Assays for determining plasma kallikrein system biomarkers

Abstract

To provide methods and assays for determining the activation level of a plasma kallikrein (pKal) system, and to provide their use for evaluating the activity of pKal regulators on the pKal system.SOLUTION: Provided is an ex vivo activation method that includes incubating a plasma sample obtained from a subject with an activator of a plasma kallikrein (pKal) system, measuring the concentration of intact high molecular weight kininogen (HMWK), cleaved HMWK, or both, in the plasma sample before and after the incubation, and determining the reduction of intact HMWK in the sample after activation.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to the filing date of U.S. Provisional Patent Application No. 61 / 893,505, filed October 21, 2013, and to the filing date of U.S. Provisional Patent Application No. 61 / 939,837, filed February 14, 2014, both of which are incorporated by reference in their entireties. [Background technology]

[0002] Background technology Plasma kallikrein (pKal) is the major bradykinin-generating enzyme in the circulatory system. Activation of pKal occurs via the contact system, which is involved in disease pathology associated with hereditary angioedema (HAE). Bradykinin is an important mediator of pain, inflammation, edema, and angiogenesis. Summary of the Invention [Problem to be solved by the invention]

[0003] SUMMARY OF THE DISCLOSURE Plasma kallikrein (PKal) is a serine protease component of the contact system and the main bradykinin-generating enzyme in the circulatory system. The contact system is activated by factor XIIa upon exposure to foreign bodies or negatively charged surfaces, or on the endothelial cell surface by prolylcarboxypeptidase (Sainz IM et al., Thromb Haemost 98, 77-83, 2007). Activation of plasma kallikrein amplifies intrinsic coagulation through its feedback activation of factor XII and potentiates inflammation through the production of the proinflammatory nonapeptide bradykinin. As the main kininogenase in the circulation, plasma kallikrein plays a major role in the production of bradykinin in the vascular system. Genetic deficiency of the C1-inhibitor protein, the main natural inhibitor of plasma kallikrein, results in hereditary angioedema (HAE). Patients affected by HAE often suffer from acute attacks of painful edema caused by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). Through the use of drugs and genetic studies in animal models, the plasma kallikrein-kinin system (plasma KKS) has been implicated in a variety of diseases. [Means for solving the problem]

[0004] The present disclosure is based on the development of a number of biomarker assays, including the HMWK ex vivo activation assay, the endogenous cleaved HMWK assay, and the pKal ex vivo activation assay as described herein. Such assays can be used to measure the activation level of the plasma kallikrein (pKal) system in the plasma of a subject. They can also be used to evaluate candidate compounds for their activity in modulating (inhibiting or activating) the pKal system.

[0005] In one aspect, the present disclosure features an ex vivo activation method, which includes (i) incubating a plasma sample obtained from a subject with an activator of plasma kallikrein (pKal) system (e.g., factor XIIa (FXIIa)); (ii) measuring the concentration of intact high molecular weight kininogen (HMWK), cleaved HMWK, or both in the plasma sample before and after incubation; and (iii) determining the reduction of intact HMWK in the sample after activation. Optionally, the plasma sample and the activator are incubated in the presence of a candidate pKal regulator (e.g., a candidate inhibitor or activator such as DX2930). The method can further include evaluating the activity of the candidate pKal regulator. In some embodiments, the concentration of intact HMWK and cleaved HMWK is measured by Western blot analysis, such as Protein Simple Western blot analysis.

[0006] In another aspect, the present disclosure features a method for evaluating plasma activation in a subject, the method includes providing a plasma sample from a subject and measuring the concentration of truncated HMWK in the plasma sample.The concentration of truncated HMWK can be measured by Western blot, such as Protein Simple Western blot analysis.In some embodiments, the subject has or is suspected of having a disease related to pKal system.Alternatively or additionally, the subject is treated with a pKal inhibitor.Any of the methods described herein further include evaluating the effectiveness of the pKal inhibitor, where a reduction in the concentration of truncated HMWK after treatment compared to the concentration before treatment indicates that the pKal inhibitor is effective.

[0007] In yet another embodiment, the present disclosure provides an ex vivo assay for determining pKal activity in a sample (e.g., a plasma sample from a subject), the assay comprising (i) incubating the sample with a pKal system activator (e.g., factor XIIa or FXIIa) in the presence of a pKal substrate, and (ii) measuring the activity of pKal based on the cleavage rate of the substrate. In some examples, the substrate is bound to a label that can release a detectable signal after cleavage by pKal, and the cleavage rate of the substrate is determined based on the magnitude of the detectable signal. In some examples, the plasma sample is incubated with an activator, a pKal substrate, and a candidate pKal regulator (e.g., an inhibitor regulator or an activator regulator). The method further comprises evaluating the activity of the candidate pKal regulator (e.g., DX-2930), and a change in pKal activity level in the presence of the candidate pKal regulator compared to the level of pKal activity in the absence of the candidate pKal inhibitor indicates that the candidate inhibitor is effective. For example, a decrease in the level of pKal activity indicates that the candidate pKal modulator is a pKal inhibitor, while an increase in the level of pKal activity indicates that the candidate pKal modulator is a pKal activator. Any of the assays described herein can be performed in a microplate.

[0008] In any of the assay methods described herein, the method may further comprise evaluating whether the subject has or is at risk of pKal-related disease (e.g., HAE), and an increase in the concentration of truncated HMWK compared to a predetermined value indicates that the subject has or is at risk of the disease.In some embodiments, the subject may be a human patient with a pKal-related disease (e.g., HAE) and undergoes treatment for the disease (e.g., a pKal inhibitor such as DX2930).The plasma sample may be obtained after or during the course of treatment.In some embodiments, the method may further comprise evaluating the effectiveness of the treatment, and a decrease in the concentration of truncated HMWK compared to before treatment (e.g., a pKal inhibitor such as DX2930) or a decrease in the concentration of truncated HMWK over the course of treatment (e.g., a pKal inhibitor such as DX2930) indicates that the treatment is effective.

[0009] The disclosure further provides methods for evaluating a subject at risk for or suffering from, for example, a pKal-mediated or bradykinin-mediated disorder, which may include any of the assays described herein. The methods provided allow for analyses useful in the evaluation and treatment of patients with plasma kallikrein-mediated angioedema (KMA) or other diseases mediated by pKal.

[0010] Furthermore, embodiments of the present disclosure provide biomarkers and their use in identifying and treating patients, such as those suffering from edema caused by bradykinin produced by plasma kallikrein. The methods, compositions and devices disclosed herein are useful in many ways. For example, the concentration of pKal markers can be used to identify disorders associated with increased contact system activation. After initial screening, in vitro or in vivo testing can be performed with plasma kallikrein inhibitors (e.g., DX-88, EPIKAL2 or DX-2930), for example in preclinical models of disease. The markers disclosed herein can also be used as pharmacodynamic biomarkers or otherwise to monitor the subject's response to kallikrein inhibitors. The markers disclosed herein are useful in treating conditions including, for example, HAE, non-histamine dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burn injury, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, ventricular assist device or stent associated thrombosis, heparin-induced thrombocytopenia with thrombosis, thromboembolism, coronary heart disease with unstable angina, edema, eye disease, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis degenerative spinal disease, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute The present invention can be used in companion diagnostics to enable the treatment of diseases mediated by plasma kallikrein, such as middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive and diabetic nephropathy, allergies and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis) and tissue injury (e.g., burn or chemical injury), and to direct dosing in prophylactic treatment of pKal- or bradykinin-mediated disorders.

[0011] In one embodiment, the present disclosure provides a method for evaluating or treating a subject, for example, for distinguishing pKal-mediated disorders such as bradykinin-mediated angioedema from histamine-mediated disorders, or for predicting future episodes of pKal-mediated disorders, comprising obtaining, for example, determining, the concentration of one or more markers that correlate with pKal activation (pKal markers) disclosed herein, for example, prekallikrein, active pKal, α2M-pKal, Cl-INH-pKal, intact kininogen, and truncated kininogen, thereby evaluating or treating the subject.In one embodiment, the method comprises obtaining, for example, detecting, the concentration of one or more markers that correlate with histamine-mediated inflammatory response (H markers), for example, tryptase.

[0012] In some embodiments, the pKal-mediated disorder is HAE, IAE, IBD or IBS. In some embodiments, the pKal-mediated disorder is non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burn injury, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, ventricular assist device or stent-associated thrombosis, heparin-induced thrombocytopenia with thrombosis, thromboembolism, coronary heart disease with unstable angina, edema, eye disease, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal canal stenosis degenerative spinal disease, postoperative ileus, aortic aneurysm, osteoarthritis The present invention relates to a method for treating or preventing atherosclerosis, including the treatment of atherosclerosis, rheumatoid artery disease, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive and diabetic nephropathy, allergies and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis) and tissue injury (e.g., burn or chemical injury).

[0013] In another aspect, the disclosure provides a method of evaluating or treating a subject, the subject having a symptom consistent with both a pKal-mediated disorder, e.g., bradykinin-mediated angioedema, and a histamine-related disorder, the method comprising: a) optionally determining that the subject has a symptom, e.g., edema or abdominal discomfort, consistent with one or both of a pKal-mediated disorder and a histamine-related disorder; b) if the subject has not been treated with antihistamine therapy for the symptom, treating the subject with antihistamine therapy; c) obtaining, e.g., detecting, the concentration of one or more markers associated with pKal activation (pKal markers), e.g., prekallikrein, active pKal, α2M-pKal, Cl-INH-pKal, intact kininogen, and cleaved kininogen; and d) if the concentration meets a predetermined criterion, e.g., is at or above a reference concentration, selecting the subject for kallikrein inhibitor therapy or administering a kallikrein inhibitor to the subject, thereby evaluating or treating the subject. In some embodiments, the method comprises selecting the subject for kallikrein inhibitor therapy.In some embodiments, the method comprises administering a kallikrein inhibitor to the subject.In some embodiments, selecting the subject for kallikrein inhibitor therapy or administering a kallikrein inhibitor to the subject occurs before determining that the subject has an acceptable response to the antihistamine therapy, for example, within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours of treatment with the antihistamine therapy.In some embodiments, determining that the subject has symptoms consistent with both pKal-mediated disorder and histamine-related disorder and obtaining a sample from the patient to determine the concentration of pKal marker are performed within 30 minutes, 1 hour, 2 hours, or 3 hours of each other, or during the same visit to a medical provider.

[0014] In certain embodiments, the pKal inhibitor is selected from DX-88, DX-2930, or EpiKal-2.

[0015] In some embodiments, the method includes obtaining, e.g., determining, the concentration of one or more markers (H markers) that correlate with histamine-mediated inflammatory response. In some embodiments, the subject is evaluated for susceptibility to pKal-mediated disorder. In some embodiments, the subject has symptoms, e.g., consistent with pKal-mediated disorder, e.g., edema, e.g., HAE. In some embodiments, the subject has symptoms of a disorder characterized by unwanted pKal activation, and the subject is administered antihistamine therapy. In certain embodiments, the antihistamine therapy is administered within 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours before or after the determining step disclosed herein. In certain embodiments, the method further includes administering antihistamine therapy to the subject, e.g., before, after, or during the evaluation or determination disclosed herein.

[0016] In some embodiments, the subject is administered a kallikrein inhibitor in response to said determination or evaluation.In some embodiments, the subject has one or more or all of the following symptoms or characteristics: recurrent bouts of swelling; swelling that is entirely or mostly peripheral, for example, the subject does not have abdominal swelling or airway swelling; hives; swelling without redness, pain, and evidence of infection; non-response to antihistamine or corticosteroid therapy; or has non-histamine-mediated edema. In certain embodiments, the subject does not have a history of a pKal mediated disorder, e.g., HAE, IAE, IBD, or IBS; the subject has a history of a pKal mediated disorder, e.g., HAE, IAE, IBD, or IBS; the subject does not have a history of HAE; the subject has a history of HAE; the subject does not have a history of IAE; the subject has a history of IAE; the subject does not have a history of IBD or IBS; the subject has a history of IBD or IBS; the subject does not have a history of a histamine mediated disorder, e.g., food allergy; the subject has a history of a histamine mediated disorder, e.g., food allergy; the subject has a history of a histamine mediated disorder, e.g., HAE, IAE, IBD, or or the subject does not have a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and has a history of a histamine mediated disorder, such as food allergy; the subject has a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and does not have a history of a histamine mediated disorder, such as food allergy; or the subject has a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and has a history of a histamine mediated disorder, such as food allergy.

[0017] In some embodiments, the subject is undergoing treatment with a kallikrein inhibitor, e.g., as a preventive treatment, e.g., for HAE, and the subject's response to the kallikrein inhibitor is evaluated or monitored, and optionally, in response to said monitoring, a treatment is selected or administered, e.g., in response to said determination, the dose of the kallikrein inhibitor is adjusted.In some embodiments, the determination of pKal marker is performed in the context of a companion diagnostic, and optionally, based on said determination, a therapeutic agent is administered or discontinued.In some embodiments, in response to said obtaining, an impending acute attack, e.g., an attack of HAE or IEA, is identified.In certain embodiments, said subject is evaluated for sudden angioedema susceptibility.In certain embodiments, said evaluation comprises determining whether said subject suffers from a pKal-mediated disorder, e.g., a bradykinin-mediated disorder, e.g., a pKal-mediated angioedema, or a histamine-mediated disorder, e.g., an allergic food reaction.

[0018] In some embodiments, the subject does not have a history of a pKal-mediated disorder, such as HAE or IAE. In some embodiments, the subject has a history of a pKal-mediated disorder, such as HAE or IAE. In some embodiments, the subject does not have a history of a pKal-mediated disorder, such as HAE, IAE, IBD or IBS; the subject has a history of a pKal-mediated disorder, such as HAE, IAE, IBD or IBS; the subject does not have a history of HAE; the subject has a history of HAE; the subject does not have a history of IAE; the subject has a history of IAE; the subject does not have a history of IBD or IBS; the subject has a history of IBD or IBS; the subject does not have a history of a histamine-mediated disorder, such as food allergy; the subject has a history of a histamine-mediated disorder, such as food allergy; the subject does not have HAE, IAE, IBD, or the subject does not have a history of a pKal mediated disorder, such as IBS, and does not have a history of a histamine mediated disorder, such as food allergies; or the subject does not have a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and has a history of a histamine mediated disorder, such as food allergies; the subject has a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and does not have a history of a histamine mediated disorder, such as food allergies; or the subject has a history of a pKal mediated disorder, such as HAE, IAE, IBD or IBS, and has a history of a histamine mediated disorder, such as food allergies.

[0019] In some embodiments, the pKal marker, such as the pKal marker as disclosed herein, is detected by an antibody-based reagent. In some embodiments, the pKal marker is detected by a sandwich immunoassay. In some embodiments, the method comprises obtaining, e.g., detecting, the concentration of α2M-pKal and C1-INH-pKal, e.g., by a sandwich immunoassay. In some embodiments, the method comprises obtaining, e.g., detecting, the concentration of one or both of the kininogens, e.g., intact or cleaved kininogens, by an electrophoretic separation assay, e.g., Western blot. In some embodiments, the pKal marker is detected by a Simple Western™ assay. Simple Western™ is known in the art (see, e.g., Rustandi et al., Qualitative and quantitative evaluation of Simon™, a new CE-based automated Western blot system as applied to vaccine development., Electrophoresis, 2012, September;33(17):2790-2797). Simple Western™ products are also commercially available (see, eg, ProteinSimple, Santa Clara, Calif.).

[0020] In some embodiments, a first pKal marker, such as prekallikrein, active preKal, α2M-pKal or C1NH-pKal, is detected by sandwich immunoassay, and a second pKal marker, such as kininogen, is detected in an assay based on separation of sample from other products, such as electrophoretic separation, such as Western blot or Simple Western™ separation. In some embodiments, detection of pKal marker is qualitative. In some embodiments, detection of pKal marker is quantitative. In certain embodiments, the method comprises determining the concentration of C1-INH-pKal and α2M-pKal. In certain embodiments, the method comprises determining the concentration of active pKal. In certain embodiments, the concentration of prekallikrein, active pKal, α2M-pKal, C1-INH-pKal, intact kininogen and cleaved kininogen are detected, respectively.

[0021] In some embodiments, the method includes comparing the concentration of a pKal marker, such as prekallikrein, active pKal, α2M-pKal, C1-INH-pKal, intact kininogen or truncated kininogen, with a reference value. In some embodiments, the reference value is a function of the concentration of the pKal marker in HAE, such as in one or more HAE subjects. In some embodiments, the reference value is a function of the concentration of the pKal marker in HAE during an attack, such as in one or more HAE subjects during an acute attack. In some embodiments, the reference value is a function of the concentration of the pKal marker in IAE, such as in one or more IAE subjects. In some embodiments, the reference value is a function of the concentration of the pKal marker in IAE during an acute attack, such as in one or more IAE subjects during an acute attack. In some embodiments, the reference value is a function of the concentration of the pKal marker in the absence of HAE or IAE, such as in one or more subjects with no history of HAE or IAE.

[0022] In some embodiments, the method comprises, for example, according to the comparison, classifying the subject, for example, classifying the subject with respect to the risk of pKal-mediated disorder, or administering or stopping treatment to the subject.In some embodiments, the method comprises, for example, according to the comparison, selecting a treatment for the subject.In some embodiments, the method comprises, for example, according to the comparison, administering or stopping treatment to the subject, such as kallikrein binding agent, bradykinin B2 receptor antagonist or C1-INH supplement.In some embodiments, the treatment is administration of a pKal inhibitor, for example, a pKal inhibitor selected from DX-88, EpiKal-2 and DX-2930.

[0023] In one embodiment, a sample from the subject is contacted with a substrate comprising a capture agent for two or more of the following markers disclosed herein, e.g., prekallikrein, e.g., an anti-prekallikrein antibody; active pKal, e.g., an anti-active pKal antibody; α2M-pKal, e.g., an anti-α2M-pKal antibody; C1INH-pKal, e.g., an anti-C1INH-pKal antibody; or an H marker, e.g., an anti-H marker antibody, and optionally at least one capture agent is a capture agent for the pKal marker.

[0024] In certain embodiments, the method includes obtaining a sample, such as a blood or plasma sample, from the subject.

[0025] In some embodiments, the substrate comprises a capture agent for C1-INH-pKal and a capture agent for α2M-pKal, hi some embodiments, the substrate further comprises one or both of a capture agent for prekallikrein, e.g., an anti-prekallikrein antibody, and a capture agent for active pKal, e.g., an anti-activated pKal antibody.

[0026] In some embodiments, the first capture agent (of the first marker) and the second capture agent (of the second marker) are arranged on the substrate such that the signal indicating the presence of the first marker can be distinguished from the signal indicating the presence of the second marker. In some embodiments, the first capture agent (of the first marker) is arranged at a first location or address, and the second capture agent (of the second marker) is arranged at a second location or address. In some embodiments, the first location or address and the second location or address do not overlap on the substrate. In some embodiments, the first capture agent is a capture agent of the first pKal marker. In some embodiments, the first capture agent is a capture agent of the first pKal marker, and the second capture agent is a capture agent of the second pKal marker. In some embodiments, the first capture agent is a capture agent of the pKal marker, and the second capture agent is a capture agent of the H marker. In some embodiments, the first marker is 2M-pKal and the second marker is C1INH-pKal. In one embodiment, the capture agent for 2M-pKal is an anti-2M-pKal antibody, and the capture agent for C1INH-pKal is an anti-C1INH-pKal antibody.

[0027] In some embodiments, the method includes contacting a detectable, e.g., labeled, anti-2M-pKal antibody and a detectable, e.g., labeled, anti-C1INH-pKal antibody with a substrate to measure the presence or amount of pKal marker. In some embodiments, the antibodies are labeled with a moiety that produces a colored product, emits photons, absorbs photons, alters the substrate, or alters the conductivity of the substrate. In some embodiments, the antibodies are labeled with a moiety that utilizes electrochemiluminescence. In some embodiments, the antibodies are labeled with resinium. In some embodiments, the substrate is provided in a meso scale discovery device. In some embodiments, the substrate is provided as a dip-stick device suitable for use with one or both of blood and plasma. In some embodiments, the first capture agent and the second capture agent are disposed in a common or fluidly connected chamber, e.g., a chamber, e.g., a well or well in a multi-chamber device, e.g., a multi-well plate. In certain embodiments, the first capture agent and the second capture agent are printed onto a substrate.

[0028] In one embodiment, the capture agent of a first pKal marker is at a first position on the substrate, and the capture agent of a second pKal marker is at a second position on the substrate, and the first and second positions are arranged such that a signal indicating the presence of the first pKal marker is distinguishable from a signal from the second pKal marker. In one embodiment, the substrate comprises a capture agent of a third marker at a third position, and the third position is arranged on the substrate such that a signal indicating the presence of the third marker is distinguishable from signals from the first and second markers. In one embodiment, the first capture agent is specific for α2M-pKal or C1INH-pKal. In one embodiment, the first capture agent is specific for α2M-pKal, and the second capture agent is specific for C1INH-pKal.

[0029] In some embodiments, the determination of the concentration of the pKal marker in a sample can be performed within 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours of contacting the substrate with the sample. In some embodiments, the determination of the concentrations of two pKal markers in a sample can be performed within 1 hour, 2 hours, 3 hours, 4 hours, or 5 hours of contacting the substrate with the sample. In some embodiments, the determination of the concentrations of two pKal markers can be performed in assays performed simultaneously, for example in assays where the incubations or other intervals of the tests overlap with each other.

[0030] In other aspects, the disclosure provides a substrate that includes capture agents for multiple pKal markers, eg, as described herein.

[0031] In a further aspect, the disclosure provides a method for determining whether a disorder is susceptible to treatment with a pKal inhibitor, the method comprising, for example, assessing the concentration of one or more pKal markers, e.g., as described herein, in a subject suffering from the disorder or in an animal model of the disorder, comparing the determined concentration to a standard, and if the concentration meets a predetermined standard, e.g., if the concentration is at or above the standard concentration, it indicates a disorder susceptible to treatment with a pKal inhibitor. In some embodiments, the method comprises assessing the effect of a kallikrein inhibitor in vitro or in vivo, or in an animal model of the disorder.

[0032] In another aspect, the disclosure provides a method of treating a subject with a pKal-mediated disorder, e.g., a bradykinin-mediated disorder, comprising assessing, determining, the concentration of a pKal marker described herein, e.g., by a method described herein, and selecting a treatment, e.g., selecting one or both of a dose or frequency of administration of a kallikrein inhibitor, in response to said assessment. In some embodiments, the method comprises administering a kallikrein inhibitor to said subject. In some embodiments, said patient has been administered a kallikrein inhibitor prior to said assessment. In some embodiments, the method comprises administering a kallikrein inhibitor at said selected dose or frequency.

[0033] In a further aspect, the disclosure provides a method for determining whether a disorder is susceptible to treatment with a pKal inhibitor, the method comprising: assessing the concentration of one or more pKal markers, e.g., as described herein, in a subject suffering from the disorder or in an animal model of the disorder; comparing the determined concentration with a reference; and indicating a disorder susceptible to treatment with a pKal inhibitor if the concentration meets a predefined reference, e.g., if the concentration is at or above the reference concentration. In some embodiments, the method comprises assessing the effect of a kallikrein inhibitor in vitro or in vivo, or in an animal model of the disorder.

[0034] In other aspects, the disclosure features methods and devices for collecting samples, e.g., blood, with minimal contact activation. In certain embodiments, the disclosure features a container having therein a capture reagent, e.g., a kallikrein inhibitor, e.g., a polypeptide similar in sequence to DX-88, e.g., a polypeptide differing from DX-88 by one, two or less than five amino acid residues, e.g., EPIKAL-2, as described herein. The container is configured, e.g., with an opening, a mouth, a septum, etc., to allow collection of a sample, e.g., blood, from a subject and binding of a pKal-related marker, e.g., pKal1, in the sample to the capture agent in the same container. Measurement of the bound species, e.g., pKal, can be performed in the same container, or in some embodiments, the substrate can be removed from the container prior to measurement, e.g., the measurement can be performed on or in another device. In certain embodiments, the volume of the container is 0.5-100 ml, 0.5-50 ml, 0.5-10 ml, 1-100 ml, 1-50 ml, 1-25 ml. In some embodiments, the capture agent, such as the pKal capture agent, is placed on the inner surface of the vessel. The capture agent can be bound to a surface to which a first specific binding partner is bound, and a second specific binding partner can be bound to the capture agent. Examples of specific binding partners are biotin and avidin. In some embodiments, the biotinylated capture agent, such as the pKal capture agent, such as a kallikrein inhibitor, such as a polypeptide with sequence similarity to DX-88, such as a polypeptide with 1, 2 or less than 5 amino acid residues different from DX-88, such as Epikal-2, is placed on the surface of the vessel coated with avidin.

[0035] The present disclosure provides biomarkers capable of identifying patients with plasma kallikrein-mediated angioedema (KMA) or other diseases mediated by pKal, which are useful for assessment and treatment.

[0036] Patients shown to exhibit pKal activation via biomarkers are candidates for treatment with pKal inhibitors, such as DX-88, a small molecule protein inhibitor of pKal approved for the treatment of acute edema attacks associated with HAE. Other pKal inhibitors include DX-2930, a fully human antibody inhibitor. In some embodiments, patients shown to exhibit pKal activation via biomarkers are candidates for treatment with bradykinin B2 receptor antagonists, such as incatibant (Firazyr®). In some embodiments, patients shown to exhibit pKal activation via biomarkers are candidates for treatment with C1-INH supplements, such as purified human pasteurized nanofiltered C1-INH concentrate (Berinert®).

[0037] The embodiments of the present disclosure provide biomarkers and their use in identifying and treating patients, for example, patients suffering from edema caused by bradykinin produced by plasma kallikrein. The methods, compositions and devices disclosed herein are useful in many ways. For example, the concentration of pKal markers can be used to identify disorders associated with increased contact system activation. Initial screening can be followed by in vitro or in vivo testing, for example in preclinical models of disease, with plasma kallikrein inhibitors (e.g., DX-88, EPIKAL-2 or DX-2930). The markers disclosed herein can also be used as pharmacodynamic biomarkers or to otherwise monitor the subject's response to kallikrein inhibitors. The markers disclosed herein can be used in companion diagnostics to enable treatment of diseases mediated by plasma kallikrein, to manage dosage during prophylactic treatment of HAE, or to identify impending acute HAE attacks.

[0038] Another exemplary embodiment includes a microplate-based pKal activity assay, which includes: (a) providing a plasma sample from a subject; (b) incubating the plasma sample with an activator of the pKal system in the presence of a pKal substrate that is linked to a label that can emit a detectable signal after cleavage by pKal; and (c) measuring the activity of pKal based on the concentration of the detectable signal. The activator can be factor XIIa. In some examples, the plasma sample can be incubated with the activator, the pKal substrate, and a candidate pKal inhibitor. In some examples, the method further includes evaluating the activity of the candidate pKal inhibitor, and a reduction in the level of pKal activity in the presence of the candidate pKal inhibitor compared to the level of pKal activity in the absence of the candidate pKal inhibitor indicates that the candidate inhibitor is effective.

[0039] The details of one or more embodiments of the disclosure are set forth in the description below. Other features and advantages of the disclosure will become apparent from the following drawings and detailed description of certain embodiments, as well as the appended claims. [Brief description of the drawings]

[0040] [Figure 1] Depiction of components involved in contact system activation of plasma kallikrein. Truncated HMWK can be determined by Western blot. α2M-pKal and C1INH-pKal can be determined by immunoassay (MSD platform). [Diagram 2]Detection of cleaved kininogen by Western blot analysis. Samples were analyzed using SDS-PAGE (3-8% Tris-acetate) under reducing conditions, then transferred to a PVDF membrane and immunoblotted. Lane 1: 50 nM intact kininogen, lane 2: 50 nM cleaved kininogen, lane 3: 50 nM low molecular weight kininogen, lane 4: 1:20 sodium citrate-added human plasma (glass blood collection tube), lane 5: 1:20 sodium citrate-added human plasma (plastic blood collection tube) treated with kallikrein, lane 6: 1:20 sodium citrate-added human plasma (plastic blood collection tube), lane 7: 1:20 sodium citrate-added human plasma (plastic blood collection tube) with 20 nM two-chain kininogen. [Diagram 3] Figure 1 shows purification of C1INH-pKal complex. Cation exchange chromatogram (Panel A) shows separation of C1INH, pKal and the complex. Fractions from cation exchange were collected and analyzed by SDS-PAGE (Panel B). [Figure 4] Sandwich ELISA standard curve for detecting C1INH-pKal in human plasma. Mouse anti-pKal (clone 13G11) was solid-phase and goat anti-C1 inhibitor was used for detection. [Diagram 5] Sandwich ELISA standard curve for detecting C1INH-pKal in human plasma. Goat anti-C1-INH was solid-phased and mouse anti-pKal (clone 13G11) was used for detection. [Figure 6] Figure 1 shows purification of the α2M-pKal complex. The complex was purified by size-exclusion chromatography (Panel A) and fractions were analyzed by SDS-PAGE (Panel B). [Figure 7] Quantification of α2M-pKal complex. Plasma kallikrein activity was shown to decrease to a plateau level upon addition of excess α2M ​​(Panel A). A standard curve was generated using a 10-fold excess of α2M over pKal, and the molar concentration of our purified α2M-pKal complex was determined according to the concentration of pKal in the complex. [Figure 8] Sandwich ELISA standard curve for detecting α2M-pKal in human plasma. Anti-α2M was solid-phased and mouse anti-pKal (clone 13G11) was used for detection. [Figure 9] Sandwich ELISA standard curve for detecting α2M-pKal in human plasma. Mouse anti-pKal (clone 13G11) was immobilized and anti-α2M was used for detection. [Figure 10] 1 shows the detection of complexes of C1INH-pKal and α2M-pKal in normal human plasma after activation with dextran sulfate. [Figure 11] Detection of intact kininogen (ie, single chain) in patient samples obtained at the time of stroke.Patient plasma samples were collected in citrated blood collection tubes containing an anti-protease cocktail. [Figure 12] 1 is a schematic representation of a microplate-based pKal activity assay; the experiment depicted shows the inhibitory activity of DX-2930 on pKal activity in treated monkeys. [Figure 13] Experiments in plasma from placebo / vehicle treated monkeys as well as DX-2930 controls spiked into this same plasma are shown. [Figure 14] Shown are HMWK in plasma samples from HAE patients (CM, DG, BB and GR). Samples were taken in anti-protease inhibitor cocktail. MW: marker, C: 1 strand (6.44 μg / ml) + 2 strand (1.54 μg / ml), 1: CM basal, 2: CM attack, 3: DG basal, 4: DG attack, 5: BB basal, 6: BB attack, 7: GR basal, 8: GR attack, and 9: pooled normal plasma. [Figure 15] 1 shows the concentration of single-chain HMWK in samples from HAE patients as determined by Western blot analysis. [Figure 16]Figure 1 shows the inhibitory activity of DX-2930 against pKal activation in cynomolgus monkeys.Multiple doses of DX-2930 were used in Western blot analysis of cynomolgus monkey kininogen from day 4 APTT plasma samples (with or without plasma activation with kaolin or 15 μg / ml dextran sulfate). [Figure 17] 1 illustrates an exemplary HMWK ex vivo activation assay for testing the inhibitory activity of an exemplary pKal inhibitor, Dx-2930, using both Protein Simple Western and conventional Western blot analysis. The HMWK ex vivo activation assay described herein showed that the lowest concentration of DX-2930 inhibited pKal activation, which was superior to endogenous C1 inhibitor alone in normal human plasma. [Figure 18] FIG. 1 shows HMWK ex vivo activation assay showing Protein Simple raw data for Factor XIIa activated plasma with titration of DX2930. [Figure 19] 1 shows detection of human HMW kininogen in plasma after 30 min activation as determined by Western blot analysis. [Figure 20] 1 shows a comparison between Protein Simple Western analysis and conventional Western blot analysis in detecting plasma activation. [Figure 21] 1 shows a comparison between Protein Simple Western analysis and conventional Western blot analysis in detecting plasma activation. [Figure 22] Figure 1 shows the percentage reduction of single-chain HMWK 30 min after Factor XIIa activation in undiluted plasma sample BRH745047. [Figure 23] Figure 1 shows the percentage reduction of single-chain HMWK 30 min after Factor XIIa activation in undiluted plasma samples BRH745048. [Figure 24] Figure 1 shows the percentage reduction of single-chain HMWK 30 min after Factor XIIa activation in undiluted plasma samples BRH745064. [Diagram 25]Figure 1 shows the percentage reduction of single-chain HMWK after 30 min of Factor XIIa activation in undiluted plasma samples BRH745062. [Figure 26] 1 shows the percentage reduction of single-chain HMWK 30 min after Factor XIIa activation in undiluted plasma samples BRH745049, BRH745062 and BRH745063. [Figure 27] 1 is a graph showing the reduction of single-chain HMWK in plasma containing various concentrations of factor XIIa. [Figure 28] 1 shows the concentrations of two-chain HMWK in normal, baseline and HAE attack patients as determined by the endogenously cleaved kininogen assay described herein. [Figure 29] FIG. 1 is a graph showing endogenously cleaved kininogen in HAE samples as determined by Protein Simle Western Blot and conventional Western Blot. [Diagram 30] Concentrations of endogenous truncated kininogen in samples from HAE patients are shown. Lane 1: Purified single and double chain HMWK. Lane 2: Normal human plasma sample collected with citrate. [Diagram 31] 1 is a graph showing the percent inhibition of pKal activity in plasma samples from monkeys treated with DX-2930, and in plasma samples spiked with DX-88 (ecallantide), C1-INH, or DX-2930 in vitro. Percent inhibition was measured using an exemplary ex vivo pKal activation assay. Error bars represent standard error of the mean. [Diagram 32] 1 is a graph showing the ex vivo percent inhibition of pKal activity in plasma from humans and monkeys treated with different doses of DX-2930.Percent inhibition was measured using an exemplary ex vivo pKal activation assay.The dashed line shows the percent inhibition observed for 80nM DX-2930, a concentration that matches the Cmax of the therapeutically effective dose of ecallantide. [Diagram 33]1 is a series of plots showing "% inhibition of pKal activity" in plasma samples from a DX-2930 Phase 1 study comparing the mean inhibition after each day of dosing for each dose group to the corresponding samples in the vehicle control group. Error bars are standard error. [Diagram 34] 1 shows Western blot data from DX-2930 Phase 1a subjects after treatment with Factor XIIa. Error bars were calculated using standard deviation between subjects. Group 1 subjects were administered 0.1 mg / kg, Group 2 subjects were administered 0.3 mg / kg, Group 3 subjects were administered 1 mg / kg, and Group 4 subjects were administered 3 mg / kg. [Diagram 35] Graph of Western blot data from DX-2930 Phase 1a subjects not treated with Factor XIIa. Error bars were calculated using standard deviation between subjects. Group 1 subjects were administered 0.1 mg / kg, Group 2 subjects were administered 0.3 mg / kg, Group 3 subjects were administered 1 mg / kg, and Group 4 subjects were administered 3 mg / kg. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0041] Described herein is an assay for measuring the concentration of biomarkers related to the plasma kallikrein (pKal) system, such as intact high molecular weight kininogen (HMWK), cleaved HMWK, and / or pKal activity, in a sample from a subject. In one embodiment, the assay comprises incubating a sample from a subject with an activator of the pKal system, and measuring the level of intact high molecular weight kininogen (HMWK), cleaved HMWK, and / or pKal activity. Such assays are useful, for example, for assessing whether a subject has or is at risk for a pKal-related disease, for evaluating treatments for pKal-related diseases, and for identifying drug candidates, such as pKal modulators, such as pKal inhibitors. Such assays are also useful for selecting patients for treatment, such as treatments for pKal-related diseases.

[0042] definition For convenience, before further description of the present disclosure, certain terms employed in the specification, examples and appended claims are defined here. Other terms are defined as they appear in the specification.

[0043] The singular forms "a," "an," and "the" include plural references unless the context clearly dictates otherwise.

[0044] As used herein, "obtaining" or "obtaining" refers to gaining possession of a physical entity or value, e.g., a numerical value, by "directly obtaining" or "indirectly obtaining" the physical entity or value. "Direct obtaining" means performing a process (e.g., performing an assay or test on a sample, or "analyzing a sample" as that term is defined herein) to obtain the physical entity or value. "Indirect obtaining" refers to receiving a physical entity or value from another party or source (e.g., a laboratory of a third party party that directly obtained the physical entity or value). Direct obtaining of a physical entity includes performing a process that involves a physical change of a physical substance, e.g., a starting substance, e.g., analyzing a sample. Exemplary changes include creating a physical entity from two or more starting substances, shearing or fragmenting a substance, separating or purifying a substance, mixing two or more separated entities into a mixture, and performing a chemical reaction that involves breaking or forming a covalent or non-covalent bond. Obtaining a value directly includes performing a process involving a physical change of a sample or another substance, e.g., performing an analytical process involving a physical change of a substance, e.g., a sample, analyte, or reagent (sometimes referred to herein as a "physical analysis"), performing an analytical method, e.g., a method involving one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from other substances; mixing an analyte or a fragment or other derivative thereof with another substance, e.g., a buffer, solvent, or reactant; or altering the structure of an analyte or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or altering the structure of a reagent, or a fragment or other derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent.

[0045] As used herein, "analyzing" a sample includes performing a process that involves a physical change to the sample or other material, such as a starting material. Exemplary changes include creating a physical entity from two or more starting materials, shearing or fragmenting a material, separating or purifying a material, combining two or more separate entities into a mixture, and performing a chemical reaction that involves breaking or forming covalent or non-covalent bonds. Analysis of a sample may include performing an analytical process that involves physical change of a substance, e.g., a sample, analyte, or reagent (often referred to herein as "physical analysis"), performing an analytical method, e.g., a method that involves one or more of the following: separating or purifying a substance, e.g., an analyte, or a fragment or other derivative thereof, from other substances; mixing the analyte or a fragment or other derivative thereof with other substances, e.g., a buffer, solvent, or reactant; or altering the structure of the analyte or a fragment or derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the analyte; or altering the structure of a reagent, or a fragment or derivative thereof, e.g., by breaking or forming a covalent or non-covalent bond between a first atom and a second atom of the reagent.

[0046] As used herein, the term "agonist" refers to an agent that mimics or upregulates (e.g., enhances or complements) the biological activity of a protein. An agonist can be a wild-type protein or a derivative thereof that has at least one biological activity of the wild-type protein. An agonist can be a compound that increases at least one biological activity of a protein. An agonist can be a compound that increases the interaction of a polypeptide with another molecule, such as a target peptide or nucleic acid.

[0047] As used herein, the term "antagonist" refers to an agent that downregulates (e.g., suppresses or inhibits) at least one of the biological activities of a protein. An antagonist can be a compound that inhibits or reduces the interaction of a protein with other molecules, such as a target peptide or an enzyme substrate. An antagonist can be a compound that reduces or inhibits the amount of expressed protein present. Typically, inhibiting a protein or gene refers to reducing the expression or associated activity of a protein or gene by at least 10% or more, such as 20%, 30%, 40% or 50%, 60%, 70%, 80%, 90% or more, or reducing the expression or associated activity by 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more, as measured by one or more methods described herein or recognized in the art.

[0048] As used herein, "binding affinity" refers to the apparent association constant or K a Refers to. a is the dissociation constant (K d ) is the reciprocal of the binding affinity of the binding protein to a particular target molecule. 5 M -1 , 10 6 M -1 , 10 7 M -1 , 10 8 M -1 , 10 9 M -1 , 10 10 M -1 and 10 11 M -1 A higher affinity binding of a binding protein to a first target compared to a second target can have a binding affinity of K a is the K for binding to the second target a (or the number K d ) higher (or lower) than K dIn such cases, the binding protein has specificity for a first target (e.g., a protein or a mimetic thereof in a first conformation) compared to a second target (e.g., the same protein or a mimetic thereof in a second conformation, or a second protein). The difference in binding affinity (e.g., specificity or other comparison) may be at least 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 15-fold, 20-fold, 37.5-fold, 50-fold, 70-fold, 80-fold, 91-fold, 100-fold, 500-fold, 1000-fold, or 10 5 It could be double.

[0049] Binding affinity can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for evaluating binding affinity are in TRIS buffer (50 mM TRIS, 150 mM NaCl, 5 mM CaCl2, pH 7.5). Using these techniques, the concentrations of bound and free binding protein can be measured as a function of the binding protein (or target) concentration. The concentration of bound binding protein ([bound]) is calculated by combining the concentration of free binding protein ([free]) and the concentration of binding sites for the binding protein on the target with the following formula: [bound] = N [free] / ((1 / Ka) + [free]) where (N) is the number of binding sites per target molecule.

[0050] However, K a It is not always necessary to precisely determine K, because sometimes it is determined using methods such as ELISA or FACS analysis, and K a and thus it is sufficient to obtain a quantitative measure of affinity that can be used for comparison, such as determining whether a higher affinity is, for example, two-fold higher, to obtain a qualitative measure of affinity, or to obtain an estimate of affinity by activity in a functional assay, for example an in vitro or in vivo assay.

[0051] The term "binding protein" refers to a protein that can interact with a target molecule. This term is used interchangeably with "ligand." "Plasma kallikrein binding protein" refers to a protein that can interact with (e.g., bind to) plasma kallikrein, and includes in particular proteins that selectively or specifically interact with and / or inhibit plasma kallikrein. A protein inhibits plasma kallikrein if it causes a reduction in the activity of plasma kallikrein compared to the activity of plasma kallikrein under the same conditions in the absence of the protein. In some embodiments, the plasma kallikrein binding protein is an antibody.

[0052] The term "capture agent" refers to a moiety that specifically binds to its ligand. As used herein, the term "complex" or "complex formation" refers to a complex between elements that have specific affinity for each other.

[0053] A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues with similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).

[0054] The motif sequence of a biopolymer may include positions that may be variant amino acids. For example, the symbol "X" in such a context refers to any amino acid (e.g., any of the 20 natural amino acids) unless otherwise specified, e.g., any non-cysteine ​​amino acid. Other permissible amino acids may also be indicated, e.g., using brackets and slashes. For example, "A / W / F / N / Q" means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are permissible at that particular position.

[0055] As used herein, a "detection reagent" refers to a moiety that binds to a moiety to be detected. Typically, the detection reagent generates a signal, such as fluorescence, or produces a measurable compound.

[0056] "Epitope" refers to a site on a target compound to which a binding protein (e.g., an antibody such as a Fab or full length antibody) binds. When the target compound is a protein, the site can be composed entirely of amino acid building blocks, or the site can be composed entirely of chemical modifications of the amino acids of the protein (e.g., glycosyl moieties), or a combination thereof. Overlapping epitopes contain at least one common amino acid residue, glycosyl group, phosphate group, sulfate group, or other molecular structure.

[0057] A first binding protein (e.g., an antibody) "binds to the same epitope" as a second binding protein (e.g., an antibody) if the first binding protein binds to the same site on the target compound as the second binding protein or if the first binding protein binds to a site that overlaps with the site to which the second binding protein binds (e.g., overlaps by 50%, 60%, 70%, 80%, 90% or 100% in terms of amino acid sequence or other molecular features such as glycosyl groups, phosphate groups or sulfate groups).

[0058] A first binding protein (e.g., an antibody) "competes for binding" with a second binding protein (e.g., an antibody) if binding of the first binding protein to its epitope reduces (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more) the amount of the second binding protein that binds to its epitope. Competition can be direct (e.g., the first binding protein binds to the same epitope as the second binding protein or to an epitope that overlaps with the epitope bound by the second binding protein) or indirect (e.g., binding of the first binding protein to its epitope causes a conformational change in the target compound, thereby reducing the ability of the second binding protein to bind to its epitope).

[0059] As used herein, a "functional" biological molecule is one that is in a form in which it exhibits a property and / or activity by which it is characterized.

[0060] Calculation of "homology" or "sequence identity" between two sequences (these terms are used interchangeably herein) is performed as follows: The sequences are aligned for optimal comparison purposes (e.g., for optimal alignment, gaps can be introduced into one or both of the first and second amino acid or nucleic acid sequences, and non-homologous sequences can be ignored for comparison purposes). Optimal alignment is determined as the best score using the GAP program in the GCG software package with a Blossum62 scoring matrix with a gap penalty of 12, a gap extension penalty of 4, and a frameshift gap penalty of 5. The amino acid residues or nucleotides at corresponding amino acid or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, the molecules are identical at that position (as used herein, amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between two sequences is a function of the number of identical positions shared by the sequences.

[0061] In preferred embodiments, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 92%, 95%, 97%, 98% or 100% of the length of the reference sequence. For example, the reference sequence may be the length of an immunoglobulin variable domain sequence.

[0062] As used herein, the term "hybridize under low stringency, medium stringency, high stringency or very high stringency conditions" describes conditions for hybridization and washing. Guidance for performing hybridization reactions can be found in Current Protocols in Molecular Biology, John Wiley & Sons, NY (1989), 6.3.1-6.3.6. Aqueous and non-aqueous methods are described in that reference and either can be used. Specific hybridization conditions referred to herein are: (1) low stringency hybridization conditions in 6× sodium chloride / sodium citrate (SSC) at about 45° C., followed by two washes in 0.2× SSC, 0.1% SDS at at least 50° C. (under low stringency conditions, the wash temperature can be increased to 55° C.); (2) low stringency hybridization conditions in 6× SSC at about 45° C., followed by one or more washes in 0.2× SSC, 0.1% SDS at 60° C. (3) high stringency hybridization conditions in 6×SSC at about 45° C. followed by one or more washes in 0.2×SSC, 0.1% SDS at 65° C., and (4) very stringency hybridization conditions in 0.5 M sodium phosphate, 7% SDS at 65° C. followed by one or more washes in 0.2×SSC, 1% SDS at 65° C. Very stringency conditions (4) are the preferred conditions and should be used unless otherwise specified. The present disclosure includes nucleic acids that hybridize to the nucleic acids described herein or their complements at low, medium, high or very high stringency, such as nucleic acids encoding the binding proteins described herein. The nucleic acids may be the same length as the reference nucleic acid or may be within 30%, 20% or 10% of its length. The nucleic acids may correspond to regions encoding immunoglobulin variable domain sequences described herein.

[0063] An "isolated composition" refers to a composition in which at least 90% of at least one component of the natural sample from which the isolated composition is obtained has been removed. An artificially or naturally produced composition can be a "composition" that is "at least" a particular degree of pure if the species or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure by weight.

[0064] As used herein, the term "in vitro" refers to events that take place in an artificial environment, such as in a test tube or reaction vessel, such as in cell culture, but not within a multicellular organism.

[0065] As used herein, the term "in vivo" refers to events that take place within a multicellular organism, such as a human or non-human animal.

[0066] An "isolated composition" refers to a composition in which at least 90% of at least one component of the natural sample from which the isolated composition is obtained has been removed. An artificially or naturally produced composition can be a "composition" that is "at least" a particular degree of pure if the species or population of species of interest is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure by weight.

[0067] An "isolated" protein refers to a protein that has been removed from at least 90% of at least one component of the natural sample from which it is obtained. A protein can be "at least" pure to a particular degree if the subject species or population of species is at least 5, 10, 25, 50, 75, 80, 90, 92, 95, 98, or 99% pure by weight.

[0068] The term "kallikrein" (e.g., plasma kallikrein) refers to a subgroup of the serine protease family, peptidases (enzymes that cleave peptide bonds in proteins). Plasma kallikrein cleaves kininogen to produce kinins, which are potent proinflammatory peptides.

[0069] The term "kallikrein inhibitor" refers to any agent or molecule that inhibits kallikrein. For example, DX-88 (also referred to herein as "PEP-1") is a potent (Ki<1 nM) and specific inhibitor of plasma kallikrein (NP_000883) (see, for example, WO95 / 21601 or WO2003 / 103475).

[0070] As used herein, the term "DX-2922" is used interchangeably with the term "X101-A01." Other variants of this antibody are described below. [Table 1]

[0071] As used herein, the term "DX-2930" is used interchangeably with the term "X124-G01." Other variants of this antibody are described below. [Table 2]

[0072] The term "modulator" refers to a polypeptide, nucleic acid, macromolecule, complex, molecule, small molecule, compound, species, etc. (natural or non-natural) that may be capable of modulating, or an extract made from a biological material such as a cell or tissue of bacteria, plant, yeast, or animal. Modulators can be included in an assay to evaluate their potential activity as (direct or indirect) inhibitors or activators (e.g., agonists, partial antagonists, partial agonists, inverse agonists, antagonists, antibacterial agents, inhibitors of bacterial infection or proliferation, etc.) of functional properties, biological activities or processes, or combinations thereof. In such an assay, many modulators can be screened at once. The activity of a modulator can be known, unknown, or partially known.

[0073] A "non-essential" amino acid residue is one that can be altered from the wild-type sequence of a binding agent, e.g., an antibody, without eliminating, or more preferably, without substantially altering, the biological activity, whereas an "essential" amino acid residue results in a significant loss of activity.

[0074] A "patient," "subject," or "host" (these terms are used interchangeably) treated by the method can mean either a human or a non-human animal. In certain embodiments, the subject is at risk for or suffers from a kallikrein-mediated disorder, such as a bradykinin-mediated disorder, such as hereditary angioedema (HAE). In certain embodiments, the subject is diagnosed with a condition, such as non-histamine dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burn injury, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, ventricular assist device or stent associated thrombosis, heparin-induced thrombocytopenia with thrombosis, thromboembolism, coronary heart disease with unstable angina, edema, eye disease, gout, bowel disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis degenerative spinal disease, post-operative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, At risk for or suffering from thrombosis, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive and diabetic nephropathy, allergies and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis) and tissue injury (e.g., burn or chemical injury).

[0075] The terms "prekallikrein" and "pre-plasma kallikrein" are used interchangeably herein and refer to the zymogen form of active plasma kallikrein, also known as prekallikrein.

[0076] The term "prevention" of or "preventing" a disease in a subject refers to administering a medical treatment, such as, for example, administration of a drug, to a subject such that at least one symptom of the disease is prevented, i.e., prior to the manifestation of clinical symptoms of an undesirable condition (e.g., a disease or other undesirable condition in a host animal) to prevent the host from developing the undesirable condition. "Preventing" a disease can also be referred to as "prophylaxis" or "prophylactic treatment."

[0077] As used herein, the term "substantially identical" (or "substantially homologous") is used herein to refer to a first amino acid or nucleic acid sequence that contains a sufficient number of identical or equivalent amino acid residues or nucleic acids (e.g., having similar side chains, e.g., conservative amino acid substitutions) as a second amino acid or nucleic acid sequence such that the first and second amino acid or nucleic acid sequences have (or encode proteins that have) a similar activity, e.g., binding activity, binding selectivity or biological activity. In the case of antibodies, the second antibody has the same specificity for the same antigen and at least 50%, at least 25%, or at least 10% of the affinity for the same antigen.

[0078] Sequences similar or homologous to the sequences disclosed herein are also part of this application. In some embodiments, the sequence identity may be about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more.

[0079] Furthermore, substantial identity exists when the nucleic acid segment will bind to a complementary strand under selective hybridization conditions (e.g., highly stringent hybridization conditions). The nucleic acids may be present in whole cells, in a cell lysate, or in a partially purified or substantially pure form.

[0080] The motif sequence of a biopolymer may include positions that may be variant amino acids. For example, the symbol "X" in such a context refers to any amino acid (e.g., any of the 20 natural amino acids) unless otherwise specified, e.g., any non-cysteine ​​amino acid. Other permissible amino acids may also be indicated, e.g., using brackets and slashes. For example, "A / W / F / N / Q" means that alanine, tryptophan, phenylalanine, asparagine, and glutamine are permissible at that particular position.

[0081] Statistical significance can be measured by any method known in the art. Exemplary statistical tests include Student's t-test, Mann-Whitney U nonparametric test, and Wilcoxon nonparametric test. A statistically significant relationship has a P value of less than 0.05 or 0.02. The terms "induce," "inhibit," "enhance," "elevate," "increase," "decrease," and the like refer to a distinct qualitative or quantitative difference, for example, between two states, and can mean that there is a difference between the two states, for example, that there is a statistically significant difference.

[0082] As used herein, a "sample" refers to a composition comprising tissue, e.g., blood, plasma, or protein, from a subject. Samples include both the original unprocessed sample taken from a subject and those that have been subsequently processed, e.g., in partially purified or preserved forms. Exemplary samples include blood, plasma, tears, or mucus.

[0083] A "therapeutically effective amount" preferably modulates a measurable parameter, such as plasma kallikrein activity, to a statistically significant extent or at least about 20%, more preferably at least about 40%, even more preferably at least about 60%, and even more preferably at least about 80%, compared to untreated subjects. The ability of a compound to modulate a measurable parameter, such as a disease-related parameter, can be evaluated in an animal model system that is predictive of efficacy in human disorders and conditions. Alternatively, this property of a composition can be evaluated by testing the ability of the compound to modulate an in vitro parameter.

[0084] "Treatment" of a disease (or condition) in a subject or "treatment" of a subject with a disease refers to administering medical therapy to the subject, such as administering a medication, such that at least one symptom of the disease is cured, alleviated or reduced.

[0085] The term "preventing" a disease in a subject refers to administering a medical treatment, such as administering a drug, to a subject such that at least one symptom of the disease is prevented, i.e., prior to the onset of clinical symptoms of an undesirable condition (e.g., a disease or other undesirable condition in a host animal) to prevent the host from becoming undesirable. "Preventing" a disease is also referred to as "prophylaxis" or "prophylactic treatment."

[0086] A "prophylactically effective amount" refers to an amount effective, at a dosage, and for a period of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be lower than the therapeutically effective amount.

[0087] Headings, including alphabetical or numeric headings, are for ease of understanding and reading only and do not impose any chronological order or hierarchy of preference unless otherwise indicated.

[0088] Contact biomarkers Plasma kallikrein circulates as an inactive enzyme precursor called prekallikrein, which is mostly bound to its substrate, high molecular weight kininogen (HMWK). In response to a stimulus, factor XII is activated to factor XIIa. Factor XIIa cleaves prekallikrein to form active plasma kallikrein (Figure 1). Approximately 75-90% of circulating prekallikrein binds to HMWK through a non-active site interaction with domain 6 of HMWK. Free and active pKal bound to HMWK produces cleaved HMWK and bradykinin. Biomarkers of plasma kallikrein activation are shown in Table 2. The suitability of the biomarkers can be demonstrated by tracking their concentrations in the presence and absence of acute attacks of HAE. The concentrations of these biomarkers may also vary during attacks of bradykinin-mediated edema or other diseases mediated by pKal activity.

[0089] Table 2 below provides markers that can be evaluated by the methods described in Table 2 and elsewhere herein to evaluate a subject for pKal or bradykinin-mediated disorders. Table 2 shows the direction of change in concentration of markers associated with pKal or bradykinin-mediated disorders.

[0090] Biomarker assays The concentration (e.g., amount) of the biomarkers disclosed herein, or changes in the concentration of the biomarkers disclosed herein, can be evaluated using the assays described herein and / or known in the art. Assays that can be used to evaluate the concentration of biomarkers include, for example, immunoassays, such as Western blots, enzyme-linked immunosorbent assays (ELISAs) (e.g., sandwich ELISAs), radioimmunoassays, electrochemiluminescence-based detection assays and related techniques. Mass spectrometry-based approaches can also be used. Assays that rely on chromogenic substrates can also be used.

[0091] In certain embodiments, electrochemiluminescence detection assays or assays that rely on a combination of electrochemiluminescence and patterned array technology (eg, ECL or MULTI-ARRAY technology assays from Meso Scale Discovery (MSD)) are used.

[0092] (i) Prekallikrein The concentration of prekallikrein can be assessed using existing assays for prekallikrein, such as immunoassays, such as prekallikrein ELISA. For example, a prekallikrein ELISA kit is commercially available from antibodiesonline.com (catalog number ABIN858073). Antibodies that bind to prekallikrein are known in the art and can be used to detect prekallikrein, for example, in immunoassays. For example, mouse monoclonal anti-human prekallikrein antibodies have been generated. See, for example, Veloso, D. et al., Blood, 1987, 70(4):1053-1062. Sheep anti-human prekallikrein antibodies are commercially available from GenWay Biotech, Inc. (GenWayID: GWB-58AC79).

[0093] In one embodiment, the concentration of pKal in the sample aliquot is determined.Then, all prekallikrein in the sample aliquot is converted to pKal to determine the concentration of pKal.The first concentration is subtracted from the second concentration to obtain the amount of prekallikrein.The concentration can be determined by enzyme activity.

[0094] (ii) Active plasma kallikrein Active plasma kallikrein (activated pKal) can be detected, for example, using an immunoassay. The immunoassay can use an antibody that binds only to active plasma kallikrein, for example, DX-2930 or DX-2922. In one embodiment, the assay relies on the binding of active pKal to a capture agent, for example, a kallikrein inhibitor, for example, a polypeptide that is similar in sequence to DX-88, for example, a polypeptide that differs from DX-88 by one, two or less than five amino acid residues, for example, EPIKAL-2.

[0095] In some embodiments, the capture reagent is provided on a substrate, contacted with the sample, and the amount of the capture reagent bound is determined, for example, by anti-pKal antibody.In some embodiments, to reduce the level of contact activation, sample manipulation, such as, for example, transfer from one container to another, is minimized.In some embodiments, the capture reagent is placed in the same device, for example, collection container, for example, blood collection tube, that is used to collect sample, for example, blood, from a subject.

[0096] In the present disclosure, an ex vivo activation assay (e.g., microplate-based pKal activity assay) for determining pKal activity is also provided, which includes (i) providing a plasma sample from a subject; (ii) incubating the plasma sample with a pKal-based activator in the presence of a pKal substrate that is linked to a label that can emit a detectable signal after cleavage by pKal; and (iii) measuring the activity of pKal based on the concentration of detectable signal.In some examples, the activator is factor XIIa.In some examples, the plasma sample is incubated with the activator, the pKal substrate and a candidate pKal inhibitor.In some examples, the method further includes evaluating the activity of the candidate pKal inhibitor, and if the level of pKal activity in the presence of the candidate pKal inhibitor is reduced compared to the level of pKal activity in the absence of the candidate pKal inhibitor, the candidate inhibitor is effective.

[0097] [Table 3] TIFF2024150637000005.tif212162

[0098] The method may further include assessing whether the subject has or is at risk of having a disease associated with pKal, where an elevated level of pKal activity compared to a predetermined value (e.g., the level of pKal activity in a sample from a healthy subject or a population of healthy subjects) indicates that the subject has or is at risk of having the disease.

[0099] The method may further include evaluating the effectiveness of the treatment, such as the treatment of a disease associated with pKal. A number of biological samples (e.g., plasma samples) can be obtained from a patient having a disease associated with pKal, such as HAE, and being treated with a therapeutic agent, such as a pKal inhibitor, before, during, and / or after the treatment. The level of pKal activity or a biomarker indicative of pKal activity in the biological samples can be measured using any of the assay methods disclosed herein. A reduction in the level of pKal activity compared to before treatment or a reduction in the level of one or more biomarkers indicative of pKal activity, or a reduction in the level of pKal activity / biomarkers over the course of treatment indicates that the treatment is effective.

[0100] (iii)α2M-pKal complex Plasma kallikrein α2 macroglobulin complex (α2M-pKal complex) can be detected, for example, using immunoassays. For example, a sandwich-based ELISA assay has been developed as described in Example 2. A quantitative sandwich ELISA is also reported in Kaufman, N. et al., Blood, 1991, 77(12):2660-2667 and Wachtfogel, YT et al., 1989, Blood, 73:468-471. An immunofixed enzyme assay is also reported in Harpel, PC et al., J Biol Chem, 1985, 260(7):4257-4263. A chromogenic substrate assay is also available, for example, using the chromogenic substrate S-2302, available at chromogenicsubstrates.com, whose protocol is provided at chromogenicsubstrates.com / methods / chromogenic_substrates_methods_kallikrein-like.htm.

[0101] (iv)C1INH-pKal complex C1INH-pKal complexes can be detected, for example, using immunoassays, such as ELISA methods, for example, as described in Example 3. For example, a sandwich ELISA can be used in which an anti-pKal antibody (e.g., mouse mAb 13G11) is used as the capture antibody and an antibody against C1INH is used as the detection antibody, or a sandwich ELISA in which an antibody against C1INH is used as the capture antibody and an anti-pKal antibody (e.g., mouse mAb 13G11) is used as the detection antibody. Antibodies against C1INH are known in the art. For example, a mouse monoclonal antibody against human C1 inhibitor is available from ABBIOTEC (catalog number 250122). Another mouse monoclonal antibody against human C1 inhibitor (4G12) is available from pierce-antibodies.com (product number LF-MA0136). A goat anti-human C1 inhibitor antibody is available from Quidel (catalog number A300). Another ELISA sandwich assay for detection of C1INH-pKal complexes was used in Wachtfogel, YT et al., 1989, Blood, 73:468-471.

[0102] (v) Intact HMWK Intact high molecular weight kininogen (HMWK) can be assayed using immunological methods such as, for example, agglutination or radioimmunoassay (see, for example, Kerbiriou-Nabias, DM, Br J Haematol, 1984, 56(2):2734-2786). Monoclonal antibodies against human HMWK light chains are known (see, for example, Reddigari, SR and Kaplan, AP, Blood, 1999, 74:695-702). Chromogenic substrate-based assays for HMWK can also be used (see, for example, Scott, CF et al., Thromb Res, 1987, 48(6):685-700; Gallimore, MJ et al., Thromb Res, 2004, 114(2):91-96).

[0103] The human gene encoding HMWK is kininogen 1 (KNG1). KNG1 is transcribed and alternatively spliced ​​to form mRNAs encoding either HMWK or low molecular weight kininogen (LMWK). Exemplary protein sequences for HMWK are provided below: >gi|156231037|ref|NP_001095886.1| Kininogen-1 isoform 1 precursor [Homo sapiens] (Sequence ID No. 5)

[0104] (vi) Cutting type HMWK Truncated high molecular weight kininogen (HMWK), also referred to herein as "truncated kininogen", can be assayed using methods such as those described in Example 1, such as Western blot. An antibody that binds to truncated HMWK can be used, such as mouse mAb clone 11H05. Additionally, truncated HMWK can be assessed using mass spectrometry. Immunoblot techniques for assessing the concentration of truncated HMWK are known in the art (see, for example, Buhler R. et al., Blood Coagul Fibrinolysis, 1995, 6(3):223-232).

[0105] Exemplary sequences for the heavy and light chains of truncated kininogen are provided below. > Truncated kininogen-1 heavy chain QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITY SIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISLMK (Sequence identification number 6) > Truncated kininogen-1 light chain SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS (Sequence Identification Number 7)

[0106] In some embodiments, the present disclosure provides an ex vivo activation method. Such a method includes (i) incubating a plasma sample obtained from a subject with an activator of the plasma kallikrein (pKal) system (e.g., factor XIIa); (ii) measuring the concentration of intact high molecular weight kininogen (HMWK), cleaved HMWK, or both in the plasma sample before and after incubation; and (iii) determining the decrease in intact HMWK in the sample after activation. In some examples, the concentration of intact HMWK and cleaved HMWK is measured by Western blot analysis, for example, Simple Western™ Protein Simple™ Western blot analysis. Simple Western™ assays are known in the art (see, for example, Rustandi et al., Electrophoresis, 2012, September;33(17):2790-2797). Simple Western™ products are also commercially available (see, for example, ProteinSimple™, Santa Clara, Calif.).

[0107] The ex vivo activation method described herein can be used to evaluate the activity of pKal inhibitor candidates in inhibiting plasma activation.More specifically, plasma samples can be incubated with activator in the presence of pKal inhibitor candidates.If activity level is reduced in the presence of inhibitor candidates, it indicates that the candidate is effective in inhibiting plasma activation.

[0108] In another embodiment, the present disclosure provides a method for measuring endogenous truncated HMWK.Such a method can include (i) providing a plasma sample from a subject, and (ii) measuring the concentration of truncated HMWK in the plasma sample.In some instances, the concentration of truncated HMWK is measured by Protein Simple Western blot analysis.

[0109] Endogenous truncated HMWK assay can be applied to identify subjects who have or suspect that they have diseases related to pKal system, such as the diseases described herein.In some examples, plasma samples are obtained from subjects who have or suspect that they have diseases related to pKal system.If the endogenous truncated HMWK in a subject is elevated compared to the endogenous truncated HMWK in a healthy subject, it indicates that the subject has or suspect that they have the disease.

[0110] Alternatively or additionally, endogenous cleaved HMWK assay can be used to evaluate the effectiveness of treating diseases related to the pKal system. In this case, plasma samples are obtained from subjects with the disease and treated with pKal inhibitors. If the concentration of cleaved HMWK is reduced after treatment compared to before treatment, it indicates that the pKal inhibitor is effective.

[0111] Alternatively or additionally, the assay can also be used to assess whether a subject has or is at risk of having a pKal-related disease, where an elevated concentration of truncated HMWK compared to a predetermined value (e.g., the concentration of truncated HMWK in a sample from a healthy subject or a population of healthy subjects) indicates that the subject has or is at risk of having the disease.

[0112] The method may also include evaluating the effectiveness of a treatment, such as treating a disease associated with pKal. A number of biological samples (e.g., plasma samples) can be obtained from a patient with a disease associated with pKal, such as HAE, and treated with a therapeutic agent, such as a pKal inhibitor, before, during, and / or after treatment. The concentration of intact HMWK and / or truncated HMWK in the biological samples can be measured using any of the methods disclosed herein. A reduction in the concentration of truncated HMWK compared to before treatment or a reduction in the concentration of truncated HMWK over the course of treatment indicates that the treatment is effective.

[0113] antibody Antibody and antigen-binding fragment can be used in the provided method.In some embodiments, the capture agent is or comprises an antibody or antigen-binding fragment.In some embodiments, the detection agent is or comprises an antibody or antigen-binding fragment.In some embodiments, the therapeutic composition for treating pKal-mediated or bradykinin-mediated disorder is or comprises an antibody or antigen-binding fragment.

[0114] As used herein, the term "antibody" refers to a protein that contains at least one immunoglobulin variable domain or immunoglobulin variable domain sequence. For example, an antibody can contain a heavy (H) chain variable region (abbreviated herein as VH) and a light (L) chain variable region (abbreviated herein as VL). In other examples, an antibody contains two heavy (H) chain variable regions and two light (L) chain variable regions. The term "antibody" encompasses 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), 629-639)) as well as complete antibodies. Antibodies can have structural characteristics of IgA, IgG, IgE, IgD, IgM (and their subtypes). Antibodies may be from any source, but primate (human and non-human primates) and primatized are preferred.

[0115] The VH and VL regions can be further subdivided into hypervariable regions called "complementarity determining regions ("CDRs")" interspersed with more conserved regions called framework regions ("FRs"). The extent of the framework regions and CDRs has been precisely defined (see Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242 and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, also see www.hgmp.mrc.ac.uk). The Kabat definition is used herein. Each VH and Vl typically consists of three CDRs and four FRs, arranged from amino terminus to carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0116] The VH or VL chain of the antibody may comprise all or a portion of the heavy or light chain constant region, respectively, thus forming an immunoglobulin heavy or light chain, respectively. In one embodiment, the antibody is a tetramer of two immunoglobulin heavy chains and two immunoglobulin light chains, where the immunoglobulin heavy and light chains are interconnected, for example, by disulfide bonds. In IgG, the heavy chain constant region comprises three immunoglobulin domains, CH1, CH2 and CH3. The light chain constant region comprises a CL domain. The variable regions of the heavy and light chains comprise binding domains that interact with antigens. The constant region of the antibody typically mediates the binding of the antibody to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The light chain of the immunoglobulin may be of type kappa or lambda. In one embodiment, the antibody is glycosylated. The antibody may be functional for antibody-dependent cellular cytotoxicity and / or complement-dependent cytotoxicity.

[0117] One or more regions of the antibody can be human or effectively human. For example, one or more of the variable regions can be human or effectively human. For example, one or more of the CDRs can be human, e.g., HC CDR1, HC CDR2, HC CDR3, LC CDR1, LC CDR2 and LC CDR3. Each of the light chain CDRs can be human. The HC CDR3 can be human. One or more of the framework regions can be human, e.g., HC or LC FR1, FR2, FR3 and FR4. For example, the Fc region can be human. In one embodiment, all of the framework regions are human, e.g., with the framework sequences of an antibody produced by a human somatic cell, e.g., a hematopoietic cell that produces immunoglobulins or a non-hematopoietic cell. In one embodiment, the human sequences are germline sequences, e.g., encoded by a germline nucleic acid. In one embodiment, framework (FR) residues of a selected Fab can be converted to the amino acid type of the corresponding residues in the most similar primate germline gene, particularly a human germline gene. One or more of the constant regions can be human or effectively human. For example, at least 70%, 75%, 80%, 85%, 90%, 92%, 95%, 98% or 100% of the immunoglobulin variable domain, constant region, constant domain (CH1, CH2, CH3, CL1) or entire antibody can be human or effectively human.

[0118] All or part of an antibody may be encoded by an immunoglobulin gene or a fragment thereof. Exemplary human immunoglobulin genes include the kappa, lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta, epsilon and mu constant region genes, as well as many immunoglobulin variable region genes. A full-length immunoglobulin "light chain" (about 25 kDa or about 214 amino acids) is encoded by a variable region gene (about 110 amino acids) at the NH2-terminus and a kappa or lambda constant region gene at the COOH-terminus. A full-length immunoglobulin "heavy chain" (about 50 kDa or about 446 amino acids) is similarly encoded by a variable region gene (about 116 amino acids) and one of the other constant region genes mentioned above, such as gamma (which encodes about 330 amino acids). The length of the human HC varies greatly, with the HC CDR3 varying from about 3 amino acid residues to more than 35 amino acid residues.

[0119] The term "antigen-binding fragment" of a full-length antibody refers to one or more fragments of a full-length antibody that retains the ability to specifically bind to a target of interest. Examples of binding fragments encompassed by the term "antigen-binding fragment" of a full-length antibody include (i) a Fab fragment, which is a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by disulfide bridges in the hinge region; (iii) an Fd fragment consisting of VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of the VH domain (Ward et al. (1989) Nature 341:544-546), and (vi) an isolated complementarity-determining region (CDR) that retains functionality. Furthermore, although the two domains of the Fv fragment, the VL and VH, are encoded by separate genes, they can be recombinantly produced as a single protein chain joined by a synthetic linker such that the VL and VH regions pair to form a monovalent molecule known as a single-chain Fv (scFv) (see, e.g., U.S. Pat. Nos. 5,260,203, 4,946,778 and 4,881,175; Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883).

[0120] Antibody fragments can be obtained using any suitable technique, including conventional techniques known to those skilled in the art. The term "monospecific antibody" refers to an antibody that exhibits a single binding specificity and binding affinity for a particular target, such as an epitope. This term includes "monoclonal antibody" or "monoclonal antibody composition," which are used herein to refer to preparations of antibodies or fragments thereof of single molecular composition, regardless of how the antibody is produced.

[0121] As used herein, a "humanized" immunoglobulin variable region is an immunoglobulin variable region that has been modified to contain a sufficient number of human framework amino acid positions such that the immunoglobulin variable region does not provoke an immunogenic response in normal humans. Descriptions of "humanized" immunoglobulins include, for example, U.S. Patent No. 6,407,213 and U.S. Patent No. 5,693,762.

[0122] The inhibition constant (Ki) provides a measure of inhibitory potency; it is the concentration of inhibitor required to reduce enzyme activity by half and is independent of the enzyme or substrate concentration. The apparent Ki (K i,app ) is obtained at different substrate concentrations by measuring the inhibitory effect of different concentrations of an inhibitor (e.g., an inhibitory binding protein) on the extent of a reaction (e.g., enzyme activity), and an estimate of the apparent Ki value is obtained by fitting the change in the pseudo-first-order rate constant as a function of inhibitor concentration to the Morrison equation (Equation 1). Ki is expressed as K i,app It is obtained from the Y-intercept derived from a linear regression analysis of a plot of versus substrate concentration.

number

[0123] Hereditary Angioedema (HAE) In some embodiments, the disease or condition involving plasma kallikrein activity is hereditary angioedema (HAE).Hereditary angioedema (HAE) is also known as "Quincke's edema", C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE).HAE is characterized by recurrent episodes of severe swelling (angioedema), which can affect, for example, the limbs, face, genitals, digestive tract, and airway.Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; airway obstruction, which may be accompanied by throat swelling or sudden hoarseness; repeated episodes of abdominal cramps without obvious cause; and / or intestinal swelling, which may be severe and lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock.Approximately one-third of people with HAE develop a non-itchy rash during attacks, called erythema marginatum.

[0124] Airway swelling can be life threatening and cause death in some patients. Mortality is estimated at 15-33%. HAE accounts for approximately 15,000-30,000 emergency department visits annually.

[0125] Trauma or stress, such as dental procedures, illness (e.g., viral illnesses such as colds and influenza), menstruation, and surgery, can trigger attacks of angioedema. To prevent acute attacks of HAE, patients may try to avoid certain stimuli that have previously caused attacks. However, in many cases, attacks occur without a known trigger. Typically, HAE symptoms first appear during childhood and worsen during adolescence. On average, untreated individuals experience attacks every 1-2 weeks, with most episodes lasting about 3-4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary greatly among people with hereditary angioedema, even among members of the same family.

[0126] There are three types of HAE, known as types I, II, and III. HAE affects 1 in 50,000 people, with type I accounting for approximately 85% of cases, type II accounting for approximately 15% of cases, and type III being very rare. Type III is the most recently described type and was originally thought to occur only in women, but families with affected males have been identified.

[0127] HAE is inherited in an autosomal dominant pattern, so that an affected person may inherit a mutation from one affected parent. De novo mutations in the gene may also occur, so HAE may also occur in people who have no family history of the disorder. It is estimated that 20-25% of cases arise from de novo spontaneous mutations.

[0128] Mutations in the SERPING1 gene cause types I and II hereditary angioedema. The SRPING1 gene provides instructions for the production of the C1 inhibitor protein, which is important in controlling inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause type I hereditary angioedema reduce the concentration of C1 inhibitor in the blood. In contrast, mutations that cause type II produce an abnormally functioning C1 inhibitor. Without adequate concentrations of functional C1 inhibitor, excessive amounts of bradykinin are produced. Bradykinin promotes inflammation by increasing the seepage of fluid through blood vessel walls into body tissues. Excessive accumulation of fluid in body tissues causes the swelling episodes seen in people with types I and II hereditary angioedema.

[0129] Mutations in the F12 gene are associated with some cases of hereditary angioedema type III. The F12 gene provides instructions for the production of blood clotting factor XII. In addition to its important role in blood clotting (clotting), factor XII is also an important stimulator of inflammation and is involved in the production of bradykinin. Certain mutations in the F12 gene result in the production of factor XII with increased activity. As a result, more bradykinin is produced and the blood vessel walls become more exudative, which leads to episodes of swelling. The cause of other cases of hereditary angioedema type III remains unknown. One or more mutations in genes yet to be identified may be responsible for the disorder in these cases.

[0130] Although HAE may show similarities to other types of angioedema caused by allergies or other medical conditions, the causes and treatments are significantly different. When hereditary angioedema is misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and / or epinephrine, which are typically ineffective in HAE, although epinephrine can be used for life-threatening reactions. Misdiagnosis can also result in unnecessary exploratory laparotomy in patients with abdominal swelling, and in some HAE patients, abdominal pain is incorrectly diagnosed as psychogenic.

[0131] C1 inhibitor therapy, as well as other treatments for HAE, is described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0132] Acute treatment of an HAE attack is provided to halt the progression of edema as quickly as possible. One acute treatment is the intravenous administration of C1 inhibitor concentrates from donor blood, but this treatment is not available in many countries. In emergency situations where C1 inhibitor concentrates are not available, fresh frozen plasma (FFP), which also contains C1 inhibitor, can be used as an alternative.

[0133] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are currently available in the United States, and two C1 inhibitor products are currently available in Canada. Pasteurized Berinert P (CSL Behring) was approved by the FDA for acute attacks in 2009. Nanofiltered Cinryze (ViroPharma) was approved by the FDA for prophylaxis in 2008. Rhucin (Pharming) is an investigational recombinant C1 inhibitor that does not pose the risk of infectious disease transmission by human blood-borne pathogens.

[0134] Treatment of an acute HAE attack may also include administration of pain-relieving medications and / or intravenous fluids.

[0135] Other therapeutic modalities can stimulate the synthesis of C1 inhibitor or reduce its consumption. Androgenic drugs such as danazol can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0136] Helicobacter pylori can cause abdominal attacks. Antibiotics to treat h. pylori will reduce abdominal attacks.

[0137] Newer therapies target the contact cascade. Ecallantide (KALBITOR®, DX-88, Dyax) inhibits plasma kallikrein and is approved in the United States. Icatibant (FIRAZYR®, Shire) inhibits the bradykinin B2 receptor and is approved in Europe and the United States.

[0138] Diagnosis of HAE may depend, for example, on family medical history and / or blood tests. Laboratory findings associated with types I, II, and III HAE are described, for example, in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, the concentration of C1 inhibitor is decreased, as is the concentration of C4, while the concentration of C1q is normal. In type II HAE, the concentration of C1 inhibitor is normal or elevated, but the function of C1 inhibitor is abnormal. The concentration of C4 is decreased and the concentration of C1q is normal. In type III, the concentrations of C1 inhibitor, C4, and C1q may all be normal.

[0139] Symptoms of HAE can be assessed using questionnaires, such as questionnaires completed by the patient, clinician, or family members. Such questionnaires are known in the art and include, for example, visual analog scales. See, for example, McMillan, CV et al., Patient. 2012;5(2):113-126.

[0140] Other pKal- or bradykinin-mediated disorders Other exemplary diseases or conditions associated with plasma kallikrein activity include non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burn injury, cerebral ischemia / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, ventricular assist device or stent-associated thrombosis, heparin-induced thrombocytopenia with thrombosis, thromboembolism, coronary heart disease with unstable angina, edema, eye disease, gout, intestinal disease, oral mucositis, neuropathic pain, inflammatory pain, spinal stenosis degenerative spinal disease, postoperative ileus, aortic aneurysm. , osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral cerebral edema, sepsis, acute middle cerebral artery (MCA) ischemic events (stroke), restenosis (e.g., after angioplasty), systemic lupus erythematosus nephritis, autoimmune diseases, inflammatory diseases, cardiovascular diseases, neurological diseases, diseases associated with protein misfolding, diseases associated with angiogenesis, hypertensive and diabetic nephropathy, allergies and respiratory diseases (e.g., anaphylaxis, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis) and tissue injury (e.g., burn or chemical injury).

[0141] treatment Subjects who are at risk of or suffering from pKal-mediated or bradykinin-mediated disorders identified using assay methods such as those described herein can be treated with any suitable therapeutic agent.In some embodiments, the methods provided include selecting a treatment for a subject based on the results of the assays provided, such as biomarker detection.

[0142] In certain embodiments, the methods include one or both of selecting or administering a therapeutic agent, e.g., a kallikrein binding agent as described herein, e.g., a bradykinin B2 receptor antagonist as described herein, e.g., a C1-INH supplement as described herein, for administration to the subject based on the results of an assay, e.g., biomarker detection.

[0143] In some embodiments, plasma kallikrein binding protein or polypeptide is administered to the subject.In some embodiments, the kallikrein binding agent is kallikrein inhibitor, such as peptide, small molecule inhibitor, kallikrein antibody or its fragment.In some embodiments, bradykinin B2 receptor antagonist is administered to the subject.In some embodiments, C1-INH replacement therapy is administered to the subject.

[0144] Therapeutic agents, such as kallikrein inhibitors, such as bradykinin B2 receptor antagonists, such as C1-INH supplements, can be administered as part of a combination therapy with other therapies to treat diseases or conditions involving plasma kallikrein and / or bradykinin activity. For example, the combination therapy with one or more of kallikrein inhibitors, bradykinin B2 receptor antagonists, or C1-INH supplements, such as one or more of kallikrein inhibitors, bradykinin B2 receptor antagonists, or C1-INH supplements and other therapies, can be provided in a number of different forms. The first agent can be administered before or after the administration of the other treatment. In some situations, the first agent and other treatments (e.g., therapeutic agents) are administered simultaneously or in close time proximity (e.g., with a short time interval between injections, such as the same treatment session). The first agent and other treatments can also be administered with a larger time interval.

[0145] Plasma kallikrein binding agents Plasma kallikrein binding agents (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding agents, e.g., small molecules) are useful therapeutic agents for various diseases and conditions, such as diseases and conditions involving plasma kallikrein activity.For example, in some embodiments, the disease or condition involving plasma kallikrein activity is hereditary angioedema (HAE).In some embodiments, plasma kallikrein binding proteins or polypeptides are administered to subjects at risk of or suffering from pKal-mediated or bradykinin-mediated disorders.

[0146] Many useful protein inhibitors of kallikrein, either tissue or plasma kallikrein, contain a Kunitz domain. As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and containing at least two, preferably three disulfides. The domain folds so that the first and sixth cysteines, the second and fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a Kunitz domain having 58 amino acids, cysteines can be present at positions corresponding to the 5th, 14th, 30th, 38th, 51st, and 55th amino acids according to the numbering of the BPTI homology sequence provided below, and disulfides can be formed between the 5th and 55th, 14th and 38th, and 30th and 51st cysteines), or, if two disulfide bonds are present, they form between the corresponding subsets of those cysteines. The spacing between each cysteine ​​can be within 7, 5, 4, 3, 2, 1 or 0 amino acids in the intervals corresponding to positions 5 to 55, 14 to 38 and 30 to 51 according to the numbering of the BPTI sequence provided below. The BPTI sequence can be used as a reference to refer to specific positions in any general Kunitz domain. Comparison of the Kunitz domain of interest with BPTI is performed by identifying the optimal alignment that maximizes the number of aligned cysteines.

[0147] The 3D structure (high resolution) of the Kunitz domain of BPTI is known. One X-ray structure has been deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of several BPTI homologues (Eigenbrot et al. (1990) Protein Engineering, 3(7):591-598; Hynes et al. (1990) Biochemistry, 29:10018-10022) are known. At least 81 Kunitz domain sequences are known. Known human homologues include the three Kunitz domains of LACI, also known as tissue factor pathway inhibitor (TFPI) (Wun et al. (1988) J. Biol. Chem. 263(13):6001-6004; Girard et al. (1989) Nature, 338:518-520; Novotny et al. (1989) J. Biol. Chem., 264(31):18832-18837), the two Kunitz domains of inter-alpha trypsin inhibitor, APP-I (Kido et al. (1988) J. Biol. Chem., 263(34):18104-18107), the Kunitz domains from collagen, and the three Kunitz domains of TFPI-2 (Sprecher et al. (1994) PNAS USA, 91:3353-3357), the Kunitz domain of hepatocyte growth factor activator inhibitor type I, the Kunitz domain of hepatocyte growth factor activator inhibitor type II, and the Kunitz domain described in U.S. Patent Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa that contains three Kunitz domains (amino acid sequence in Table 1).

[0148] [Table 4] TIFF2024150637000008.tif94165

[0149] The Kunitz domains mentioned above are called LACI-K1 (residues 50-107), LACI-K2 (residues 121-178), and LACI-K3 (residues 213-270). The cDNA sequence of LACI is reported by Wun et al. (J.Biol.Chem., 1988, 263(13):6001-6004). Girard et al. (Nature, 1989, 338:518-520) report a mutation study in which the P1 residue of each of the three Kunitz domains was altered. LACI-K1 inhibits factor VIIa (F.VIIa) when it is complexed with tissue factor, and LACI-K2 inhibits factor Xa.

[0150] Exemplary Kunitz domain-containing proteins include the following, with SWISS-PROT accession numbers in parentheses: A4_HUMAN(P05067)、A4_MACFA(P53601)、A4_MACMU(P29216)、A4_MOUSE(P12023)、A4_RAT(P08592)、A4_SAISC(Q95241)、AMBP_PLEPL(P36992)、APP2 _HUMAN(Q06481)、APP2_RAT(P15943)、AXP1_ANTAF(P81547)、AXP2_ANTAF(P81548)、BPT1_BOVIN(P00974)、BPT2_BOVIN(P04815)、CA17_HUMAN(Q0238) 8)、CA36_CHICK(P15989)、CA36_HUMAN(P12111)、CRPT_BOOMI(P81162)、ELAC_MACEU(O62845)、ELAC_TRIVU(Q29143)、EPPI_HUMAN(O95925)、EPPI_MO USE(Q9DA01)、HTIB_MANSE(P26227)、IBP_CARCR(P00993)、IBPC_BOVIN(P00976)、IBPI_TACTR(P16044)、IBPS_BOVIN(P00975)、ICS3_BOMMO(P07481) 、IMAP_DROFU(P11424)、IP52_ANESU(P10280)、ISC1_BOMMO(P10831)、ISC2_BOMMO(P10832)、ISH1_STOHE(P31713)、ISH2_STOHE(P81129)、ISIK_HEL PO(P00994)、ISP2_GALME(P81906)、IVB1_BUNFA(P25660)、IVB1_BUNMU(P00987)、IVB1_VIPAA(P00991)、IVB2_BUNMU(P00989)、IVB2_DABRU(P00990) 、IVB2_HEMHA(P00985)、IVB2_NAJNI(P00986)、IVB3_VIPAA(P00992)、IVBB_DENPO(P00983)、IVBC_NAJNA(P19859)、IVBC_OPHHA(P82966)、IVBE_DENPO O(P00984)、IVBI_DENAN(P00980)、IVBI_DENPO(P00979)、IVBK_DENAN(P00982)、IVBK_DENPO(P00981)、IVBT_ERIMA(P24541)、IVBT_NAJNA(P20229)、MCPI_MELCP(P82968)、SBPI_SARBU(P26228)、SPT3_HUMAN(P49223)、TKD1_BOVIN(Q28201)、TKD1_SHEEP(Q29428)、TXCA_DENAN(P81658)、UPTI_PIG(Q29100)、AMBP_BOVIN(P00978)、AMBP_HUMAN(P02760)、AMBP_MERUN(Q62577)、AMBP_MESAU(Q60559)、AMBP_MOUSE(Q07456)、AMBP_PIG(P04366)、AMBP_RAT(Q64240)、IATR_HORSE(P04365)、IATR_SHEEP(P13371)、SPT1_HUMAN(O43278)、SPT1_MOUSE(Q9R097)、SPT2_HUMAN(O43291)、SPT2_MOUSE(Q9WU03)、TFP2_HUMAN(P48307)、TFP2_MOUSE(O35536)、TFPI_HUMAN(P10646)、TFPI_MACMU(Q28864)、TFPI_MOUSE(O54819)、TFPI_RABIT(P19761)、TFPI_RAT(Q02445)、YN81_CAEEL(Q03610)、

[0151] Various methods can be used to identify Kunitz domains from sequence databases. For example, known amino acid sequences, consensus sequences or motifs (e.g., ProSite motifs) of Kunitz domains can be searched against the GenBank sequence database (National Center for Biotechnology Information, National Institutes of Health, Bethesda, MD), for example, using BLAST; against the Pfam database of HMMs (hidden Markov models) (e.g., using default parameters for Pfam searches); against the SMART database; or against the ProDom database. For example, Pfam release 9, Pfam accession number PF00014, provides numerous Kunitz domains and HMMs for Kunitz domain identification. A description of the Pfam database can be found in Sonhammer et al. (1997) Proteins 28(3):405-420, and a detailed description of HMM can be found, for example, in Gribskov et al. (1990) Meth. Enzymol. 183:146-159; Gribskov et al. (1987) Proc. Natl. Acad. Sci. USA 84:4355-4358; Krogh et al. (1994) J. Mol. Biol. 235:1501-1531; and Stultz et al. (1993) Protein Sci. 2:305-314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, Del.) is as described in Schultz et al. (1998), Proc. Natl. Acad. Sci. USA 95:5857 and Schultz et al. (2000) Nucl. Acids Res 28:231.The SMART database contains domains identified by hidden Markov model profiling with the HMMer2 search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models of proteins and nucleic acids. Cambridge University Press). The database is also annotated and monitored. The ProDom protein domain database consists of an automated compilation of homologous domains (Corpet et al. (1999) Nucl. Acids Res. 27:263-267). The current version of ProDom is constructed using recursive PSI-BLAST searches of the SWISS-PROT38 and TREMBL protein databases (Altschul et al. (1997) Nucleic Acids Res. 25:3389-3402; Gouzy et al. (1999) Computers and Chemistry 23:333-340). The database automatically generates a consensus sequence for each domain. Prosite lists the Kunitz domain as a motif and identifies proteins that contain the Kunitz domain. See, e.g., Falquet et al., Nucleic Acids Res. 30:235-238 (2002).

[0152] Kunitz domains interact with target proteases primarily using amino acids in two loop regions ("binding loops"). The first loop region is between approximately residues corresponding to amino acids 13-20 of BPTI. The second loop region is between approximately residues corresponding to amino acids 31-39 of BPTI. An exemplary library of Kunitz domains is diverse at one or more amino acid positions in the first and / or second loop regions. When screening for Kunitz domains that interact with kallikrein or selecting for mutants with improved affinity, particularly useful positions to vary include positions 13, 15, 16, 17, 18, 19, 31, 32, 34 and 39 relative to the sequence of BPTI. At least some of these positions are predicted to be in close contact with the target protease. It is also useful to vary other positions, such as, for example, the above-mentioned positions and nearby positions in the three-dimensional structure.

[0153] The "framework regions" of the Kunitz domain are defined as residues that are part of the Kunitz domain but that explicitly exclude residues in the first and second binding loop regions, i.e., approximately corresponding to amino acids 13-20 of BPTI and 31-39 of BPTI. Conversely, residues that are not in the binding loops can tolerate a wide range of amino acid substitutions (e.g., conservative and / or non-conservative substitutions).

[0154] In one embodiment, these Kunitz domains are mutant forms of the loop structures that comprise Kunitz domain 1 of human lipoprotein-associated coagulation inhibitor (LACI) protein. LACI contains three distinct internal peptide loop structures that are typical of Kunitz domains (Girard, T. et al., 1989. Nature, 338:518-520). Mutants of Kunitz domain 1 of LACI described herein have been screened and isolated, and bind to kallikrein with enhanced affinity and specificity (see, for example, U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be applied to other Kunitz domain frameworks to obtain other Kunitz domains that interact with kallikrein, such as plasma kallikrein. Useful modulators of kallikrein function typically bind to and / or inhibit kallikrein, as determined using kallikrein binding and inhibition assays.

[0155] In some embodiments, the kallikrein binding agent (e.g., a binding protein, e.g., a polypeptide, e.g., an inhibitory polypeptide, e.g., an antibody, e.g., an inhibitory antibody, or other binding agent, e.g., a small molecule) binds to the active form of plasma kallikrein. In some embodiments, the kallikrein binding agent binds to and inhibits plasma kallikrein, e.g., human plasma kallikrein and / or mouse kallikrein.

[0156] The plasma kallikrein binding protein may be full length (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA, (e.g., IgA1, IgA2), IgD, and IgE) or may comprise only an antigen-binding fragment (e.g., Fab, F(ab')2, or scFv fragment). The binding protein may comprise two heavy chain immunoglobulins and two light chain immunoglobulins, or may be a single chain antibody. The plasma kallikrein binding protein may be a recombinant protein, such as a humanized, CDR-grafted, chimeric, deimmunized, or in vitro produced antibody, and may optionally comprise a constant region derived from a human germline immunoglobulin sequence. In certain embodiments, the plasma kallikrein binding protein is a monoclonal antibody.

[0157] In some embodiments, the kallikrein binding protein binds to and inhibits plasma kallikrein, such as human plasma kallikrein and / or mouse kallikrein.Exemplary plasma kallikrein binding proteins are disclosed in US Patent Publication No. 2012021756, the entire contents of which are incorporated herein by reference.In some embodiments, the kallikrein binding protein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X11 and an antibody (e.g., a human antibody) having a light chain and / or a heavy chain of an antibody selected from the group consisting of DX-2930, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In certain embodiments, the plasma kallikrein binding protein is selected from the group consisting of M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X It competes with or binds to the same epitope as 115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to as DX-2930 herein), X115-G04, M29-D09, M145-D11, M06-D09 and M35-G04.In some embodiments, the plasma kallikrein binding protein is DX-2930.See US20110200611 and US20120201756, which are incorporated by reference herein.

[0158] In some embodiments, the kallikrein binding polypeptide (e.g., inhibitory polypeptide) binds to the active form of plasma kallikrein. Exemplary polypeptide plasma kallikrein agents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983 and No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991 and U.S. Patent Publication No. 20110086801, the entire contents of each of which are incorporated herein by reference. In one embodiment, the kallikrein binding polypeptide is DX-88 (a non-naturally produced kallikrein inhibitor, also known as KALBITOR® (ecallantide), SEQ ID NO:3). In one embodiment, the kallikrein inhibitor is a DX-88 polypeptide comprising or consisting of about the 58 amino acid sequence of amino acids 3-60 of SEQ ID NO:3, or having the 60 amino acid sequence of SEQ ID NO:3. Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp (SEQ ID NO:3)

[0159] In one embodiment, the plasma kallikrein binding protein is EPIKAL-2 (SEQ ID NO:4), a non-naturally occurring kallikrein inhibitor having an amino acid sequence of 58 residues (corresponding to residues 3-60 of SEQ ID NO:3) with an Ile to Ser amino acid substitution at residue 34 and a Glu to Gly amino acid substitution at residue 39. The sequence of EPIKAL-2 is shown below: EpiKal2: Met His Ser Phe Cys Ala Phe Lys Ala 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 (Sequence Identification Number 4)

[0160] In some embodiments, the plasma kallikrein binding protein may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the binding proteins described herein. In some embodiments, the plasma kallikrein binding protein may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the binding proteins described herein in the HC and / or LC framework regions (e.g., HC and / or LC FR1, 2, 3 and / or 4). In certain embodiments, the plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the HC and / or LC CDRs (e.g., HC and / or LC CDR1, 2 and / or 3) to the binding proteins described herein. In certain embodiments, the plasma kallikrein binding protein can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity in the constant regions (e.g., CD1, CH2, CH3 and / or CL1) to the binding proteins described herein.

[0161] In some embodiments, the small molecule binds to the activated form of plasma kallikrein.

[0162] Bradykinin B2 receptor antagonist In some embodiments, a bradykinin B2 receptor antagonist is administered to the subject.An exemplary bradykinin B2 receptor antagonist is icatibant (FIRAZYR®), which is a peptidomimetic drug that contains 10 amino acids and blocks the binding of natural bradykinin to the bradykinin B2 receptor.

[0163] C1-INH supplements In some embodiments, a C1-INH supplement is administered to the subject. Exemplary C1-INH supplements are publicly available, such as Berinert®, a purified human pasteurized nanofiltered C1-INH concentrate. EXAMPLES

[0164] Example 1: Truncated kininogen Based on the analysis of the contact system, truncated kininogen is a suitable biomarker for measuring contact system activation. It has been previously shown that truncated kininogen is elevated during HAE attacks, in liver cirrhosis, and as a result of contact system activation in sepsis. A phage display library of antibodies was panned against truncated kininogen in combination with depletion of intact kininogen. In parallel, mice were immunized with truncated kininogen and monoclonal antibodies were obtained from hybridoma cell lines. Both attempts yielded numerous different monoclonal antibodies that bind both truncated and intact kininogen, but no antibodies that bind only to truncated kininogen were obtained.

[0165] A number of antibodies were screened for suitability in Western blot analysis and several antibodies were identified as performing well, including the mouse mAb (clone 11H05) shown in Figure 2. It is clear that this assay can detect truncated kininogen in human plasma samples. Furthermore, the data in Figure 2 confirm that plasma collection in glass tubes is sufficient to prevent contact activation and kininogen cleavage.

[0166] A mass spectrometry-based approach is also available for detecting truncated kininogen in patient plasma, in which kininogen from patient samples is immunoadsorbed, the eluted kininogen is proteolytically digested, and the peptide fragments are analyzed by LC-MS.

[0167] Example 2: Immunoassay of C1INH-pKal and α2M-pKal For the detection of these complexes, ELISA-based immunoassays have been developed, similar to sandwich-based ELISA assays previously described, see Kaufman et al., Blood 77, 2660-2667, and Wachtfogel, Blood 73, 468-471.

[0168] Example 3: C1INH-pKal Assay An ELISA for the detection of C1INH-pKal complex has been developed. This sandwich assay uses antibodies against pKal and C1INH as either capture or test reagents. The first step in the development of this assay is the preparation of the C1INH-pKal complex. This covalent complex was prepared by incubating a 2-4 fold molar excess of pKal over C1INH. The complex was then purified using cation exchange chromatography (Capto S resin) as shown in Figure 3. The complex was analyzed by ELISA using a calculated molar extinction coefficient at 280 nm (121,740 M -1 cm -1) was used to quantify C1INH-pKal. Previous reports using a similar assay to measure C1INH-pKal did not report the extinction coefficient. Accurate determination of concentration is important because the assay provides information about the dosage of the drug in order to determine how much pKal is activated during disease.

[0169] Two assay formats were investigated for the detection of C1INH-pKal by ELISA. The first format uses an anti-pKal antibody (mouse mAb 13G11) as the capture reagent and an antibody against C1INH as the detection antibody, with the signal generated by an HRP-labeled secondary antibody (Figure 4). This assay has a lower limit of quantification in the single-digit nanomolar range or below. The opposite assay format was also investigated, where anti-C1INH is the capture antibody and anti-pKal (13G11) is the detection antibody (Figure 5). This assay format has a similar lower limit of quantification. Having two assay formats provides an additional option for multiplex assays for C1INH-pKal and α2M-pKal.

[0170] Example 4: α2M-pKal complex assay An ELISA was developed for the detection of α2M-pKal complex. This sandwich assay uses antibodies against pKal and α2M as either capture or detection reagents. The first step in the development of this assay is the preparation of α2M-pKal complex. This covalent complex was prepared by incubating a 2-4 fold molar excess of pKal with α2M. The complex was then purified using size exclusion chromatography as shown in Figure 6. α2M consists of ~180 kDa subunits that exist as an oligomeric distribution up to tetramers. Upon interaction with pKal, each monomer of α2M tends to form a covalent amide bond by hydrolysis of the thioester bond in α2M with the lysine amine on pKal. As a result, a distribution of crosslinked species can be predicted that complicates quantification. A method was derived to quantify α2M-pKal complex by measuring the activity of the complex and converting it to concentration using a standard curve (Figure 7). This quantitative method is possible because it is well known in the literature that covalent complexes between α2M and target proteases do not interfere with the active site targeting of small synthetic peptide substrates.

[0171] For the C1INH-pKal assay, two assay formats were also investigated for the detection of α2M-pKal by ELISA. The first format uses an anti-pKal antibody (mouse mAb 13G11) as the capture reagent and an antibody against α2M as the detection antibody, with the signal generated by an HRP-labeled secondary antibody (Figure 8). This assay format has a lower limit of quantification in the single-digit nanomolar range or below. The opposite assay format was also investigated, where anti-α2M is the capture antibody and anti-pKal (13G11) is the detection antibody (Figure 9). This assay format has a similar lower limit of quantification. Since we are looking for a multiplex assay of C1INH-pKal and α2M-pKal, having two assay formats provides additional options.

[0172] Example 5: Detection of α2M-pKal and C1INH-pKal in activated plasma ELISA was used to detect pKal activation in normal human plasma treated with agents such as kaolin or dextran sulfate, which are known to induce contact activation. As shown in Figure 10, the assay can detect complexes formed in plasma after in vitro activation with dextran sulfate.

[0173] Example 6: Intact and truncated kininogen Western blots were used to show that plasma from patients obtained during a stroke and collected in citrated blood collection tubes containing an antiprotease cocktail showed a decreased amount of intact kininogen (i.e., single chain) (Figure 11). An increase in truncated kininogen (i.e., two chain) was observed (data not shown).

[0174] Example 7: Exemplary Assays and Assay Data (i) HMWK ex vivo activation The HMWK ex vivo activation assay can be used to evaluate the inhibitory activity of potential pKal inhibitors against contact activation. In summary, factor XIIa (e.g., 5 nM) was used to stimulate contact system activation in normal human plasma or mimic HAE attack samples to reduce the concentration of single-chain HMWK by approximately 10-50%, which can be detected in Simple Western (SBHD) or Western blot (TGA). Reduction of contact activation was observed in DX-2930-treated samples.

[0175] As shown in Figure 17, the lowest concentrations (e.g., 9-27 nM) of DX-2930 inhibitory activity were detected in the HMWK ex vivo activation assay described herein using Protein Simple Western or conventional Western blot analysis. The assay conditions used in this study were capable of detecting DX-2930 activity of 9-27 nM in 5% plasma. See also Table 2 below. [Table 5]

[0176] The raw data for Factor XIIa-activated plasma with escalating doses of DX-2930 are provided in Figure 18. See also Table 3 below. [Table 6] TIFF2024150637000011.tif13165

[0177] The reduction of single-chain HMWK was tested in various human undiluted plasma samples after activation with Factor XIIa for 30 minutes. The results are shown in Figures 22-26. See also Tables 4-8 below. [Table 7] [Table 8] [Table 9] [Table 10] [Table 11]

[0178] As shown in FIG. 27, activation of factor XIIa resulted in a concentration-dependent reduction of single-chain HMWK in plasma samples.

[0179] (ii) Endogenous truncated kininogen In this assay, the plasma from SCAT tube was analyzed by Simple Western (SBHD) and Western blot (TGA) to measure the concentration of cleaved kininogen in plasma samples.Cleaved HMWK was found to be elevated in basal and attack HAE samples compared with normal human plasma.Cleaved HMWK is expected to be reduced in HAE patients treated with DX-2930 as well as in healthy volunteers treated with DX-2930.

[0180] As shown in Figure 28, double-chain HMWK (truncated HMWK) concentrations in samples from HAE patients at baseline were found to be lower than those in patients with HAE attacks. The truncated HMWK concentrations in patients treated with DX-2930 are expected to be similar to those in normal patients.

[0181] FIG. 29 shows the levels of truncated HMWK in patients with basal HAE and those experiencing HAE attacks as determined by Protein Simple and conventional Western blot analysis.

[0182] Endogenous truncated HMWK was tested in four individuals, and the results are shown in Figure 30. Small amounts of truncated HMWK were found in normal human plasma samples collected in citrated blood collection tubes.

[0183] (iii) Ex vivo assay for measuring pKal activity This enzyme-based assay was developed to assess contact system activation in normal human plasma or mimic HAE attack samples (aiming for 10-50% reduction of single-chain HMWK) and to assess the inhibitory activity of pKal inhibitors on contact system activation. Reduction of contact activity was observed in subjects treated with DX-2930. This assay is useful for evaluating pKal inhibitors such as DX-2930 bioactivity in treated subjects (e.g. monkeys or human patients).

[0184] An example of this assay is illustrated in Figure 12. Briefly, plasma samples were placed in a 96-well microplate. An exemplary pKal inhibitor, DX-2930, and an exemplary contact system activator, Factor XIIa, were added to the plasma samples. The mixture was incubated on ice in the presence of a labeled peptide substrate of pKal for a suitable period (e.g., 2 minutes), and corn trypsin inhibitor (CTI) was added to the mixture to stop the activation reaction. The mixture was diluted as necessary, and proteolytic activity was determined by measuring the concentration of the fluorescent peptide substrate.

[0185] As shown in FIG. 12, monkeys treated with DX-2930 showed reduced levels of contact activation (reduced pKal activity).

[0186] (iv) Western blot analysis to determine truncated HMWK The concentration of cleaved HMWK was measured by Western blot analysis, which may be accompanied by LiCor detection. See also Example 8 below. Where necessary, citrate or antiprotease cocktail was used as an anticoagulant in this assay. Increased cleaved HMWK was observed in HAE samples compared to normal plasma. This assay is useful for evaluating pKal inhibitors such as DX-2930 bioactivity in treated subjects (e.g. monkeys or human patients).

[0187] Plasma samples from HAE patients in anti-protease inhibitor cocktail were tested using Western blot analysis with LiCor detection, and the results are shown in Figure 14. As shown in Figure 15, intact kininogen was reduced from 10% to 50% (Western blot analysis with LiCor detection), as previously reported. Cynomolgus monkeys treated with DX-2930 showed a concentration-dependent reduction in the levels of pKal activation (by kaolin or dextran sulfate) (Figure 16). See also Table 9 below. [Table 12]

[0188] Human HMWK in various plasma samples after 30 min activation was determined by Western blot analysis as described herein and is shown in Figure 19. Reduced samples were boiled for 5 min before loading onto the gel. Samples were diluted 20-fold and run on a 4-12% Bis-Tris gel at 150v for 90 min. Proteins on the gel were transferred to a membrane using iBlot for 7 min. Mouse anti-human kininogen (1:1,000 dilution) was used. Exposure time of the blot was 5 s.

[0189] Protein Simple Western was compared with conventional Western blot analysis in detecting plasma activation, and as shown in Figures 20 and 21, the former (right panel) is more sensitive than the latter (left panel).

[0190] Example 8: Ex vivo activation of pKal as a biomarker Ex vivo activation of prekallikrein to plasma kallikrein (pKal) in plasma can be used both as a pharmacodynamic (PD) biomarker to provide evidence of the biological activity of therapeutic inhibitors of pKal, such as DX-2930, and for the detection of activated pKal in disease samples.

[0191] In the first experiment, plasma was obtained from cynomolgus monkeys given a single SC injection of DX-2930 (5 mg / kg) and activated with 10 nM factor XIIa to generate active pKal, which was monitored using a synthetic substrate (Pro-Phe-Arg-AMC). Corn trypsin inhibitor was added to stop factor XIIa activation prior to the addition of the substrate. The percent inhibition observed in plasma samples from dosed cynomolgus monkeys matched the inhibition of plasma samples prepared with molar equivalents of either DX-2930 or ecallantide (Figure 31). It is clear that the plasma concentration of DX-2930 reached a drug level (~265 nM) that inhibited approximately 80% of the pKal generated by ex vivo addition of factor XIIa. Figure 31 also shows that the same amount of pKal inhibition was observed with an equivalent concentration of ecallantide. In contrast, addition of equivalent concentrations of C1-INH to plasma did not inhibit pKal activated by factor XIIa in this ex vivo activation assay.

[0192] In a second experiment, plasma was obtained from cynomolgus monkeys receiving 5 weeks of SC injections of different doses of DX-2930, and citrated plasma was obtained from humans (n=6) in a Phase 1a clinical trial receiving a single SC injection of different doses of DX-2930. Plasma samples were activated with factor XIIa and the resulting pKal activity was measured using a synthetic substrate (Pro-Phe-Arg-AMC). Corn trypsin inhibitor was added to stop the activation of factor XIIa prior to the addition of the substrate. Percent inhibition was determined using the pKal activity present in the pre-dose plasma from each individual as a baseline. Figure 32 shows that as the dose of DX-2930 increased, the percent inhibition increased in both human and monkey plasma samples.

[0193] Results from two experiments indicate that the ex vivo activation assay is useful as a PD biomarker for the biological activity of therapeutic inhibitors of pKal.

[0194] Example 9: Ex vivo inhibition of pKal activity in plasma samples from a DX-2930 Phase 1A study The ex vivo inhibition (bioactivity) of DX-2930 in plasma from human subjects administered DX-2930 subcutaneously was examined in this study.

[0195] material ·DX-2930 (106.7 mg / ml = 732 μM) ·Human Factor XIIa - ERL HFXIIa 2790P (1.72 mg / ml = 25.3 μM) Corn trypsin inhibitor (CTI) - ERL CTI 360 (1.54 mg / ml = 123 μM) · Peptide substrate = PFR-AMC, Sigma Cat. No. 99273, Lot 037K1207. Assay buffer = 20 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% Triton X-100, 0.1% PEG-8000 Corning 96-well white polystyrene microplate, Catalog No. 3789 Spectramax M2 plate reader Citrated plasma collected in the DX-2930 Phase 1A study

[0196] method Plasma diluted 1:40 was activated by the addition of 10 nM factor XIIa for 2 min at room temperature. Factor XIIa was quenched by the addition of 100 nM CTI and plasma kallikrein (pKal) proteolytic activity was assessed by further diluting sample plasma 1:10 and adding 10 μM of the fluorescent peptide substrate PFR-AMC. Each plasma sample was reported as a percentage of pKal activity, which was converted to "% inhibition" based on pre-dose controls for each individual.

[0197] result Citrated plasma samples were obtained from healthy subjects in the DX-2930 Phase 1a study and analyzed using an ex vivo bioactivity assay in plasma. Significant inhibition of pKal activity was observed in dose group 3 (1.0 mg / kg DX-2930) and dose group 4 (3.0 mg / kg DX-2930), achieving a maximum inhibition of pKal activity of approximately 19% and 36%, respectively (Figure 33). The inhibition achieved in groups 3 and 4 was sustained and consistent with the apparent half-life of approximately 20 days. Inhibition of pKal activity in dose group 1 (0.1 mg / kg DX-2930) and dose group 2 (0.3 mg / kg DX-2930) was not significant.

[0198] These results indicate that the ex vivo activation assay is useful as a PD biomarker for the biological activity of therapeutic inhibitors of pKal.

[0199] Figure 10: Analysis of DX-2930 biological activity in Phase 1a study samples by Western blot analysis. The bioactivity of DX-2930 in the plasma of human subjects treated with DX-2930 was examined using Western blot analysis as described herein.

[0200] Western blot analysis was performed on citrated plasma samples obtained on day 1 (before DX-2930 or placebo administration), day 5, or day 28 after administration of DX-2930 or placebo. Samples were analyzed by Western blot using an antibody that detects high molecular weight kininogen (HMWK), a substrate for activated plasma kallikrein, the target enzyme inhibited by DX-2930. Plasma kallikrein acts on HMWK to produce the proinflammatory peptide bradykinin and a two-chain form known as the cleaved form of HMWK, which is detected by Western blot analysis. Plasma samples treated with and without activated blood coagulation factor XIIa (FXIIa) were analyzed by Western blot analysis. Factor XIIa converts inactive prekallikrein in plasma to activated plasma kallikrein.

[0201] The results from this study show that factor XIIa-treated samples from subjects administered 3 mg / kg DX-2930 (Group 4) showed a statistically lower percentage of two-chain HMDK (truncated HMWK) at Day 5 (p=0.0011) and Day 28 (p=0.0028) compared to pre-dose samples from those subjects. This is evidence of the biological activity of DX-2930 on plasma kallikrein-mediated proteolysis of endogenous substrate (HMWK) (Figure 34). The reduction in the percentage of two-chain HMWK observed in Group 4 tended to be lower than that observed in other dose groups or in the placebo-treated group.

[0202] Furthermore, subjects not treated with factor XIIa and administered DX-2930 at 0.3 mg / kg, 1 mg / kg, or 3 mg / kg showed a lower percentage of two-chain HMWK than that observed in the placebo or 0.1 mg / kg doses (Figure 35).

[0203] Taken together, these results further demonstrate the usefulness of Western blot analysis as a PD biomarker for the biological activity of therapeutic inhibitors of pKal.

[0204] Equivalents and Scope Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the present disclosure described herein. The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth in the appended claims.

[0205] In the claims, articles such as "a," "an," and "the" can mean one or more, unless the context clearly indicates to the contrary or otherwise. A claim or description containing "or" between one or more members of a group is considered to be satisfied when one or more, or all of the members of the group are present, used, or otherwise relevant in a given product or process, unless the context clearly indicates to the contrary or otherwise. The disclosure includes embodiments in which exactly one member of a group is present, used, or otherwise relevant in a given product or process. The disclosure includes embodiments in which one or more, or all of the members of a group are present, used, or otherwise relevant in a given product or process.

[0206] Furthermore, the disclosure encompasses all modifications, combinations, and permutations in which one or more limitations, elements, clauses, and descriptive terms from one or more of the enumerated claims are introduced into another claim. For example, any claim that is dependent on another claim can be modified to include one or more limitations found in any other claim that is dependent on the same base claim. When elements are present as a list, for example in Markush group format, each subgroup of elements is also disclosed, and any element can be removed from the group. In general, when the disclosure or aspects of the disclosure are described as comprising certain elements and / or features, it should be understood that a particular embodiment of the disclosure or aspects of the disclosure consists of or essentially consists of such elements and / or features. For simplicity, such embodiments have not been specifically described in these exact words herein. The terms "comprising" and "containing" are intended to be open-ended and allow for the inclusion of additional elements or steps. When ranges are given, the endpoints are included. Further, unless otherwise indicated or clear from the context and the understanding of one of ordinary skill in the art, values ​​expressed as ranges can take any specific value or subrange within the ranges set forth in other embodiments of this disclosure, to one-tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise.

[0207] This application refers to various issued patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. In the event of a conflict between any of the incorporated references and this specification, this specification will control. Furthermore, any particular embodiment of the present disclosure that falls within the prior art may be expressly excluded from any one or more of the claims. Because such embodiments are deemed known to those of skill in the art, they may be excluded even if the exclusion is not expressly described herein. Any particular embodiment of the present disclosure may be excluded from any claim for any reason, whether or not related to the existence of prior art.

[0208] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments described herein. The scope of the embodiments described herein is not intended to be limited to the above description, but rather is set forth in the appended claims. Those skilled in the art will recognize that various changes and modifications may be made to the description without departing from the spirit or scope of the disclosure, as set forth in the following claims.

Claims

1. A method for analyzing a sample, comprising: (i) providing a plasma sample obtained from a subject having, suspected of having, or at risk for an acute attack of a disease associated with the contact activation system; (ii) incubating the plasma sample with an activator of the plasma kallikrein (pKal) system; (iii) measuring the concentration of cleaved high molecular weight kininogen (HMWK) in the plasma sample; and (iv) identifying the subject as having an acute attack of the disease if the concentration of truncated HMWK deviates from the concentration of truncated HMWK in a sample from a control subject.

2. The method described in claim 1, wherein the concentration of the truncated HMWK is measured by Western blot analysis.

3. The method described in claim 2, wherein the Western blot analysis is Protein Simple Western blot analysis.

4. A method according to any one of claims 1 to 3, wherein the disease associated with the contact activation system is hereditary angioedema (HAE).

5. The method described in claim 4, wherein the HAE is type I HAE or type II HAE.

6. A method described in any one of claims 1 to 5, wherein the deviation in the concentration of truncated HMWK comprises an increase in the concentration of truncated HMWK compared to the concentration of truncated HMWK in a sample derived from a control subject.

7. A method described in any one of claims 1 to 6, wherein the subject is a human patient undergoing treatment for the disease and the plasma sample is obtained after or during the course of the treatment.

8. The method of claim 7, further comprising evaluating the effectiveness of the treatment, wherein a decrease in the concentration of truncated HMWK compared to before the treatment or a decrease in the concentration of truncated HMWK throughout the course of the treatment indicates that the treatment is effective.

9. The method described in claim 7 or 8, wherein the treatment is a pKal inhibitor.

10. The method of claim 9, wherein the pKal inhibitor is DX-88, DX-2930, or EPIKAL-2.

11. A method according to any one of claims 1 to 10, wherein the activator of the pKal system is factor XIIa.

Images