsequent experiments.

[0191] As described above, the binding of 559B-M004-B04 to double-chain HMWK was increased by supraphysiological concentrations of ZnCl2 and inhibited by metal chelation with high concentrations of EDTA. The zinc-binding site in the double-chain HMWK domain has been previously described, and a synthetic peptide (HKH20, HKHGHGHGKHKNKGKKNGKH (SEQ ID NO: 83) encompassing this site has been shown to inhibit contact system activation by weakening cell surface binding (Nakazawa et al., Int. Immunopharmacol. (2002) 2:1875-1885). Therefore, we hypothesized that the HKH20 peptide, as well as the GCP28 peptide corresponding to a sequence within domain 3, inhibited the binding of double-chain HMWK to 559B-M004-B04. We tested whether the HKH20 peptide, but not the GCP28 peptide, could inhibit the binding of double-chain HMWK to M004-B04 by ELISA. As shown in Figure 15, the HKH20 peptide inhibited the binding of double-chain HMWK to M004-B04, but the GCP28 peptide did not, suggesting that the M004-B04 epitope may be located near the zinc-binding site within domain 5. To perform the assay, the kininogen peptide was diluted to 250 μg / mL and preincubated in the assay plate. Purified double-chain HMWK in deficient human plasma was then diluted to 160 and added to the plate.

[0192] The time dependence of cleaved HMWK formation in normal citrated human plasma was assessed at various time points after activation of the contact activation system by ellagic acid or FXIIa (Figure 8). Finally, an ELISA assay was used to assess the presence and amount of cleaved HMWK in plasma samples from patients with hereditary angioedema (HAE) compared with citrated plasma samples from normal patients (without HAE). Samples from HAE patients were found to contain higher levels of double-chain HMWK (1423 ± 603 ng / mL) than samples from normal donors (432.4 ± 186 ng / mL) (Figure 9), which was statistically significant by one-way ANOVA (P = 0.017).

[0193] Because M004-B04 specifically binds to a neoepitope on two-chain HMWK that is not present on single-chain or LMWK and antibody binding was found to be dependent on plasma kallikrein activity, we also tested an assay using a pair of mouse monoclonal antibodies (11H05 and 13B12) for detection (Figure 16). Because antibody 13B12 appears to bind to the heavy chain of HMWK and antibody 11H05 appears to bind to the light chain of HMWK, combining both antibodies for detection resulted in an enhanced signal, likely due to the lack of overlap in the binding epitopes of the antigens.

[0194] The importance of plasma collection for assessing the contact system has been previously documented (Suffritti et al., Clin. Exp. Allergy (2014) 44:1503-1514). It is well known that contact of plasma with glass or other polar surfaces can result in extensive ex vivo contact system activation, which can interfere with accurate measurement of endogenous contact system activation (Colman et al., Blood (1997) 90:3819-3843). The ability of the optimized sandwich ELISA to detect double-chain HMWK was compared with various types of plasma, including a customized plasma containing a mixture of protease inhibitors in acidic citrate dextrose, using plastic vacuum collection tubes called SCAT169 (HTI, Essex, VT). As shown in Figure 6, the standard curve generated with SCAT169 plasma was less sensitive than that generated with citrated plasma, likely due to the presence of 2 mM EDTA in the collected plasma. At the plasma dilution used in this assay (1:320), this concentration of EDTA (3.1 μM) does not significantly interfere with two-chain HMWK and may help stabilize plasma from proteolysis by metalloproteases.

[0195] Citrated plasma and SCAT169 plasma from healthy subjects were compared with samples from HAE patients by Western blot and sandwich ELISA assays. In Figure 17, panels A-C, Western blot assays detecting two-chain HMWK (i.e., cleaved kininogen) in citrated plasma were able to distinguish HAE patient samples from healthy subject (HV) samples, as shown by receiver operating characteristic (ROC) analysis, with area under the curve (AUC) values ​​of 0.977 for the comparison of basal state with HV and 1.0 for the comparison of attack state with HV. Citrated plasma samples from HAE patients in the quiescent (basal) state were distinguished from attack state samples with an AUC of 0.625 (Figure 17, panel D).

[0196] As shown in Figure 18, panels A-C, Western blot assays detecting double-chain HMWK in SCAT169 plasma were able to distinguish between HAE patient samples and healthy subject (HV) samples, as shown by ROC analysis with AUC values ​​of 0.915 for the comparison of basal state with HV or 0.967 for the comparison of seizure state with HV. SCAT169 samples from HAE patients in the quiescent (basal) state were distinguished from seizure state samples with an AUC of 0.597 (Figure 18, panel D).

[0197] In Figure 19, panels A-C, the double-chain ELISA assay detecting double-chain HMWK in citrated plasma was able to distinguish samples from HAE patients from healthy subjects, as shown by ROC analysis with AUC values ​​of 0.915 for the comparison of basal state with HV or 0.866 for the comparison of attack state with HV. Citrated plasma samples from HAE patients in the quiescent (basal) state were distinguished from attack state samples with an AUC of 0.709 (Figure 19, panel D).

[0198] As shown in Figure 20, panels A-C, the double-chain ELISA assay detecting double-chain HMWK in SCAT169 samples was able to distinguish between samples from HAE patients and healthy subjects, as shown by ROC analysis with AUC values ​​of 0.999 for the comparison of basal state to HV or 1.0 for the comparison of attack state to HV. Citrated plasma samples from HAE patients in the quiescent (basal) state were distinguished from attack state samples with an AUC of 0.8176 (Figure 20, panel D).

[0199] The ROC analysis demonstrated that both the double-chain HMWK Western blot and the double-chain HMWK ELISA demonstrated herein may be useful for identifying patients with or at risk of having HAE based on the level of cleaved kininogen in plasma compared with healthy subjects. The presence of protease inhibitors in SCAT169 plasma reduced ex vivo plasma activation at the time of blood collection.

[0200] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only one example of a generic series of equivalent or similar features.

[0201] Those skilled in the art can easily ascertain the essential characteristics of the present disclosure from the above description, and can make various changes and modifications to the present disclosure to adapt it to various applications and conditions without departing from the spirit and scope of the present disclosure. Accordingly, other embodiments are also within the scope of the claims.

[0202] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited by the above description, but is instead set forth in the appended claims.

[0203] In the claims, articles such as "a," "an," and "the" can mean one or more than one, unless indicated to the contrary or otherwise clear from the context. A claim or description including "or" between one or more elements of a group is considered to be satisfied when one, more than one, or all of the elements of the group are present in, employed in, or otherwise relevant to a given product or process, unless indicated to the contrary or otherwise clear from the context. The present disclosure includes embodiments in which exactly one element of a group is present in, employed in, or otherwise relevant to a given product or process. The present disclosure includes embodiments in which more than one or all elements of a group are present in, employed in, or otherwise relevant to a given product or process.

[0204] Furthermore, the present disclosure encompasses all variations, combinations, and modifications in which one or more limitations, elements, clauses, and descriptive terms from one or more recited claims are introduced into another claim. For example, any claim dependent on another claim can be amended to include one or more limitations set forth in any other claim dependent on the same base claim. Where elements are listed, for example, as a Markush group list, each subgroup of the elements is also disclosed, and any one or more can be deleted from the group. In general, when the present disclosure or aspects of the disclosure are referred to as including particular elements and / or features, it should be understood that particular embodiments of the disclosure or aspects of the disclosure consist of or consist essentially of such elements and / or features. For purposes of brevity, the above-described embodiments have not been specifically described in these terms herein. Similarly, it should be noted that the terms "comprising" and "containing" are intended to be open-ended and allow for the inclusion of other elements or steps. When ranges are stated, the endpoints are included. Furthermore, unless otherwise expressly stated or apparent from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can be considered to be any specific value or subrange, down to one-tenth of the unit of the lower limit of that range, that is included within the ranges set forth in various embodiments of this disclosure, unless the context clearly dictates otherwise.

[0205] This application references various issued patents, published patent applications, journal articles, and other publications, all of which are incorporated herein by reference. In the event of a discrepancy between any incorporated reference and this specification, this specification shall control. Furthermore, any particular embodiment of the present disclosure that falls within the scope of the prior art may be expressly excluded from any one or more claims. Such embodiments may be excluded even if the exclusion is not expressly stated herein, because they are deemed to be known to those skilled in the art. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0206] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific embodiments described herein. The scope of the embodiments of the invention described herein is not intended to be limited to the above description, but is instead set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications can be made to this description without departing from the spirit or scope of the present disclosure, as defined in the following claims.

Claims

[Claim 1] The invention as described in the specification or drawings of this application.

Images