Peptide quantitation assay for differentiating full-length high molecular weight kininogen (HMWK) and cleaved hmwk

Abstract

To provide sensitive and selective assay methods for differentiating full-length HMWK and cleaved HMWK.SOLUTION: A method for detecting cleaved high molecular weight kininogen (HMWK) in a sample comprises: (i) preparing a sample suspected of containing HMWK; (ii) contacting the sample with a protease to generate a plurality of digested peptides; and (iii) measuring the level of a signature peptide in the plurality of digested peptides, where the signature peptide is indicative of cleaved HMWK.SELECTED DRAWING: Figure 7

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of the filing date of U.S. Provisional Patent Application No. 62 / 267,734, filed December 15, 2015, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Kininogen is a precursor of kinins such as bradykinin and kallidin. Human kininogen exists as two splicing variants: high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK). HMWK is primarily a cofactor in coagulation and inflammation and is the preferred substrate for plasma kallikrein (pKal)-mediated bradykinin production. Both HMWK and LMWK are cysteine ​​protease inhibitors.

[0003] HMWK is a plasma protein in blood that is involved in the initiation of blood coagulation. It also produces the vasodilator bradykinin via the kallikrein-kinin system. HMWK localizes coagulation and bradykinin production by adhering to cell surface receptors on endothelium, monocytes, and platelets. The active peptide released from HMWK, bradykinin, exhibits a variety of physiological effects. It induces smooth muscle contraction, hypotension, diuresis, and blood glucose reduction, as well as being a mediator of inflammation and has cardioprotective effects both directly through the action of bradykinin and indirectly through the action of endothelium-derived relaxing factors. Bradykinin is an important mediator of pain, inflammation, edema, and angiogenesis.

[0004] Plasma kallikrein (pKal) is the main enzyme responsible for producing bradykinin in blood. Activation of pKal occurs via the contact pathway and is associated with the pathology of hereditary angioedema (HAE)-related diseases. pKal cleaves HMWK (a single-chain polypeptide) to produce bradykinin and a truncated form of HMWK, which contains two polypeptide chains (heavy and light chains) held together by disulfide bonds (Cugno et al., Blood (1997) 89:3213-3218). The initial light chain of the cleaved HMWK is approximately 56 kDa and can be further cleaved to form a shorter form of 46 kDa.

[0005] Cleaved HMWK has become a biomarker for monitoring hereditary angioedema (HAE) attacks because it increases to approximately 47% of total kininogen during HAE attacks (Cugno et al., 1997). Therefore, there is interest in developing a reliable and sensitive assay to detect the levels of cleaved HMWK in biological samples. Summary of the Invention

[0006] The present disclosure is based, at least in part, on the development of a sensitive and selective assay method, which may involve, for example, liquid chromatography-mass spectrometry (LC-MS) using multiple reaction monitoring (MRM), to distinguish between full-length and truncated HMWK. Such an assay method utilizes signature peptides representing cleaved HMWK (e.g., a C-terminal peptide from the heavy chain or an N-terminal peptide from the light chain) and / or full-length HMWK (e.g., compared to low molecular weight kininogen).

[0007] Thus, one aspect of the present disclosure provides a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample, the method comprising the steps of: (i) preparing a sample suspected of containing HMWK; (ii) contacting the sample with a protease to produce a plurality of digested peptides; and (iii) measuring the level of a signature peptide in the plurality of digested peptides, wherein the signature peptide represents cleaved HMWK.

[0008] In some embodiments, the signature peptide represents the 46 kDa light chain of cleaved HMWK and includes, but is not limited to, KHNLGHGH (SEQ ID NO: 1), KHNLGHGHKHE (SEQ ID NO: 2); KHNLGHGHK (SEQ ID NO: 3); or KHNLGHGHKHER (SEQ ID NO: 4).

[0009] In some embodiments, the signature peptide represents the 56 kDa light chain of cleaved HMWK, for example, SSRIGE (SEQ ID NO: 5).

[0010] The methods described herein may further include measuring the level of a signature peptide representing full-length HMWK, including, but not limited to, GHEKQRKH (SEQ ID NO: 6); KQRKHNLGHGHKHE (SEQ ID NO: 7); DWGHKQRKHNLGHGHKHER (SEQ ID NO: 8); HNLGHGHK (SEQ ID NO: 9); or SYYFDLTDGLS (SEQ ID NO: 10).

[0011] In another aspect, the disclosure features a method for distinguishing between full-length high molecular weight kininogen (HMWK) and cleaved HMWK in a sample. The method includes the steps of: (i) providing a sample suspected of containing full-length HMWK and / or cleaved HMWK; (ii) contacting the sample with a protease to produce a plurality of digested peptides; (iii) measuring a level of a first digested peptide (e.g., SSRIGE; SEQ ID NO:5) from step (ii), wherein the first digested peptide is unique to cleaved HMWK compared to full-length HMWK (e.g., a signature peptide for cleaved HMWK); and (iv) determining a level of a first digested peptide (e.g., SSRIGE; SEQ ID NO:5) from step (ii). (v) determining a ratio between the first digested peptide and the second digested peptide; and (vi) distinguishing cleaved HMWK from full-length HMWK in the sample based on the ratio determined in step (v).

[0012] In any of the assay methods described herein, the protease used to digest HMWK in the sample may be endoproteinase chymotrypsin, endoproteinase Glu-C, endoproteinase Asp-N, cathepsin G, or endoproteinase Lys-C. In some embodiments, any of the signature peptides can be measured by liquid chromatography-mass spectrometry (LC-MS), e.g., MRM-MS.

[0013] The sample analyzed by any of the assay methods described herein can be a biological sample (e.g., a blood sample, a plasma sample, or a serum sample) obtained from a human subject, such as a human patient with or suspected of having hereditary angioedema (HAE). In some examples, the biological sample can be a normal plasma sample, a plasma sample activated by FXIIa, or a non-activated plasma sample. When the biological sample is a plasma sample, the sample can be collected in an evacuated blood collection tube (e.g., a "SCAT tube" (sample collection anticoagulant tube)), which contains a liquid formulation including a mixture of protease inhibitors.

[0014] In some embodiments, the protease digestion step of any of the assay methods described herein may be performed in the presence of a reducing agent, such as DTT, BME, or TCEP. For example, the biological sample may be incubated with the reducing agent at 90°C for 1 hour. In some examples, the protease digestion step may be performed in the absence of a protease inhibitor, an anticoagulant (e.g., citrate), or both. In some examples, the protease / protein ratio is about 1:20, e.g., the GluC / protein ratio is 1:20.

[0015] Any of the present assay methods may be used for the diagnosis and / or prognosis of HAE. Accordingly, the present disclosure also provides methods for identifying a human subject who has HAE or is an HAE patient at risk for an HAE attack. In some embodiments, the level of cleaved HMWK (e.g., heavy chain-light chain dimer, heavy chain, or light chain, e.g., 46 kDa light chain or 56 kDa light chain) determined by the assay methods described herein can be an indicator for determining whether a human subject has HAE or is at risk for an HAE attack, where elevated levels of cleaved HMWK indicate HAE or the risk of an HAE attack. In other embodiments, the ratio between the cleaved HMWK signature peptide and the HMWK signature peptide determined by the assay methods described herein can be an indicator for determining whether a human subject has HAE or is at risk for an HAE attack, where elevated levels of the ratio indicate HAE or an HAE attack.

[0016] The details of one or more embodiments of the invention are set forth in the description that follows. Other features or advantages of the invention will be apparent from the following drawings and detailed description of some embodiments, and from the appended claims. [Brief explanation of the drawings]

[0017] [Figure 1]

[0023] Figure 1 shows exemplary peptides obtained from enzymatic digestion of truncated HMWK (e.g., 56 kDa light chain) found in plasma and a commercially available HMWK (HK) sample at optimal temperatures by MRM analysis. The figure shows SEQ ID NO: 5, SEQ ID NO: 19, SEQ ID NO: 24, and SEQ ID NO: 21, respectively, repeated from top to bottom. [Figure 2]FIG. 1 shows peptides produced by GluC digestion of HMWK standards obtained from Sigma (HK_Sigma) or Enzyme Research (HK_Enzyme Research) at 25° C. and 37° C. The figure represents, from top to bottom, SEQ ID NO:5, SEQ ID NO:19, SEQ ID NO:24, SEQ ID NO:30, SEQ ID NO:29, and SEQ ID NO:7, respectively. [Figure 3] 1 includes a chart showing the quantification of peptide SSRIGE (SEQ ID NO: 5) derived from protease digestion of the 56 kDa light chain in normal and HAE plasma samples using targeted assay MRM. [Figure 4] 1 includes a chart showing the quantification of HMWK long peptides in normal and HAE plasma samples using targeted assay MRM. [Figure 5] 1 is a chart showing the ratio of SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) in HMWK and plasma samples digested with GluC. [Figure 6] 1 is a chart showing the average ratio of SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) using the area under the curve of the peaks. [Figure 7] 1 is a chart showing the concentration of cleaved HMWK (represented by the 46 kDa light chain) to distinguish HAE patients from healthy individuals. DETAILED DESCRIPTION OF THE INVENTION

[0018] Plasma kallikrein (PKal) is a serine protease component of the contact system and the major bradykinin-producing enzyme in blood. The contact system is activated by factor XIIa (the active form of factor XII or FXII) when exposed to foreign or negatively charged surfaces, or by prolylcarboxypeptidase on the surface of endothelial cells (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation through the feedback activity of factor XII and proteolytically cleaves the kininogen precursor, high-molecular-weight kininogen (HMWK), releasing the pro-inflammatory nonapeptide bradykinin and the cleaved HMWK. The cleaved HMWK contains two polypeptide chains linked by disulfide bonds (also known as two-chain HMWK).

[0019] As the major kininogenase in blood, plasma kallikrein contributes significantly to the production of bradykinin in the vasculature. Genetic deficiency of C1 inhibitor protein (C1-INH) leads to hereditary angioedema (HAE). HAE patients suffer from acute attacks of painful edema, often precipitated by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). Through the use of drugs or genetic studies in animal models, the plasma kallikrein-kinin system (plasma KKS) has been implicated in a variety of diseases.

[0020] High molecular weight kininogen (HMWK) exists in plasma as a single polypeptide (1 chain), multidomain (domains 1-6) protein with a molecular weight of approximately 110 kDa. HMWK can be cleaved by pKal within domain 4 to release the nine-amino acid proinflammatory peptide bradykinin and a two-chain form of HMWK (cleaved kininogen). The two HMWK chains are a heavy chain containing domains 1-3 of HMWK and a light chain containing domains 5 and 6 of HMWK. The heavy chain has a molecular weight of approximately 65 kDa, while the light chain exists as two molecular weight species, approximately 56 kDa and approximately 46 kDa.

[0021] Levels of cleaved HMWK (e.g., two-chain HMWK) have been found to be elevated in HAE attacks and other pKal-related diseases. Thus, cleaved HMWK can serve as a biomarker for monitoring disease progression and / or treatment effectiveness. However, this technology lacks suitable agents and / or suitable assays that can effectively distinguish between cleaved versions of HMWK and intact HMWK.

[0022] The present disclosure is based, at least in part, on the discovery of signature peptides representing cleaved HMWK, e.g., the light chain of a two-chain HMWK (e.g., the 46 kDa light chain), and signature peptides representing HMWK (e.g., full-length HMWK). Unless otherwise specified, the term "cleaved HMWK" refers to the two-chain dimer, dimeric heavy chain, or dimeric light chain (including the 56 kDa light chain and the 46 kDa light chain) described herein. Based on the discovery of such signature peptides, a highly sensitive and selective assay method for measuring cleaved HMWK relative to HMWK has been developed. The assay methods described herein may be used in both clinical applications, e.g., the diagnosis or prognosis of HAE, and non-clinical applications, e.g., research and preclinical drug development.

[0023] I. Methods for measuring cleaved HMWK In some embodiments, methods are described herein for measuring cleaved HMWK in a sample, for example, for distinguishing cleaved HMWK from full-length HMWK in a sample. Such methods can include treating a suitable sample suspected of containing full-length HMWK, cleaved HMWK, or both with a suitable protease to produce a plurality of digested peptides, and measuring the level of one or more signature peptides representing cleaved HMWK and / or full-length HMWK. Either the level of cleaved HMWK or the ratio between cleaved HMWK and full-length HMWK can be used to represent the activity of pKal in the sample. This activity is correlated with the risk of HAE or HAE attack.

[0024] (i) Full-length and truncated HMWK The human gene encoding HMWK is kininogen 1 (KNG1). KNG1 is transcribed, or spliced, to form mRNA encoding HMWK or low molecular weight kininogen (LMWK). Exemplary protein sequences for human HMWK and LMWK are provided below (the bradykinin region is highlighted and shown in bold): [ka]

[0025] Exemplary sequences of the heavy and light chains of truncated kininogen are provided below. [ka] TIFF2025188193000004.tif33162

[0026] (ii) Sample preparation Any sample that may contain HMWK (e.g., full-length HMWK, truncated HMWK, or both) can be analyzed by the methods described herein. As used herein, "sample" refers to a composition that may contain the analyte of interest (in the present case, HMWK). A sample may include tissue from a subject, such as blood, plasma, or proteins. A sample can include an initial, unprocessed sample taken from a subject, as well as samples that have been subsequently processed, such as in a partially purified or preserved form via immunoprecipitation. Exemplary samples include blood, plasma, serum, tears, or mucus. In another example, a sample may be a composition for an in vitro assay.

[0027] In some embodiments, the sample is a sample of a bodily fluid, such as a serum or plasma sample. Such a sample may be a biological sample obtained from a subject in need of analysis. A "patient," "subject," or "host" (these terms are used interchangeably) treated by the subject method may refer to a human or a non-human animal. In some examples, the subject is a human patient who may have, be suspected of having, or be at risk for a disease associated with the contact system. For example, a human patient may have a history of HAE or be at risk for HAE. Such a human patient may have been previously treated or is undergoing treatment with an agent that targets a component of the contact system (e.g., pKal or FXIIa, or high molecular weight kininogen).

[0028] The biological sample may be a sample of a bodily fluid, such as a blood or plasma sample. Plasma samples for use in the methods described herein may be collected and processed in evacuated blood collection tubes (e.g., "SCAT" (sample collection anticoagulant tubes)), which are commonly used in medical practice for a variety of purposes to collect blood samples. The tubes described herein may be non-glass tubes containing a liquid formulation including a mixture of protease inhibitors (protease inhibitor cocktail).

[0029] In some embodiments, the protease inhibitor cocktail may include at least one serine proteinase inhibitor and at least one cysteine ​​proteinase inhibitor. The at least one serine proteinase inhibitor may be a plasma kallikrein inhibitor. The proteinase inhibitor cocktail may include multiple (e.g., two, three, four, or five) serine protease inhibitors, at least one of which may be an inhibitor of trypsin or human plasmin. Preferably, the proteinase inhibitor cocktails described herein are substantially free of protease inhibitors that are unstable in aqueous solution, i.e., the activity of unstable protease inhibitors in aqueous solution is negligible compared to the total inhibitory activity of the protease cocktail. In some examples, the amount of unstable protease inhibitors in aqueous solution may be less than 5% (w / w), e.g., less than 2%, less than 1%, or less than 0.5% of the total protease inhibitors in the cocktail. In some examples, the protease inhibitor cocktail is completely free of protease inhibitors that are unstable in aqueous solution (e.g., an aqueous solution at pH 4-6). An example of a protease inhibitor that is not stable in aqueous solution is PPACK II, also known as HD-Phe-Phe-Arg-chloromethylketone.

[0030] Exemplary serine protease inhibitors, cysteine ​​protease inhibitors, and trypsin protease inhibitors are listed in the table below, which can be used to make the protease inhibitor cocktails described herein. [Table 1] TIFF2025188193000006.tif215162TIFF2025188193000007.tif77162

[0031] In some examples, the protease inhibitor cocktail contained in the evacuated blood collection tube includes at least one serine proteinase inhibitor (e.g., one, two, or three), which may include at least one trypsin / plasmin inhibitor (e.g., one, two, or three), and at least one cysteine ​​protease inhibitor (e.g., one, two, or three). Such a protease inhibitor cocktail may include three serine proteinase inhibitors (e.g., benzamidine, AEBSF, and a trypsin / plasmin inhibitor, e.g., soybean trypsin inhibitor), and one cysteine ​​protease inhibitor (e.g., leupeptin).

[0032] In another example, the protease inhibitor cocktail can include at least one serine protease inhibitor (e.g., plasma kallikrein inhibitor) and at least one cysteine ​​protease inhibitor (e.g., leupeptin). The plasma kallikrein inhibitor can be EPI-KAL2 (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 Ser Tyr Gly Gly Cys Gly Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp; SEQ ID NO: 16), which is a specific recombinant protease inhibitor of plasma kallikrein that provides a tube with the ability to contain reagents that allow for the detection of activated plasma kallikrein, for example, using an immunoassay.

[0033] Any of the protease inhibitor cocktails may be dissolved in a suitable solution to form a liquid formulation. A suitable solution may be an acid-citrate-dextrose solution, which may contain trisodium citrate, citric acid, and glucose. The solution may have a pH value of about 4 to 6, e.g., 4.5. The liquid formulation may further contain a cationic polymer, such as a hexadimethrine bromide molecule (Polybrene®), which may inhibit activation of the contact system by interacting with negatively charged surfaces, and a chelating agent (e.g., EDTA) which may inhibit metalloproteases.

[0034] The concentration of each protease inhibitor in the cocktail may be 5-fold or 10-fold higher than the final concentration of that inhibitor used to inhibit the corresponding protease, depending on the dilution factor in practice. The final concentrations of specific commercially available protease inhibitors are known in the art and can be obtained from manufacturer protocols. In some examples, the concentration of EPI-KAL2 may be in the range of 5 to 15 μM (e.g., 5 to 10 μM or 10 to 15 μM), the concentration of leupeptin may be in the range of 200 to 300 μM (e.g., 200 to 250 μM, 240 to 270 μM, or 250 to 300 μM); the concentration of soybean trypsin inhibitor may be in the range of 1 to 3 mg / ml (e.g., 1 to 2 mg / ml or 2 to 3 mg / ml); the concentration of benzamidine may be in the range of 80 to 120 mM (e.g., 80 to 100 mM or 100 to 120 mM); and / or the concentration of AEBSF may be in the range of 10 to 30 mM (e.g., 10 to 20 mM or 20 to 30 mM).

[0035] When using peptide-based protease inhibitors (e.g., EPI-KAL2), they may be biotinylated according to conventional methods. For example, peptide inhibitors may be biotinylated as follows: Briefly, the peptide inhibitor can be dissolved in an appropriate solution, such as phosphate-buffered saline (PBS). Freshly prepared Sulfo-NHS-LC-Biotin can be added to the peptide inhibitor solution and incubated on ice for a suitable period of time. Excess, unreacted biotin and hydrolyzed biotin can be removed using a spin desalting column. Labeling of the peptide inhibitor can be confirmed by ELISA, and protein concentration can be determined by Bradford assay.

[0036] Any of the liquid formulations described herein can be prepared by conventional methods, such as dissolving the appropriate components in a suitable solution, and placed in a preferably non-glass evacuated blood collection tube, which may be stored at -20°C and thawed on ice or in a refrigerator within a suitable period of time before use.

[0037] In a particular example, the evacuated blood collection tubes used in the methods described herein are SCAT tubes, including SCAT169 and SCAT153, which are described in detail below. SCAT169: Evacuated plastic tube with a total volume of 5 mL containing 0.5 mL of the following: 100 mM benzamidine, 400 μg / mL Polybrene®, 2 mg / mL soybean trypsin inhibitor, 20 mM EDTA, 263 μM leupeptin, and 20 mM AEBSF (4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride) in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid, and 2% glucose, pH 4.5). SCAT153: 5 ml total volume evacuated plastic tube containing 0.5 ml of 10 μM biotinylated EPI-KAL2, 400 μg / mL Polybrene®, 20 mM EDTA, and 263 μM leupeptin dissolved in acid-citrate-dextrose (100 mM trisodium citrate, 67 mM citric acid, and 2% glucose, pH 4.5).

[0038] Within a suitable period of time after blood collection (e.g., not more than one hour), the blood sample may be processed to produce a plasma sample, which may be subjected to further analysis to assess characteristics associated with the contact system of the subject from whom the initial blood sample was obtained.

[0039] (iii) Protease digestion A biological sample described herein, e.g., a sample of whole plasma prepared according to the processes described herein, may be treated with an appropriate protease to generate multiple digested peptides. Alternatively, HMWK proteins, including both full-length and truncated forms, may be enriched from the sample, e.g., via immunoprecipitation, or denatured by methanol crash, prior to protease digestion.

[0040] Any suitable protease can be used in the methods described herein. Preferably, the protease cleaves proteins at specific motifs / residues so that digested peptides can be identified based on the amino acid sequence of the protein. In some examples, the protease used in the methods cleaves after glutamic acid residues. Exemplary proteases include, but are not limited to, endoproteinase Glu-C or cathepsin G. In other examples, proteases used in the assay methods described herein, such as endonuclease Asp-N, cleave before aspartic acid residues, or endonuclease Lys-C, cleave after lysine residues. In another example, proteases used in the assay methods described herein cleave after residues of amino acids bearing large hydrophobic side chains (e.g., tyrosine, tryptophan, or phenylalanine). One example of such a protease is chymotrypsin.

[0041] The protease cleavage reaction can be carried out under appropriate conditions to allow complete digestion of full-length and cleaved HMWK proteins in the sample. In some embodiments, the protease cleavage reaction mixture can include a reducing agent, such as dithiothreitol (DTT), b-mercaptoethanol (BME), or tris(2-carboxyethyl)phosphine (TCEP), at an appropriate concentration (e.g., 1 mM, 0.5 mM, 0.1 mM, or 0.05 mM). For example, if DTT is used, the concentration can be 0.1 mM or 0.05 mM. The protease cleavage reaction can be carried out for an appropriate period of time (e.g., 30 minutes, 60 minutes, 90 minutes, or 180 minutes) at an appropriate temperature (e.g., 25°C, 37°C, 50°C, 55°C, or 90°C).

[0042] In some examples, the mixture containing the reducing agent can be incubated at an elevated temperature (e.g., 55°C for 30 or 60 minutes, or 90°C for 30 or 60 minutes) for a suitable period of time to allow for complete denaturation of proteins in the sample. A protease can then be added to the mixture, and the digestion reaction can be carried out at an appropriate temperature for a suitable period of time. The exact temperature / time for digestion will depend on the particular protease used in the method and is within the knowledge of one of ordinary skill in the art. In one example, the enzyme GluC is used, and the digestion reaction can be carried out overnight (e.g., 12, 14, 17, or 19 hours) at 25°C or 37°C. In another example, chymotrypsin is used, and the digestion reaction can be carried out at 40-60°C (e.g., 50°C) for a suitable period of time, e.g., 2-5 hours (e.g., 3 hours).

[0043] The reaction mixture may be free of protease inhibitors, anticoagulants such as citrate, or both. The enzyme / protein ratio in the reaction may range from 1:5 to 1:30, e.g., 1:5, 1:10, 1:20, 1:25, or 1:30. The selection of specific reaction conditions, including temperature, reaction duration, enzyme / protein ratio, presence or absence of protease inhibitors, presence or absence of anticoagulants, and presence or absence of reducing agents, depends on the type of protease used and the sample being treated and can be determined by methods known in the art or described herein.

[0044] (iv) Measurement of signature peptides Signature peptides represent digested peptides produced from the protease reaction and are unique to one type of kininogen compared to another type of kininogen. Such peptides can be identified based on the specificity of the protease used in the methods described herein and the amino acid sequences of the different types of kininogens. See, e.g., the disclosures herein. Peptides (signature peptides) unique to a first type of kininogen (e.g., full-length kininogen) compared to a second type of kininogen (e.g., truncated kininogen) represent peptides that can only be produced by cleaving the first type of kininogen with a protease, but cannot be produced by cleaving the second type of kininogen with the same protease. For example, a signature peptide of a cleaved HMWK (e.g., a cleaved HMWK heavy chain or a light chain such as the 46 kDa light chain) represents a peptide that can only be produced by protease digestion of a cleaved HMWK (e.g., a cleaved HMWK heavy chain or light chain) and cannot be produced by digestion of full-length HMWK with the same protease. Also, a signature peptide of a full-length HMWK is a peptide that can only be produced by protease digestion of a single-chain HMWK, but cannot be produced by digestion of a cleaved HMWK using the same protease. A signature peptide of an HMWK represents a peptide that is produced by protease digestion of HMWK (single-chain HMWK, double-chain HMWK, or both) but cannot be produced by digestion of LMWK using the same protease.

[0045] Exemplary signature peptides for cleaved HMWK include (a) a signature peptide for the 56 kDa light chain, such as SSRIGE (SEQ ID NO: 5), which can be produced by Glu-C digestion, and (b) a signature peptide for the 46 kDa light chain, such as KHNLGHGH (SEQ ID NO: 1), KHNLGHGHKHE (SEQ ID NO: 2); KHNLGHGHKHER (SEQ ID NO: 4), or KHNLGHGHK (SEQ ID NO: 3), which can be produced by chymotrypsin digestion, Glu-C digestion, Asp-N digestion, and Lys-C digestion, respectively.

[0046] Table 1 below lists exemplary signature peptides for the cleaved HMWK 46 kDa light chain produced by different proteases. [Table 2]

[0047] Examples of signature peptides for HMWK (e.g., full-length HMWK) include, but are not limited to, GHEKQRKH (SEQ ID NO: 6), which can be produced by chymotrypsin digestion; KQRKHNLGHGHKHE (SEQ ID NO: 7) and SYYFDLTDGLS (SEQ ID NO: 10), which can be produced by Glu-C digestion; DWGHKQRKHNLGHGHKHER (SEQ ID NO: 8), which can be produced by Asp-N digestion, and HNLGHGHK (SEQ ID NO: 9), which can be produced by Lys-C digestion.

[0048] The level of the digested peptide of interest can be measured using suitable techniques known in the art or described herein. In some embodiments, the peptide of interest can be measured by immunoassay using an antibody specific for the peptide of interest, for example, an antibody specific for KHNLGHGH (SEQ ID NO: 1), SSRIGE (SEQ ID NO: 5), and / or SYYFDLTDGLS (SEQ ID NO: 10). Immunoassays that can be used to assess the level of the peptide of interest described herein include Western blots, enzyme-linked immunosorbent assays (ELISAs) (e.g., sandwich ELISAs), radioimmunoassays, electrochemiluminescence-based detection assays, and related techniques. Assays, such as Western blot assays, may further include the use of a quantitative imaging system, such as commercially available LICOR imaging technology (see, for example, the Odyssey® CLx Infrared Imaging System from LI-COR Biosciences). In some embodiments, electrochemiluminescence detection assays or assays that rely on a combination of electrochemiluminescence and patterned array technologies are used (e.g., ECL or MULTI-ARRAY technology assays from Mesoscale Discovery (MSD)).

[0049] As used herein, the terms "determining" or "measuring" or alternatively "detecting" or "detection" mean assessing the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample, including deriving a qualitative or quantitative concentration level of such substance, or assessing a value or classification of interest.

[0050] The phrase "antibody that specifically binds" to a peptide of interest is well understood in the art, and methods for determining such specific binding are also well known in the art. An antibody is said to exhibit "specific binding" if it reacts or binds to a particular target peptide with a higher frequency, speed, duration, and / or affinity compared to its reaction or binding to another peptide. It is also understood by reading this definition that, for example, an antibody that specifically binds to a first target peptide may or may not specifically or preferentially bind to a second target peptide. Similarly, "specific binding" or "preferential binding" does not necessarily require (although it may include) exclusive binding. Generally, but not necessarily, reference to binding implies preferential binding. In some instances, an antibody that "specifically binds" to a target peptide or its epitope may not bind to other peptides or other epitopes on the same antigen.

[0051] As used herein, the term "antibody" refers to a protein that includes at least one immunoglobulin variable domain or sequence of an immunoglobulin variable domain. For example, an antibody may include a heavy chain (H) variable region (referred to herein as V H ) and a light chain (L) variable region (abbreviated herein as V L In another example, an antibody can comprise two heavy chain (H) variable regions and two light chain (L) variable regions. The term "antibody" includes antigen-binding fragments of antibodies (e.g., single-chain antibodies, Fab and sFab fragments, F(ab')2, Fd fragments, Fv fragments, scFv, and domain antibody (dAb) fragments (de Wildt et al., Eur J Immunol. 1996; 26(3):629-39.)), as well as complete antibodies. Antibodies can have structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes). Antibodies can be from any source, although primate (human and non-human primate) and primatized antibodies are preferred.

[0052] In some embodiments, the antibodies described herein can be conjugated to a detectable label, and binding of the detection reagent to the peptide of interest can be determined based on the intensity of the signal emitted from the detectable label. Alternatively, a secondary antibody specific to the detection reagent can be used. One or more antibodies may be conjugated to a detectable label. Any suitable label known in the art can be used in the assay methods described herein. In some embodiments, the detectable label comprises a fluorophore. As used herein, the term "fluorophore" (also referred to as "fluorescent label" or "fluorescent dye") refers to a moiety that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength. In some embodiments, the detection moiety is or comprises an enzyme. In some embodiments, the enzyme is an enzyme (e.g., β-galactosidase) that produces a colored product from a colorless substrate.

[0053] In other embodiments, the peptides of interest described herein may be measured by liquid chromatography-mass spectrometry (LC-MS), an analytical technique that combines the mass analysis power of mass spectrometry (MS) with the physical separation power of liquid chromatography.

[0054] Multiple reaction monitoring (MRM)-mass spectrometry is a highly selective and sensitive method for targeted quantification of protein / peptide abundance in complex biological samples. MRM mass spectrometry is commonly used for the analysis of small molecules. Here, MRM enables the quantification of proteins in complex mixtures, providing a sensitive and selective tool for validating candidate biomarkers in disease processes. This technique can be used with appropriate instrumentation, such as an ABSciex 5500 or 6500 Qtrap mass spectrometer, and differences between various types of plasma samples were evaluated after method optimization. To obtain the cleaved HWMK or signature peptides for HWMK described herein, individual plasma samples were digested with appropriate proteases, such as chymotrypsin or Glu-C.

[0055] The level (e.g., concentration) of each signature peptide (e.g., represented by AUC) can be determined by conventional methods. Based on this, if necessary, the ratio of the signature peptide for HMWK compared to LMWK, and the ratio of the signature peptide for cleaved HMWK compared to full-length HMWK can be calculated. Such ratios can be used to distinguish between the presence of cleaved HMWK and full-length HMWK.

[0056] For example, the concentration of the signature peptide KHNLGHGH (SEQ ID NO: 1) for the 46 kDa light chain of two-chain HMWK, generated by chymotrypsin digestion, was found to be significantly higher in plasma samples from HAE patients compared to plasma samples from healthy human subjects. These results indicate that the 46 kDa light chain of two-chain HMWK, represented by this signature peptide, is a reliable biomarker for the diagnosis and prognosis of HAE.

[0057] In another example, this study found that the targeting peptides found in plasma were due to HMWK by confirming all peptides containing the HMWK vs. LMWK signature peptide, SYYFDLTDGLS (SEQ ID NO: 10), by MRM in HMWK standards and samples digested with GluC. A significant increase in the SSRIGE (SEQ ID NO: 5) peptide was observed in HAE SCAT plasma compared with normal SCAT plasma, and also between FXIIa-activated vs. non-activated plasma, with no change in HMWK peptides. The ratio of SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) was also found to be higher in HAE SCAT plasma samples compared with normal SCAT plasma samples, and similar results were found in activated vs. non-activated plasma. The relative abundance ratios using ratios for SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10) were 4.4 and 8.9 in normal versus HAE SCAT plasma, respectively, and 19.02 and 48.63 in normal citrate plasma and FXIIa-activated citrate plasma, respectively.

[0058] Kininogen-deficient plasma (negative control) samples showed low peak intensities near the detection limit for the SSRIGE (SEQ ID NO: 5) peptide, potentially predicted by the presence of LMWK. The HMWK long peptide, KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 18), in plasma samples was also measured using a targeted assay (MRM). There was little change in the HMWK long peptide between normal and HAE SCAT plasma samples, and between activated and non-activated samples.

[0059] II. Kit The present disclosure also provides kits for use in measuring levels of a signature peptide associated with cleaved HMWK and / or for distinguishing cleaved HMWK from full-length HMWK in a sample, e.g., a biological sample from a human patient. The kits can include a suitable protease (e.g., chymotrypsin or GluC), a detection agent specific for a signature peptide that can be produced by digestion with the appropriate protease, vacuum collection tubes, and, optionally, one or more of a standard cleaved kininogen and / or intact kininogen as a control. In some embodiments, the kits further include a secondary antibody and / or reagent for detecting binding of the detection agent to the peptide of interest.

[0060] In some embodiments, the kit can include instructions for use in accordance with any of the methods described herein. The included instructions can include instructions on how to use the components included in the kit to measure the level of a signature peptide in a sample treated with a protease.

[0061] The instructions associated with the use of the kits will generally include information regarding the amounts of each component and the appropriate conditions for carrying out the assay methods described herein. The instructions provided with the kits of the invention will generally be instructions written on a label or package insert (e.g., a paper sheet included in the kit), although machine-readable instructions (e.g., instructions carried on a magnetic or optical disk storage device) are also acceptable.

[0062] The label or package insert indicates that the kit is used to measure levels of a signature peptide for cleaved HMWK and / or to distinguish cleaved from full-length HMWK. Instructions may be provided for performing any of the methods described herein.

[0063] The kits of the present invention are suitably packaged, including, but not limited to, vials, bottles, jars, flexible packaging (e.g., sealed in Mylar or plastic bags), and the like.

[0064] The kit may optionally provide additional components such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert(s) on or associated with the container. In some embodiments, the disclosure provides an article of manufacture including the contents of the above-described kit.

[0065] III. Application of the assay method (i) Clinical applications: diagnosis and prognosis of disease Approximately 75-90% of circulating prekallikrein binds to HMWK via a non-active site interaction with domain 6 of HMWK. Active pKal, free and bound to HMWK, generates cleaved HMWK and bradykinin. The suitability of these biomarkers can be demonstrated by monitoring their levels in the presence and absence of acute attacks of HAE. Furthermore, levels of these biomarkers may change during attacks of bradykinin-mediated edema or other diseases mediated by pKal activity.

[0066] The assay methods and kits described herein can be used to evaluate a disease, for example, to diagnose or prognose a disease. The evaluation can include identifying a subject as being at risk for or having a disease described herein, such as a pKal-mediated disease, such as HAE. The evaluation can also include monitoring treatment of a disease, such as evaluating the effectiveness of treatment of a PKal-mediated disorder, such as HAE. The evaluation can also include identifying a disease that can be treated with a pKal inhibitor.

[0067] A. Diagnosis In some embodiments, the assay method and kit are used to determine the levels of cleaved kininogen and / or intact kininogen in a biological sample (e.g., a blood or plasma sample) collected from a candidate subject (e.g., a human patient suspected of having a PKal-mediated disorder such as HAE). The level of cleaved HMWK (e.g., two-chain HMWK, or its heavy chain and / or light chain including a 56 kDa light chain and a 46 kDa light chain) and / or the ratio between the level of cleaved HMWK and the level of intact HMWK can be determined based on the level of a signature peptide for cleaved HMWK described herein and / or the ratio between a signature peptide for cleaved HMWK described herein and a signature peptide for HMWK (e.g., compared with LMWK). The concentration or ratio of such signature peptides can be compared with a predetermined reference value or reference ratio to determine whether the subject has or is at risk for a PKal-mediated disorder, such as HAE. For example, if the concentration of a signature peptide or the ratio of two signature peptides in a sample of a candidate subject is equal to or greater than a reference value / ratio, the subject can be identified as having or at risk of having a pKal-mediated disorder, such as HAE.

[0068] The reference value / ratio can be a control value of a signature peptide or a ratio of two signature peptides described herein. In some embodiments, the control value / ratio represents the value / ratio of the signature peptide(s) in a control sample, such as a sample (e.g., a blood or plasma sample) obtained from a healthy subject or a population of healthy subjects, preferably of the same species as the candidate subject. As used herein, a healthy subject is a subject who does not have or has no history of the target disease (e.g., a PKal-mediated disorder such as HAE) at the time the levels of cleaved kininogen and / or intact kininogen are measured.

[0069] In some embodiments, the control sample can be obtained from a human HAE patient in the quiescent stage of the disease. An increase in the level of the cleaved HMWK signature peptide or an increase in the ratio between the cleaved HMWK signature peptide and the HMWK signature peptide compared to the reference value / ratio obtained from such a control sample can indicate the risk of an HAE attack.

[0070] The reference value / ratio can also be a predetermined value or ratio. Such a predetermined value / ratio can represent the value of a signature peptide for cleaved HMWK (e.g., the signature peptide for the 46 kDa light chain of two-chain HMWK) described herein or the ratio of two signature peptides in a group of subjects who do not have or are not at risk of the target disease. The predetermined value / ratio can also represent the value (e.g., concentration) of a signature peptide for cleaved HMWK (e.g., the 46 kDa light chain) described herein or the ratio of two signature peptides in a group of subjects who have the target disease (e.g., are in the quiescent stage of the disease).

[0071] The defined value / ratio can take a variety of forms. For example, it can be a single cutoff value, such as a median or mean. In some embodiments, such a defined level can be established based on comparison groups, such as one defined group known to have the target disease and another defined group known not to have the target disease. Alternatively, the defined level can be a range, for example, a range representing the ratio of two peptides of interest in a control population within a defined percentile.

[0072] The control value / ratio described herein can be determined by conventional techniques. In some examples, the control value / ratio can be obtained by performing a conventional method (e.g., the same assay as described herein for obtaining the levels of the two peptides of interest in a test sample) on a control sample as described herein. In other examples, the level of the signature peptide of interest can be obtained from a member of a control set, and the results can be analyzed, for example, by a computer program, to obtain a control level (predetermined level) that represents the level of cleaved kininogen and / or intact kininogen in the control set.

[0073] By comparing the concentration of the cleaved HMWK signature peptide described herein or the ratio of two target signature peptides similarly described herein in a sample obtained from a candidate subject with the reference ratio 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 whether an HAE patient is at risk for an HAE attack. For example, if the value of the cleaved HMWK signature peptide or the ratio of the target's two signature peptides in the candidate subject's sample deviates from the reference value or ratio (e.g., is increased compared to the reference value or ratio), the candidate subject may be identified as having or at risk for the disease, or as an HAE patient at risk for an HAE attack. If the reference value or ratio represents a range of values ​​or ratios of the target signature peptides described herein in a population of subjects with a target disease, a value or ratio of the target signature peptide in the candidate sample within this range indicates that the candidate subject has or is at risk for the target disease.

[0074] As used herein, "high value / ratio, or value / ratio exceeding a reference value / ratio" means that the value of a signature peptide or the ratio of two signature peptides is greater than the reference value or ratio, e.g., a defined threshold ratio, of the same signature peptide or the same two signature peptides in a control sample. Control levels are described in detail herein. A high value of a subject signature peptide or a high ratio of two signature peptides includes, for example, a value / ratio that is 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more greater than the reference value / ratio.

[0075] As used herein, a "low value / ratio below a reference value / ratio" means that the value of a subject's signature peptide or the ratio of two signature peptides is less than the reference value / ratio, e.g., a defined threshold, of the same subject's signature peptide or the same two signature peptides in a control sample. Control levels are described in detail herein. A low value of a signature peptide or a low ratio of two signature peptides includes, for example, a value / ratio that is 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more less than the reference ratio of the two peptides of the subject.

[0076] In some embodiments, the candidate subject is a human patient with a pKal-mediated disorder, such as HAE. For example, the subject has edema, swelling (where the swelling is entirely peripheral or mainly 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 a pKal-mediated disorder at the time of sample collection, does not have a history of symptoms of a pKal-mediated disorder, or does not have a history of a pKal-mediated disorder such as HAE. In yet other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both.

[0077] B. Evaluation of Treatment Efficacy The assay methods described herein can also be used to evaluate the effectiveness of treatment for PKal-mediated disorders (e.g., HAE). For example, multiple biological samples (e.g., blood or plasma samples) can be collected from a subject undergoing treatment before, after, or during the course of treatment. The level of a signature peptide can be measured by any of the assay methods described herein, thereby determining the level of a signature peptide related to cleaved HMWK (e.g., the 46 kDa light chain) or the ratio of a peptide specific to cleaved HMWK to a peptide specific to HMWK. A decrease in the level of a signature peptide related to cleaved HMWK (e.g., the 46 kDa light chain) or the ratio of two signature peptides after or over the course of treatment (a decrease in the level of a signature peptide related to cleaved HMWK (e.g., the 46 kDa light chain) or the ratio of two signature peptides in a sample collected later compared to the level in a sample collected earlier) indicates that the treatment is effective. In some examples, the treatment includes a therapeutic agent such as a kallikrein binding agent described herein, a bradykinin B2 receptor antagonist described herein, or a C1-INH replacement agent described herein. Examples of therapeutic agents include, but are not limited to, DX-2930, SHP643, or DX88.

[0078] If the subject is identified as not responding to treatment, the therapeutic agent is administered to the identified subject at a higher dose and / or administration frequency.In some embodiments, the dose or administration frequency of the therapeutic agent for the subject who is identified as being responsive to treatment or not needing further treatment is maintained, reduced or terminated.Alternatively, different treatment can be used for the subject who is found not responding to the first treatment.

[0079] (ii) Non-clinical applications Additionally, the assay methods described herein have non-clinical utility, e.g., for research purposes and / or preclinical drug development processes. While many diseases associated with pKal have been identified, other diseases may be mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein may be used to identify diseases associated with pKal. In some embodiments, the methods described herein may be used to study mechanisms (e.g., discovering novel biological pathways or processes involved in disease development) or disease progression.

[0080] In some embodiments, the levels or ratios of signature peptides determined by the assay methods described herein may be dependent on the development of novel treatments for diseases associated with pKal. For example, the levels or ratios of signature peptides described herein may be measured in samples from subjects undergoing a novel treatment (e.g., in a clinical trial) or in samples from in vitro assays. In some embodiments, the levels or ratios of signature peptides may represent the activity of the novel treatment in an in vitro assay, or may represent the efficacy of the novel treatment in the context of a clinical trial.

[0081] Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way whatsoever. All publications cited herein are incorporated by reference for any purpose or subject matter referenced therein.

[0082] Example Example 1: Method for distinguishing between truncated and full-length high molecular weight kininogen (HMWK) The overall objective of this example was to develop a robust LC-MS-based peptide quantification assay to distinguish between full-length kininogen (HK; single-chain or one-chain HK) and kallikrein-cleaved kininogen (the two-chain product or two-chain HK, and the heavy and / or light chains of the two-chain product). In some cases, this semi-quantitative assay measured the ratio of two-chain HK product to one-chain HK substrate to determine a cut point for two-chain HK in biological samples (e.g., plasma) from healthy volunteers compared with patients with plasma kallikrein-associated diseases, such as HAE.

[0083] Single-chain HMWK (1-chain HK) and double-chain HMWK (2HK) (Enzyme Research Laboratories and Sigma) were used as standards in the following examples. Different types of plasma samples (citrate plasma, citrate plasma + protease inhibitors (PIs), normal SCAT plasma (plasma samples from healthy subjects collected in SCAT tubes), and HAE SCAT plasma (plasma samples from HAE patients collected in SCAT tubes)) were used in the following examples. See, for example, International Patent Application No. PCT / US2016 / 046681, the relevant disclosures of which are incorporated herein by reference. Quality control was performed during experiments to address variations between different dates and experiments and to confirm reproducibility. For method optimization, EDTA-Plasma (Biochem Services) was used as a control.

[0084] The assay described and developed in this example provides a highly sensitive and selective method for validating candidate biomarkers in disease processes. In this approach, differences between various types of plasma samples were evaluated after method optimization using an ABSciex 5500 or 6500 Qtrap mass spectrometer. Individual plasma samples (including normal SCAT plasma and HAE SCAT plasma, kininogen-deficient plasma, normal citrated plasma, normal citrated plasma activated with factor XIIa, and HK standards) were digested with GluC, and multiple reaction monitoring (MRM) analysis was performed for peptides of interest. GluC was used as an exemplary protease to obtain unique peptides (signature peptides) that distinguish two-chain HK from one-chain HK. Briefly, initial experiments were designed to validate the properties of protein standards using MRM mode for specific peptides. 10 μl of sample containing 10 μg of protein was loaded onto a C18 column. Precursor and protease digestion product ions predicted by Skyline software to be unique to plasma kininogen digested with GluC were selected. Target candidate peptides, SSRIGE (SEQ ID NO: 5), SSRIGEIKE mis2 (SEQ ID NO: 19), KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 20), KKIYPTVNCQPLGMISLMK (SEQ ID NO: 21), and SYYFDLTDGLS (SEQ ID NO: 10), as well as three to five product ions, were experimentally validated for plasma kininogen. Fragmentation and collision energies were individually optimized for each peptide after using Skyline as a guide for MRM analysis. Data were analyzed using Analyst Software (ABSciex). Method development focused on optimizing sample preparation, enzymatic digestion, chromatography, and mass spectrometry parameters for different types of plasma samples and HK standards.

[0085] (i) Immunoprecipitation samples vs. whole plasma samples To validate the concept, four protein samples obtained from immunoprecipitation (IP samples) and four whole plasma samples (non-IP samples) were used in the study. The samples were eluted with SDS and then depleted using a detergent removal column. To test the peptides of interest in the samples, a targeted assay was performed on an AB Sciex 5500 Qtrap triple quad mass spectrometer.

[0086] All four peptides of interest, SSRIGE (SEQ ID NO: 5), SSRIGEIKE (SEQ ID NO: 19), KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 18), and KKIYPTVNCQPLGMISLMK (SEQ ID NO: 21), were found in non-IP samples but not detected in IP plasma samples. No differences in peaks were observed between activated (FXIIa-treated) and non-activated plasma samples. Table 2 below shows the peak intensities of peptides in IP and non-IP samples. There was no difference in peak intensity for SSRRIGE (a peptide unique to the light chain of 2HK produced by GluC digestion; SEQ ID NO: 22) or KKIYPTVNC QPLGMISLMKRPPGFSPFRSSRIGE (a peptide unique to the long peptide of HK produced by GluC digestion; SEQ ID NO: 18) in activated or non-activated samples (boxed columns).

[0087] EDTA plasma was run as a positive control with IP / non-IP samples, and all peptides of interest were found in both types of samples. [Table 3]

[0088] The results indicated that whole plasma samples digested with Glu C provided sufficient sensitivity to detect both single-chain and double-chain HK peptides. Therefore, whole plasma samples digested with an exemplary Glu C protease were used in the experiments described below.

[0089] (ii) Optimization of sample preparation (a) Digestion protocol To optimize GluC digestion of plasma, various protocols were used to optimize GluC digestion. Different parameters of the protocols were modified as summarized below. Protocol 1: Addition of a denaturation step before digestion (e.g., in the presence of DTT at 0.1 M) Protocol 2: Use of different concentrations of reducing agent (0.05 M DTT) Protocol 3: 10-fold dilution of the pre-digested sample; and Protocol 4: Increasing the enzyme incubation time to 19 hours Protocol 2 resulted in good detection of SSRIGE (SEQ ID NO: 5) and KKIYPTVNCQPLGMISLMK (SEQ ID NO: 21) based on peak intensity and peak shape, and therefore the digestion method of Protocol 2 was used in subsequent experiments.

[0090] (b) Reduction temperature and incubation time Similarly, the reduction temperature and incubation time were optimized. Briefly, HK standards and EDTA-plasma were used to evaluate the temperature (55°C vs. 90°C) and incubation time (30 min vs. 60 min) for sample reduction during digestion. HK: 1 hour at 90℃ HK: 55℃ for 30 minutes Plasma EDTA (Biochem): 90℃ for 1 hour Plasma EDTA (Biochem): 55°C for 30 minutes

[0091] These samples were analyzed using multiple reaction monitoring (MRM) analysis to detect the peptides SSRIGE (SEQ ID NO: 5) and KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 20). Higher peak intensities were found in samples (both HK standard samples and plasma samples) reduced at 90°C for 1 hour compared to samples reduced at 55°C for 30 minutes. Conditions including a 1 hour incubation at 90°C were used to reduce samples in subsequent experiments.

[0092] (c) Plasma protease inhibitors (PIs) and anticoagulants The effects of the presence of PI and anticoagulant in plasma on digestion efficiency were also evaluated. Digestion efficiency was evaluated for plasma containing PI and plasma without PI, and also between plasma containing citrate as an anticoagulant and plasma containing EDTA. Samples were evaluated by MRM analysis using an AB Sciex 5500 Qtrap LC-MS / MS System, and peak intensities were compared for SSRIGE (SEQ ID NO: 5), as indicated by the arrows in Figure 3. These results indicated that plasma EDTA without PI > plasma EDTA with PI > citrate plasma without PI > citrate plasma with PI.

[0093] These results also indicated that citrate and protease inhibitors had a negative effect on GluC digestion, as a decrease in peptide response was observed in samples containing citrate, PI, or both.

[0094] (iii) Digestion with different enzymes to confirm sequence coverage To test whether sequence coverage is enzyme-dependent, we used different enzymes, trypsin. Sigma HK standard samples and Biochem EDTA-plasma samples were digested with trypsin, run on a high-resolution accurate mass (HRAM) instrument, and analyzed using Proteome Discoverer. The results were analyzed using Proteome Discoverer™ software.

[0095] Full-length kininogen was detected with good sequence coverage in both trypsin-digested HK and plasma samples.

[0096] (iv) SIM (Single Ion Monitoring) of EDTA-Plasma and HK samples Highly concentrated HK and plasma samples were used for SIM, and peptides of interest were detected using high-resolution accurate mass (HRAM) instrumentation. 2 Data analysis was performed on EDTA plasma and HK (Sigma) samples.

[0097] Analysis of GluC-digested EDTA plasma samples on the HRAM instrument detected all four peptides, SSRIGE (SEQ ID NO: 5), KKIYPTVNCQPLGMISLMK (SEQ ID NO: 21), the HK long peptide, and bradykinin. For the HK standard sample, the peptides SSRIGE (SEQ ID NO: 5), KKIYPTVNCQPLGMISLMK (SEQ ID NO: 21), and bradykinin were found, but the HK long peptide was not.

[0098] To confirm the identity of the standards, two different sources of HK (Enzyme research and Sigma) were used. However, in this experiment, no detectable differences were observed between the two standards, and the long peptides of HK were not detected in any of the standard samples.

[0099] (v) Different types of plasma, different Glu C, and different enzyme:protein ratios To further optimize GluC digestion, different types of plasma samples (EDTA, citrate plasma with PI, and EDTA, citrate plasma without PI), different sources of GluC (Protea and Promega), and different enzyme:protein ratios (1:20 and 1:10) were evaluated. Additionally, full scan, SIM-MS, and ELISA were performed. 2Various detection platforms were used to detect the peptides of interest in different samples, including (on HRAM), MRM (QQQ; on a triple Quad instrument). Table 3 provides an overview of the various combinations of sample type, enzyme, and dilution used in this experiment. [Table 4]

[0100] (a) Full scan and SIM-MS 2 (HRAM) Samples of the different types of plasma and standards mentioned above were digested with GluC, and the digestion products were analyzed by full scan and SIM-MS. 2 The samples were run on an HRAM instrument for analysis. Sample information is shown on the right hand side of the legend color map. Analysis was performed using Proteome Discoverer™ and Xcalibur™ software to determine the peak intensity for each peptide of interest.

[0101] In plasma samples, the following peptide intensity responses were observed for SSRIGE (SEQ ID NO: 5): Plasma EDTA without PI > Plasma EDTA with PI > Citrated plasma without PI > Citrated plasma with PI.

[0102] It was determined that a 1:20 (enzyme:protein) ratio provided a better response than a 1:10 (enzyme:protein) ratio. For the HK long peptide, intensity was only minimally affected by plasma type or enzyme:protein ratio. The HK long peptide was not observed in any of the standard samples; however, the KKYIPTVNCQPLGMISLMK peptide (SEQ ID NO:23) and bradykinin were detected in all standards, indicating that the HK long peptide may be degraded. Without being bound by theory, this may explain the observation of various fragments of the HK long peptide, including KKIYPTVNCQPLGMISLMK (SEQ ID NO:21), RPPGFSPFR (SEQ ID NO:24), and SSRIGE (SEQ ID NO:5).

[0103] Table 4 below shows the results of full scan and SIM-MS on the HRAM instrument. 2 The intensities of peptides of interest are shown (sorted by sample type) using the data from [Table 5] TIFF2025188193000012.tif157162

[0104] (b) Multiple Reaction Monitoring (MRM) Targeted Assay Using the same set of samples described above, peptides of interest were detected using a targeted assay (MRM). These samples were detected using an AB Sciex Qtrap 5500 instrument and then analyzed using AB Sciex Analyst software. Sample information is presented on the right hand side of the color map.

[0105] Detection of the SSRIGE peptide (SEQ ID NO: 5) was consistently observed regardless of sample type and conditions. Using the optimized sample preparation, the SSRIGEKE (SEQ ID NO: 25) peptide was not detected. However, the HK long peptide was present only in plasma samples, but not in the HK standard samples. The KKYIPTVNCQPLGMISLMK (SEQ ID NO: 23) peptide and bradykinin peptide were present in all HK standard samples, but the HK long peptide was not detected. These results were consistent with the full scan and MS analyses described above. 2 This is similar to the data set from

[14] , suggesting that the long peptides of HK may be degraded, further explaining the observation of fragments of the long peptides of HK mentioned above.

[0106] Table 5 below shows the intensities of peptides of interest (sorted by sample listing) using MRM data on an AB Sciex Qtrap 5500 instrument. [Table 6] TIFF2025188193000014.tif192162

[0107] (c) Sequence coverage using HRAM and Proteome Discoverer™ software To determine the optimal GluC concentration for protein digestion, sequence coverage of HK standard samples from different sources (Protea and Promega) and different enzyme:protein concentrations (1:20 and 1:10) was assessed by full scan on the HRAM instrument after digestion with GluC. The 1:20 dilution of GluC from either source was optimal for digestion because it provided more sequence coverage in the region of interest. As provided below, the 1:10 dilution also provided coverage in the region of interest, but this peptide had a lower confidence score. [ka]

[0108] In the amino acid sequence of single-chain HMWK shown above, the bolded Glu-C-derived peptides were identified by a single ion, whereas the underlined peptides were identified by multiple ions. Bradykinin is shown in italics and bold.

[0109] (vi) Alignment of high molecular weight kininogen (HMWK) and low molecular weight kininogen (LMWK) sequences To identify peptides unique to HMWK compared to LMWK, an alignment of the sequences of these two kininogens was performed using UniProt software and is provided below (hHMWK: SEQ ID NO: 11; hLMWK: SEQ ID NO: 12). [ka] TIFF2025188193000017.tif103162

[0110] We found four peptides that were unique to HMWK compared with LMWK. 1. SYYFDLTDGLS (SEQ ID NO: 10) 2. INPTTQMKE (SEQ ID NO: 26) 3. KQRKHNLGHGHKHE (SEQ ID NO: 7) 4. EDSTTPSAQTQE (SEQ ID NO: 27)

[0111] a. Full scan and MS on HRAM 2 Detection of HMWK-specific peptides in HK samples using Starting with the unique peptide SYYFDLTDGLS (SEQ ID NO: 10), full scan and MS 2 was performed using a standard of HK digested with GluC. Full scan and MS 2was performed on an HRAM instrument and analyzed using Proteome Discoverer™ software. As shown below, peptides unique to HMWK were detected in the HK standard, suggesting that the HK standard contained HMWK. [ka]

[0112] The locations of HMWK-specific peptides are shown in bold and underlined in the HMWK amino acid sequence shown above.

[0113] b. Detection of HMWK-specific peptides in HK samples and EDTA plasma by MRM To determine the presence of HMWK in the standards and to compare the plasma samples with the standards, GluC-digested HK and GluC-digested plasma were subjected to targeted analysis on an AB Sciex 5500 Qtrap. A peptide unique to HMWK (SYYFDLTDGLS; SEQ ID NO: 10) was detected in both samples using an MRM assay, although the peptide peak intensity was lower in plasma compared to the HK standard. This was expected due to the complexity and high background noise of plasma. These results confirmed that the peptide was derived from HMWK.

[0114] (vii) Digestion conditions a. Digestion of HK and plasma with GluC at 25°C and 37°C The effectiveness of digesting HK and plasma samples with GluC at 25°C rather than 37°C was evaluated. HMWK-specific peptides were detected in the HK standard, but the long HK peptides were not detected in the standard. To test whether there was incomplete digestion or whether the higher temperature degraded the HK standard into bradykinin and KKYIPTVNCQPLGMISLMK (specific for the 2HK heavy peptide; SEQ ID NO: 23), plasma and HK samples were digested with GluC at 25°C rather than 37°C. MRM analysis was used to target and detect peptides of interest, including the HMWK-specific peptide. Peak intensities are as follows: HK Sigma Glu C 25℃: SSRIGE (SEQ ID NO: 5): 5960 KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 20): 4000 SYYFDLTDGLS (SEQ ID NO: 10): 7.6 e4 HK Enzyme Research Glu C 25°C: SSRIGE (SEQ ID NO: 5): 4.5 e5 ·SSRIGEIKE mis 2 (SEQ ID NO: 19): 1.5 e5 KKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGE (SEQ ID NO: 20): 2.3 e5 SYYFDLTDGLS (SEQ ID NO: 10): 4.6 e5

[0115] GluC digestion of HK standards (Enzyme Research and Sigma) at 25°C detected all three peptides of interest, including the HK long peptide, indicating that higher temperatures degrade the standards and therefore the HK long peptide is not observed when digested at 37°C. The Enzyme Research standard provided higher peak intensities than the Sigma standard. The mis2 cleavage peptide SSRIGEIKE (SEQ ID NO: 19) was only found in the Enzyme Research standard. For plasma samples, the results indicated that GluC digestion at 37°C resulted in better digestion efficiency than GluC digestion at 25°C. Figure 1. Confirmation of the long peptide and other HMWK-specific peptides using the standard at 25°C indicates that the peptides observed in the plasma samples are derived from HK.

[0116] No peptides except SSRIGE (SEQ ID NO: 5) were detected in plasma samples at 25°C; however, all of the predicted peptides of interest were detected in plasma samples digested at 37°C (Figure 1). The HK long peptide was observed in both standards at 25°C but not at 37°C.

[0117] These results suggest that digestion with GluC at 25°C worked well for the standards, while digestion at 37°C worked well for plasma.

[0118] a. Optimized digestion method using high-resolution accurate mass (HRAM) instrumentation Additionally, to determine whether an alternative detection platform could be used to detect peptides of interest in plasma samples digested at 25°C, samples (both HK and plasma) digested with GluC at 25°C and 37°C were evaluated on the HRAM instrument for a full scan to detect peptides of interest, including long peptides in HK and peptides unique to HMWK.

[0119] All of the peptides of interest and HMWK-specific peptides were detected in the HK standard digested at 25°C and the plasma sample digested at 37°C. This data set was consistent with the MRM data described above, with the exception of the mis2 cleavage peptide SSRIGEIKE (SEQ ID NO: 19), which is commonly observed in the HK standard from Enzyme Research. The LMWK-specific peptide YKGRPPKAGAE (SEQ ID NO: 28), also included in this assay, was found in the plasma sample but not in the HK standard, consistent with expectations (Figures 2 and 3).

[0120] b. Sequence coverage of HK-full scan at 25°C Sequence coverage of HK standards was assessed using optimized GluC digestion of HK, GluC source, and enzyme:protein ratio. Briefly, GluC digestion at 25°C with an enzyme:protein ratio of 1:20 resulted in significantly improved sequence coverage of HK (both HK supplied by Enzyme Research and Sigma) with high confidence scores, especially in the region of interest (long peptides of HK). [ka]

[0121] The position of the long peptide of HK is shown in bold and underlined in the amino acid sequence of HMWK shown above.

[0122] c. Full scan and SIM on HRAM instrument Additionally, SIM and full scan using the HRAM instrument were performed on HK standards digested at 25°C to detect HK long peptides. HK long peptides were observed in HK standards after digestion at 25°C by full scan and SIM on the HRAM instrument.

[0123] (viii) HMWK-specific peptides vs. LMWK-specific peptides upon GluC digestion at 25°C Two additional HMWK-specific (compared to LMWK) peptides, INPTQMKE (SEQ ID NO: 29) and SYYDDGLS (SEQ ID NO: 30), were added, and MRM analysis was performed to detect these unique peptides. Following digestion of HK standards (HK (Sigma) and HK (Enzyme Research)) at 25°C, additional HMWK-specific peptides were observed, confirming the presence of HK.

[0124] (ix) Evaluation of differences between different types of samples using optimized methods a. Quantification of SSRIGE (SEQ ID NO: 5) in plasma samples using a targeted assay (MRM) Individual plasma samples were digested with GluC and subjected to MRM analysis to detect the peptide of interest, SSRIGE (SEQ ID NO: 5). The sample types included normal SCAT and HAE SCAT plasma samples, kininogen-deficient plasma samples, normal citrated plasma samples, factor XIIa-activated normal citrated plasma samples, and HK standard samples.

[0125] As shown in Figure 3, SSRIGE (SEQ ID NO: 5) peptide was quantified using the quantitation wizard of the analysis software. A significant increase in SSRIGE (SEQ ID NO: 5) peptide was detected in HAE SCAT plasma samples compared to normal SCAT plasma samples. Also, SSRIGE (SEQ ID NO: 5) peptide was significantly increased in activated plasma compared to non-activated plasma samples. Kininogen-deficient plasma samples showed minimal SSRIGE (SEQ ID NO: 5) peptide, which was expected due to the absence of HMWK.

[0126] b. Quantification of HK long peptides in plasma samples using a targeted assay (MRM) Plasma samples were digested with GluC and subjected to MRM analysis to detect the HK long peptide. Sample types included normal SCAT and HAE SCAT plasma samples, kininogen-deficient plasma samples, normal citrated plasma samples, normal citrated plasma samples activated with factor XIIa, and HK standards. Very little change in the HK long peptide was detected between the normal SCAT and HAE SCAT plasma samples. Furthermore, no differences were observed between activated and unactivated samples. (Figure 4).

[0127] (x) Evaluation of consistency and reproducibility of GluC digestion To assess the reproducibility of GluC digestion from day to day, HK standards and plasma (citrated plasma + PI) samples were digested on three consecutive days and subjected to MRM analysis to detect peptides of interest (see Tables 6A and 6B below). All peptides of interest were present in the HK standards and plasma samples at the peak intensities shown. Tables 6A and 6B. Each of the three days of GluC digestion data for HK standards and plasma (citrated plasma + PI) had consistent results. [Table 7] Table 6A: HK digested with GluC for three consecutive days

[0128] (xi) the ratio between SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10) a. Calculation of the ratio of SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10) peptides Using HMWK and plasma samples digested with GluC, the ratios between SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10) peptides were calculated by targeted assay (MRM) in normal plasma versus plasma from HAE patients. Measuring the ratio of SSRIGE (SEQ ID NO: 5) peptide to peptides unique to HMWK may be used, for example, in diagnostic assays. All ratios and ratio means were calculated by taking the area under the peptide peaks in the quantification using Analyst® software.

[0129] An overall increase in the SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) ratio was observed in HAE SCAT plasma samples compared to normal SCAT plasma samples, and the SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) ratio was significantly increased in activated versus non-activated plasma (Figure 5).

[0130] b. Mean ratio between SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10) peptides The average ratio of SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) was calculated for different types of samples as the area under the peak using the MRM targeted assay ( FIG. 6 ). The average ratio between SSRIGE (SEQ ID NO: 5) / SYYFDLTDGLS (SEQ ID NO: 10) was found to be higher in HAE SCAT plasma samples compared to normal SCAT plasma samples. This ratio of the SSRIGE (SEQ ID NO: 5) peptide to HMWK vs. the LMWK-specific peptide SYYFDLTDGLS (SEQ ID NO: 10) has the potential to be used in assays such as diagnosing diseases or disorders associated with cleaved kininogen or distinguishing between samples from healthy individuals and those from diseased individuals.

[0131] (xii) Optimization of LC-MS testing using HK standards To increase the efficiency of this method, the LC-MS experiments were optimized by optimizing the gradient and shortening the experiment time. LC-MS optimization was primarily performed using HK standards. As shown in Figure 6, the results of several methods are presented using various LC gradients and experiment times. The LC-MS method was successfully shortened to a 10-minute experiment. The chromatogram on the bottom left represents the "optimized method," since the SYYFDLTDGLS peptide (SEQ ID NO: 10) appears as a single peak instead of a split peak. Both peptides required for the ratio calculation, SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10), were found in significantly less experiment time.

[0132] (xiii) Optimization of LC-MS experiments using plasma samples The short LC-MS experiment described above was validated using plasma samples, where the peptides of interest (SSRIGE (SEQ ID NO: 5) and SYYFDLTDGLS (SEQ ID NO: 10)) were detected. Detection of the peptides of interest in both HK standards and plasma samples was achieved using a 16.5 minute experimental method, which is a significant improvement over the original method (42 minutes). The intensity of both peptides decreased as the complexity of the plasma samples increased: EDTA plasma > citrate plasma > SCAT plasma.

[0133] Example 2: Quantification of cleaved HWMK 46 kDa light chain in HAE plasma using signature peptides and LC-MS / MS The exemplary method described in this example was designed to determine the concentration of endogenous double-chain HMWK (which can be represented by the level of the 46 kDa light chain) in human plasma. The method involved crushing 25 μL of plasma, digesting the pellet, further purifying using MCX SPE, and analyzing by liquid chromatography-tandem mass spectrometry (LC-MS / MS). A second-dimensional HPLC (Agilent Metasil AQ C18 column, 5.0 μm, 2.1 mm × 100 mm (C / N A0530100X020) and an AB Sciex QTrap 6500 mass spectrometer were used to isolate the double-chain HMWK signature peptide, HN-KHNLGHGH-OH (SEQ ID NO: 1), and a stably labeled internal standard (SLIS) (KHNL[ 13 The system was operated in SRM (Selected Reaction Monitoring) mode under conditions optimal for the detection of [C6]GHGH.

[0134] A 25 μL aliquot of human plasma was mixed with 2.5 μL of 10% SDS and 5 μL of DTT (500 mM), and the proteins were reduced at 37°C for 60 minutes. 75 μL and 600 μL of methanol were added sequentially to the solution to precipitate the proteins. The supernatant was discarded, and the protein pellet was washed again with 700 μL of methanol. After centrifugation, the pellet was reconstituted in 500 μL of ammonium bicarbonate buffer (100 mM) with vigorous vortexing for 10 minutes. A 25 μL aliquot of the internal standard solution (KHNL[ 13 [C6]GHGH (30 ng / mL) and 5.0 μL of iodoacetamide solution (500 mM) were added, and the mixture was stored in the dark for 30 min. After that, the protein was digested by adding 10 μL of chymotrypsin (8 mg / mL) and kept at 50°C for 3 h with gentle vortexing.

[0135] The resulting mixture was acidified and loaded onto an MCX 96-well cartridge (30 μm, 10 mg) for desalting. The cartridge was washed with 900 μL of 2.0% formic acid solution, 900 μL of water, and 900 μL of methanol, respectively, and the resulting peptides were eluted with 600 μL of 5.0% ammonium hydroxide in methanol. The eluted solution was dried with nitrogen gas and reconstituted in 100 μL of 1.0% HFBA in methanol:water (10:90; v / v).

[0136] For sample analysis, peptides were loaded onto an LC-MS / MS instrument operated in MRM mode, where the signature peptide KHNLGHGH (SEQ ID NO: 1) and its internal standard KHNL[ 13 The ionic intensity of [C6]GHGH was recorded, which allowed the calculation of the relative amount of the 46 kDa light chain of the two-chain HMWK in the plasma samples.

[0137] (i) Assay validation (precision and accuracy) The intraday (n=6) and interday (n=18) precision (RSD (%)) and accuracy (RE (%)) for quantifying 46 kDa light chain in human plasma (SCAT169) were determined using the methods described herein. As shown in Table 7 below, the levels of 46 kD determined in both the intraday and interday assays using the methods described herein are very close to the theoretical concentration of 46 kDa in the samples tested. These results demonstrate the accuracy of the assay methods described herein using the 46 kDa light chain signature peptide described above as a biomarker. [Table 8]

[0138] (ii) Stability assessment Human plasma samples were collected in SCAT169 tubes according to the method described herein. These samples were subjected to four freeze-thaw cycles or stored on the benchtop (4°C) for 8 hours. The assay method described above using the signature peptide of the 46 kDa light chain was performed to measure the level of the 46 kDa light chain in both samples. The results thus obtained indicate that the two-chain HMWK (represented by the 46 kDa light chain) is stable in human plasma samples. Table 8. [Table 9]

[0139] (iii) Use of the assay method in disease diagnosis and prognosis Plasma samples were collected in SCAT169 tubes from healthy human subjects and HAE patients. The assay method described above was performed to measure the levels of double-chain HMWK in these human plasma samples. As shown in Figure 7, the levels of double-chain HMWK in HAE patients were significantly higher than those in healthy human subjects. This result indicates that double-chain HMWK and any of its components (e.g., the 46 kDa light chain) can serve as reliable biomarkers for the diagnosis and / or prognosis of HAE.

[0140] 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 an example of a generic series of equivalent or similar features.

[0141] From the foregoing description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to adapt it to various applications and conditions without departing from the spirit and scope of the present invention. Accordingly, other embodiments are within the scope of the appended claims.

[0142] Doctrine of Equivalents While several embodiments of the present invention have been described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions described herein and / or obtaining one or more of the results and / or advantages described herein, and each such variation and / or modification is intended to be within the scope of the embodiments of the present invention described herein. In a broader sense, those skilled in the art will readily recognize that all parameters, dimensions, materials, and configurations described herein are intended to be exemplary, and that the actual parameters, dimensions, materials, and / or configurations will depend on the specific application(s) for which the teachings of the present invention are used. 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 invention described herein. Thus, it is understood that the above-described embodiments are presented by way of example only, and that, within the scope of the appended claims and their equivalents, embodiments of the present invention can be practiced other than as specifically described and claimed. The inventive embodiments of the present disclosure are directed to each individual feature, system, article, material, kit, and / or method described herein. Furthermore, combinations of any two or more of such features, systems, articles, materials, kits, and / or methods are included within the inventive scope of the present disclosure, provided that such features, systems, articles, materials, kits, and / or methods are not mutually inconsistent.

[0143] All definitions herein and as used should be understood to go beyond dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the terms defined.

[0144] As used in this specification and the claims, the indefinite articles "a" and "an" should be understood to mean "at least one," unless expressly stated to the contrary.

[0145] The term "and / or," as used in the specification and claims, should be understood to mean conjunctive association of multiple elements, i.e., "either or both" of multiple elements that are presented conjunctively in some cases and disjunctively in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" of the multiple elements listed conjunctively. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to the elements specifically identified. Thus, as a non-limiting example, when used in conjunction with open-ended language such as "comprising," a designation of "A and / or B" can, in one embodiment, refer to A only (optionally including elements other than B); in another embodiment, B only (optionally including elements other than A); in yet another embodiment, both A and B (optionally including other elements), etc.

[0146] As used herein and in the claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating multiple items in a list, "or" or "and / or" should be construed as inclusive, i.e., including at least one of a number or list of elements, as well as more than one element, and optionally additional unlisted items. Only when terms to the contrary are specified, such as "only one of" or "exactly one of," or when used in the claims, "consisting of" refers to the inclusion of exactly one element of a number or list of elements. Generally, as used herein, the term "or" should be construed simply to represent exclusive alternatives (i.e., "one or the other, but not both") when preceding exclusive terms such as "either," "one of," "only one of," or "exactly one of." When used in the claims, "consisting essentially of" shall have its ordinary meaning as used within patent law.

[0147] As used in this specification and claims, the phrase "at least one" in reference to a list of one or more elements should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements in the list of elements. This definition also allows for the optional presence of elements other than those specifically identified in the list of elements referred to by the phrase "at least one," whether related or unrelated to the specifically identified element. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer to, in one embodiment, at least one A, optionally including more than one A (where B is absent and optionally including elements other than B); in another embodiment, at least one B, optionally including more than one B (where A is absent and optionally including elements other than A); in yet another embodiment, at least one A, optionally including more than one A, and at least one B, optionally including more than one B (and optionally including other elements), etc.

[0148] Also, unless expressly stated to the contrary, it should be understood that in any method claimed herein that includes more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the method steps or acts are described.

[0149] In the claims and the foregoing specification, all transitional phrases such as "comprising," "including," "carrying," "having," "containing," "involving," "holding," "consisting of," and the like, are understood to be open-ended, i.e., to mean including, but not limited to, the recited items. Only the transitional phrases "consisting of" and "consisting essentially of" are intended to be closed or semi-closed transitional phrases, respectively, as set forth in the United States Patent Office Manual of Patent Examining Procedures, Section 2111.03.

Claims

1. 1. A method for evaluating a treatment of a plasma kallikrein (pKal)-mediated disorder, comprising: (i) contacting a biological sample obtained from a patient being treated for a pKal-mediated disorder before, after, and / or during the course of treatment with a protease to generate a plurality of digested peptides; (ii) measuring the level of a signature peptide representing cleaved high molecular weight kininogen (HMWK) in the plurality of digested peptides in each of the biological samples; (iii) identifying the patient as not responding to the treatment if the level of the signature peptide in a biological sample taken after or during the course of treatment increases or remains the same compared to the level of the signature peptide in a biological sample taken before the treatment; A method comprising:

2. 2. The method of claim 1, wherein the signature peptide represents the 46 kDa light chain of cleaved HMWK.

3. the signature peptide representing the 46 kDa light chain is KHNLGHGH (SEQ ID NO: 1), KHNLGHGHKHE (SEQ ID NO: 2); KHNLGHGHK (SEQ ID NO: 3); or KHNLGHGHKHER (SEQ ID NO: 4) The method of claim 2, wherein

4. 2. The method of claim 1, wherein the signature peptide represents the 56 kDa light chain of cleaved HMWK.

5. 5. The method of claim 4, wherein the signature peptide representing the 56 kDa light chain is SSRIGE (SEQ ID NO: 5).

6. 6. The method of any one of claims 1 to 5, wherein the protease is selected from the group consisting of chymotrypsin, endoproteinase Glu-C, endoproteinase Asp-N, cathepsin G, and endoproteinase Lys-C.

7. The method of any one of claims 1 to 6, further comprising measuring the level of a signature peptide representing full-length HMWK.

8. The signature peptide representing the full-length HMWK is GHEKQRKH (SEQ ID NO: 6); KQRKHNLGHGHKHE (SEQ ID NO: 7); DWGHEKQRKHNLGHGHKHER (SEQ ID NO: 17); HNLGHGHK (SEQ ID NO: 9); or SYYFDLTDGLS (SEQ ID NO: 10) The method of claim 7, wherein

9. 9. The method of claim 7 or 8, wherein the level of the signature peptide representing the truncated HMWK, the level of the signature peptide representing the full-length HMWK, or both, is measured by liquid chromatography-mass spectrometry (LC-MS).

10. 10. The method of any one of claims 1 to 9, wherein the level of the signature peptide in a biological sample taken after or during the course of treatment from a patient identified as non-responding in step (iii) is higher than the level of the signature peptide in a biological sample taken from a healthy subject.

11. 1. A method for evaluating a treatment of a plasma kallikrein (pKal)-mediated disorder, comprising: (i) contacting a biological sample obtained from a patient being treated for a pKal-mediated disorder before, after, and / or during the course of treatment with a protease to generate a plurality of digested peptides; (ii) measuring the level of a first digested peptide from step (i), wherein said first digested peptide is unique to cleaved high molecular weight kininogen (HMWK) compared to full-length HMWK; (iii) measuring the level of a second digested peptide from step (i), wherein the second digested peptide is unique to HMWK compared to low molecular weight kininogen (LMWK); (iv) determining the ratio between the first digested peptide and the second digested peptide; (v) identifying the patient as not responding to the treatment if the ratio of the first digested peptide to the second digested peptide in a biological sample collected after or during the course of treatment increases or remains the same compared to the ratio of the first digested peptide to the second digested peptide in a biological sample collected before the treatment; A method comprising:

12. 12. The method of claim 11, wherein the first digested peptide is SSRIGE (SEQ ID NO: 5).

13. 13. The method of claim 11 or 12, wherein the second digested peptide is SYYFDLTDGLS (SEQ ID NO: 10).

14. The method of any one of claims 11 to 13, wherein the protease cleaves after glutamic acid residues.

15. 15. The method of claim 14, wherein the protease is endoproteinase Glu-C or cathepsin G.

16. 16. The method of claim 15, wherein the protease is endoproteinase Glu-C.

17. 17. The method of any one of claims 11 to 16, wherein the first digested peptides and the second digested peptides are measured by liquid chromatography-mass spectrometry (LC-MS).

18. 18. The method of any one of claims 11 to 17, wherein the ratio of the first digested peptide to the second digested peptide in a biological sample taken after or during the course of treatment from a patient identified as non-responding in step (iii) is higher than the ratio of the first digested peptide to the second digested peptide in a biological sample taken from a healthy subject.

19. The method of any one of claims 1 to 18, wherein the plurality of biological samples are blood samples or plasma samples.

20. The method according to any one of claims 1 to 19, wherein the biological sample is a plasma sample collected in an evacuated blood collection tube containing a liquid formulation comprising a mixture of protease inhibitors.

21. 21. The method of claim 20, wherein the evacuated blood collection tube contains an anticoagulant.

22. A method according to any one of claims 1 to 21, wherein step (i) is carried out in the presence of a reducing agent.

23. 23. The method of claim 22, wherein the biological sample is incubated with the reducing agent at 90°C for 1 hour.

24. 24. The method of any one of claims 1 to 23, wherein step (i) is carried out in the absence of a protease inhibitor, an anticoagulant, or both a protease inhibitor and an anticoagulant.

25. 25. The method of any one of claims 16 to 24, wherein in step (i) the ratio of protease / HMWK is about 1:

20.

26. The method of any one of claims 1 to 25, wherein the pKal-mediated disorder is hereditary angioedema (HAE).

27. 27. The method of any one of claims 1 to 26, wherein the patient is resistant to antihistamine therapy, corticosteroid therapy, or both.

28. The method of any one of claims 1 to 27, wherein the patient has symptoms of the pKal-mediated disorder.

29. 29. The method of claim 28, wherein the symptom is edema; recurrent attacks of swelling; swelling that is entirely or predominantly peripheral; hives; redness, pain, and swelling in the absence of evidence of infection; or non-histamine-mediated edema.

30. The patient is does not have symptoms of said pKal-mediated disorder at the time said sample is taken; have no history of symptoms of said pKal-mediated disorder; or have no history of said pKal-mediated disorder; 30. The method according to any one of claims 1 to 29.

Images