Immunoassay for the detection of cleaved high molecular weight kininogens

Abstract

The present invention is entitled "Immunoassay for detecting cleaved high molecular weight kininogen". The present disclosure provides an immunoassay for detecting cleaved high molecular weight kininogen (HMWK) with high sensitivity and specificity and an isolated antibody that specifically binds to cleaved HMWK.

Description

[0001] The present application is a divisional application of a Chinese application filed on October 19, 2016, with application number 201680069922.5 (PCT / US2016 / 057640) and invention name “Immunoassay Method for Detecting Cleaved High Molecular Weight Kininogen”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 U.S.C. §119 of U.S. Provisional Application No. 62 / 243,505, filed on October 19, 2015, and U.S. Provisional Application No. 62 / 335,311, filed on May 12, 2016, the entire contents of each of which are incorporated herein by reference. Background Art

[0004] Kininogens are precursors of kinins such as bradykinin and kallikrein. There are two types of human kininogens, high molecular weight kininogens (HMWK) and low molecular weight kininogens (LMWK), which are splice variants. HMWK is mainly used as a cofactor for coagulation and inflammation, and is a preferred substrate for bradykinin generation mediated by plasma kallikrein (pKal).

[0005] Plasma kallikrein (pKal) is the major bradykinin-generating enzyme in the circulation. Activation of pKal occurs via contact systems associated with the disease pathology associated with hereditary angioedema (HAE). pKal cleaves HMWK (a single-chain polypeptide) to produce bradykinin and a cleaved form of HMWK containing two polypeptide chains held together by disulfide bonds. Cugno et al., Blood (1997) 89: 3213-3218.

[0006] During an attack of hereditary angioedema (HAE), cleaved HMWK increases to approximately 47% of total kininogen (Cugno et al., Blood (1997) 89:3213-3218), making it a biomarker for monitoring HAE attacks. Therefore, it is of interest to develop a sensitive and reliable assay to detect the level of cleaved HMWK in biological samples. Summary of the invention

[0007] Some aspects of the present disclosure provide an immunoassay method for detecting cleaved high molecular weight kininogen (HMWK) with high sensitivity and specificity. The method includes: (i) providing a support member, on which a first reagent (e.g., an antibody such as 559B-M004-B04) that specifically binds to cleaved HMWK is attached; (ii) contacting the support member of (i) with a biological sample suspected of containing cleaved HMWK; (iii) contacting the support member obtained in (ii) with a second reagent that binds HMWK, wherein the second reagent is conjugated to a marker; and (iv) directly or indirectly detecting a signal released from a marker of the second reagent bound to the support member to determine the level of cleaved HMWK in the biological sample. In some cases, step (ii) may be performed in the presence of ZnCl2.

[0008] In some embodiments, prior to step (ii), the support member of (i) is incubated with a blocking buffer.

[0009] In some embodiments, the second reagent is a mixture of polyclonal antibodies, monoclonal antibodies or two or more monoclonal antibodies that bind to HMWK. Two or more monoclonal antibodies in the mixture can bind to different epitopes in HMWK. In some embodiments, the marker is a signal release agent. In some embodiments, the marker is a member of a receptor-ligand pair. In this case, the immunoassay method may also include contacting the second reagent fixed on the support member in (iii) with another member of the receptor-ligand pair before step (iv), wherein the other member is conjugated to the signal release agent. In one example, the receptor-ligand pair is biotin and streptavidin.

[0010] Another aspect of the present disclosure provides a method for detecting a high molecular weight kininogen (HMWK) cleaved in a sample, the method comprising: (i) contacting a sample suspected of containing cleaved HMWK with any of the antibodies described herein (e.g., 559B-M004-B04); (ii) measuring the complex of the cleaved HMWK and the antibody formed in step (i); and (iii) determining the level of cleaved HMWK in the sample based on the result of step (ii). In some embodiments, step (i) is performed in the presence of ZnCl2. In some embodiments, step (i) is performed using an enzyme-linked immunosorbent assay (ELISA) or an immunoblot assay.

[0011] In any of the methods described herein, the sample can be a biological sample obtained from a subject (e.g., a human subject), such as a plasma sample or a serum sample. In some embodiments, the method further comprises collecting the sample into a vacuum blood collection tube comprising one or more protease inhibitors.

[0012] Any of the assays described herein (eg, immunoassays) can be an ELISA assay, a Western blot assay, or a lateral flow assay.

[0013] In some embodiments, the biological sample is obtained from a subject (e.g., a human patient) having a disease. The assay method may also include determining whether the disease is mediated by plasma kallikrein based on the level of cleaved HMWK, the deviation of the level of cleaved HMWK in the sample from the level of cleaved HMWK in the control sample indicating that the disease is mediated by plasma kallikrein.

[0014] Any of the assay methods described herein may also include identifying patients with a disease or disorder mediated by plasma kallikrein based on the level of cleaved HMWK, or evaluating the therapeutic efficacy of a disease or disorder. In some embodiments, the method may also include: if the subject is identified as having the disorder, administering to the subject an effective amount of a therapeutic agent for treating the disorder, such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R) inhibitor, and / or a C1 esterase inhibitor. In some embodiments, the pKal inhibitor is an anti-pKal antibody. In some embodiments, the therapeutic agent is lanadelumab, ecallantide, icatibant, or a human plasma-derived C1 esterase inhibitor.

[0015] In some embodiments, the subject is a human patient receiving treatment for a disorder, and wherein the method further comprises evaluating the efficacy of the treatment based on the level of the cleaved HMWK determined in step (iii), and the deviation of the level of the cleaved HMWK in the sample from the subject from the level of the control sample indicates the efficacy of the treatment. In some embodiments, the method further comprises confirming the appropriate treatment of the subject based on the level of the cleaved HMWK. In some embodiments, the method further comprises identifying the subject as a candidate for treating the disease based on the level of the cleaved HMWK.

[0016] In some embodiments, the human patient has a history of a disease (e.g., HAE). In some embodiments, the method further comprises evaluating the risk of a subject's disease onset based on the level of cleaved HMWK, and the deviation of the level of cleaved HMWK in the sample from the subject from the level of the control sample indicates the risk of a disease onset. In some embodiments, the method further comprises administering a therapeutic agent to the subject if the subject is at risk of a disease onset.

[0017] On the other hand, a kit for detecting cracked high molecular weight kininogen (HMWK) is provided, the kit comprising a first reagent (e.g., an antibody described herein) that specifically binds to cracked HMWK. In some embodiments, the kit further comprises a second reagent, a support member, or both a second reagent and a support member that bind to HMWK, and optional instructions for detecting cracked HMWK. In some embodiments, the support member is a 96-well plate.

[0018] In another aspect of the present disclosure, there is provided an isolated antibody that specifically binds to the cracked high molecular weight kininogen (HMWK). In some embodiments, the antibody binds to the same epitope as 559B-M004-B04, or competes with 559B-M004-B04 for binding to the cracked HMWK. In some embodiments, the antibody includes the same heavy chain complementary determining region and light chain complementary determining region as 559B-M004-B04, such as the same heavy chain variable region and light chain variable region as 559B-M004-B04. In an example, the antibody is 559B-M004-B04.

[0019] Any of the antibodies specific for cleaved HMWK described herein can be used in a method for detecting cleaved high molecular weight kininogen (HMWK) in a sample. This method may include: (i) contacting a sample suspected of containing cleaved HMWK with an antibody; (ii) measuring the complex of cleaved HMWK and the antibody formed in step (i); and determining the level of cleaved HMWK in the sample based on the result of step (ii). In some embodiments, the sample is a biological sample, such as a serum sample or a plasma sample obtained from a human subject. The results obtained by the method can be relied upon to determine the risk of the subject from whom the sample was obtained developing a disorder mediated by plasma kallikrein, such as HAE. In some embodiments, step (i) may be performed in the presence of ZnCl2.

[0020] Any of the immunoassay methods described herein can be in a Western blot format or an ELISA format.

[0021] In yet another aspect, isolated antibodies are provided that bind both intact high molecular weight kininogen (HMWK) and cleaved HMWK.

[0022] In some embodiments, the antibody that binds both intact HMWK and cleaved HMWK does not bind to low molecular weight kininogen (LMWK). In some embodiments, the antibody binds to the same epitope as 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08. In some embodiments, the antibody competes with 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08 for binding to intact HMWK and / or cleaved HMWK.

[0023] In some embodiments, the antibody comprises the same heavy chain CDRs and light chain CDRs as 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08. In some examples, the antibody is selected from the group consisting of 559B-M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-M0042-E06, 559B-M0070-H10, 559B-M0068-D01, and 559B-M0004-E08.

[0024] In other embodiments, antibodies that bind both intact HMWK and cleaved HMWK also bind LMWK. In some embodiments, the antibodies bind to 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038- H03, 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 or 559B-M0065-B10 bind to the same epitope. In some embodiments, the antibody is compatible with 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M 0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559-M0064-H02 or 559B-M0065-B10 compete for binding to intact HMWK, cleaved HMWK and / or LMWK.

[0025] In some embodiments, the antibodies include 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03 , 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 or 559B-M0065-B10. In some examples, the antibody is selected from the group consisting of: 559B-M0069-C09, 559B-M0038-F04, 559B-M0044-C05, 559B-M0047-H01, 559B-M0019-E12, 559B-X0004-B05, 559B-M0048-D12, 559B-M0053-G01, 559B -M0038-H03, 559B-M0017-H08, 559B-M0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 559B-M0064-H02 and 559B-M0065-B10.

[0026] The details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure will be apparent from the following drawings and detailed description of several embodiments, and from the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] The following drawings form part of this specification and are included to further illustrate certain aspects of the present disclosure, which may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

[0028] Figure 1 are graphs showing binding of 559B-M0004-B04 to intact HMWK (dark grey bars) or cleaved HMWK (light grey bars) under the indicated ELISA conditions.

[0029] Figure 2Figures showing various Fab clones binding to complete 1-chain (intact) HMWK, 2-chain (cleaved) HMWK or LMWK are presented. A: Fab clones identified using the phage display screening method described herein. Intact HWMK is shown in dark grey bars, cleaved HMWK is shown in light grey bars, and LMWK is shown in medium grey bars. B: Binding for several example Fab clones. LWMK is shown in dark grey bars, intact HMWK is shown in light grey bars, and cleaved HWMK is shown in medium grey bars.

[0030] Figure 3 559B-M0004-B04 is a graph showing the specificity of intact HMWK, cleaved HMWK or LMWK. Purified cleaved HMWK was spiked into SBT assay buffer (circles) or HMWK deficient plasma (squares). Purified intact HMWK was spiked into SBT assay buffer (triangles). Purified LMWK was spiked into SBT assay buffer (diamonds). The y-axis represents the ELISA signal in absorbance units, and the x-axis presents the kininogen concentration in μg / mL.

[0031] Figure 4 Is a detection diagram showing 2-chain HMWK (cleaved HMWK) in plasma or assay buffer. The purified cleaved HMWK was incorporated into SBT assay buffer (open circles), SBT assay buffer and analyzed in the presence of 10% plasma (squares), or incorporated into HMWK-deficient plasma and analyzed in the presence of 10% plasma (triangles). The purified cleaved HMWK was also incorporated into assay buffer and analyzed in the presence of 2.5% plasma (diamonds) or incorporated into HMWK-deficient plasma and analyzed in the presence of 2.5% plasma (solid circles). The y-axis represents the ELISA signal in absorbance units, and the x-axis represents the kininogen concentration in μg / mL.

[0032] Figure 5 are graphs showing the levels of cleaved HMWK in the indicated human plasma samples before and after activation of the contact system. A: Before and after activation of the contact system with FXIIa or ellagic acid. B: Before and after activation of the contact system with FXIIa, pKal or ellagic acid.

[0033] Figure 6 is a graph showing the levels of cleaved HMWK in plasma samples from 12 normal human donors before and after activation of the contact system with ellagic acid.

[0034] Figure 7Graphs showing cleaved HMWK levels after inhibition with pKal inhibitors are presented. A: Inhibition with landarumab / DX-2930 or C1-INH prior to activation of the contact system with ellagic acid. B: Pooled sodium citrate plasma samples were inhibited with landarumab / DX-2930 prior to activation of the contact system with 10 nM FXIIa.

[0035] Figure 8 is a graph showing the production of cleaved HMWK at the indicated time points after activation of the contact system with FXIIa or ellagic acid.

[0036] Fig. 9 is a graph showing the levels of 2-chain HMWK in plasma samples from normal subjects and subjects with HAE.

[0037] Fig.10 : is a photograph showing the results obtained by HMWK Western blot analysis, which is consistent with the results obtained by the 2-chain HMWK ELISA assay described herein. Human citrated plasma samples (normal plasma, FXII-deficient plasma, and prekallikrein-deficient plasma) were probed with mouse monoclonal anti-HMWK light chain antibody and then with goat anti-mouse detection antibody. The plasma samples analyzed were untreated or activated with 100nM pKal, 10nM FXIIa, or 10% ellagic acid.

[0038] Fig.11 Graph showing that addition of ZnCl2 to citrated or EDTA plasma samples increases the signal of 2-chain HMWK in an ELISA assay. The x-axis shows the concentration of ZnCl2 in the assay wells after 40-fold dilution.

[0039] Fig.12 Schematic diagrams presenting the discovery and development of assays using the antibodies described herein. A: Schematic diagram of the phage display method for discovering 2-chain HMWK binding antibodies. B: Exemplary sandwich ELISA assay in which 2-chain HMWK-specific antibodies / Fabs (e.g., 559B-M0004-B04) are immobilized in 96-well plates to capture 2-chain HMWK in citrated plasma, followed by washing and detection with an anti-HMWK antibody conjugated to a marker (anti-HMWK-HRP).

[0040] Fig.13 is a graph showing the results from a 2-chain HMWK sandwich ELISA standard curve in which citrated plasma samples were spiked with 2-chain HMWK (10% final dilution).

[0041] Fig.14Show that 2-chain HMWK specific antibodies are selected and screened by phage display. A: For each antibody (Fab) tested, the ratio of the results of the 2-chain HWMK binding assay on the y-axis to the results of the LMWK binding assay is plotted relative to the ratio of the 2-chain HMWK binding assay results to the 1-chain HMWK binding assay results on the x-axis. Before adding biotinylated 2-chain HMWK, 1-chain HMWK or LMWK, then streptavidin-HRP, the recombinant Fab fragments are passively fixed to 384-well plates. B: Show that the Fab fragments separated for the separation are bound to 1-chain HMWK, 2-chain HMWK or LMWK.

[0042] Fig.15 is a graph showing competition between 2-chain HMWK and kininogen peptides (HKH20 and GCP28) for binding to 559B-M0004-B04.

[0043] Fig.16 is a graph showing the standard curve of the optimized sandwich ELISA for detecting 2-chain HMWK in human plasma samples.

[0044] Fig.17 Western blot analysis comparing 2-chain HMWK levels in citrated plasma samples from healthy subjects and HAE patients is presented. A: Scatter plot comparing the percentage of 2-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Base") and during HAE attacks ("Attack"). B: ROC (Receiver Operating Characteristic) analysis comparing the sensitivity and specificity of detection for HAE Base samples with healthy subject samples (AUC = 0.977). C: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with healthy subject samples (AUC = 1). D: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with HAE Base samples (AUC = 0.625).

[0045] Fig.18Western blot analysis comparing 2-chain HMWK levels in SCAT169 plasma samples from healthy subjects and HAE patients is presented. A: Scatter plot comparing the percentage of 2-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Base") and HAE attacks ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection for HAE Base samples with healthy subject samples (AUC = 0.915). C: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with healthy subject samples (AUC = 0.967). D: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with HAE Base samples (AUC = 0.597).

[0046] Fig.19 ELISA analysis graphs comparing 2-chain HMWK levels in citrated plasma samples from healthy subjects and HAE patients are presented. A: Scatter plot comparing the percentage of 2-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Base") and during HAE attacks ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection for HAE Base samples with healthy subject samples (AUC = 0.795). C: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with healthy subject samples (AUC = 0.866). D: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with HAE Base samples (AUC = 0.709).

[0047] Fig. 20 ELISA analysis graphs comparing 2-chain HMWK levels in SCAT169 samples from healthy subjects and HAE patients are presented. A: Scatter plot comparing the percentage of 2-chain HMWK in samples from healthy subjects ("HV") and HAE patients between HAE attacks ("Base") and HAE attacks ("Attack"). B: ROC analysis comparing the sensitivity and specificity of detection for HAE Base samples with healthy subject samples (AUC = 0.999). C: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with healthy subject samples (AUC = 1). D: ROC analysis comparing the sensitivity and specificity of detection for HAE Attack samples with HAE Base samples (AUC = 0.8176). DETAILED DESCRIPTION

[0048] Plasma kallikrein (PKal) is the serine protease component of the contact system, and is the main bradykinin generating enzyme in the circulation. When exposed to external or negatively charged surface, by factor XIIa (active form of factor XII or FXII) or on the endothelial cell surface, by prolyl carboxypeptidase, the contact system is activated (Sainz IM etc., Thromb Haemost 98,77-83,2007). The activation of plasma kallikrein amplifies intrinsic coagulation by the feedback activation of its factor XII, and proteolytic cleavage of kininogen precursor, high molecular weight kininogen (HMWK), releases proinflammatory nonapeptide bradykinin and the HMWK of cracking, which contains two polypeptide chains (also referred to as 2-chain HMWK) connected by disulfide bonds.

[0049] As the main kininogenase in the circulation, plasma kallikrein is primarily responsible for the generation of bradykinin in the vasculature. Genetic defects in C1 inhibitory protein (C1-INH) lead to hereditary angioedema (HAE). Patients with HAE suffer from acute attacks of painful edema that are usually precipitated by unknown triggers (Zuraw BL et al., N Engl J Med 359, 1027-1036, 2008). The plasma kallikrein-kinin system (plasma KKS) has been implicated in various diseases by using pharmacological agents or genetic studies in animal models.

[0050] The level of cleaved HME was found to increase in HAE attacks and other pKal-related diseases. Therefore, cleaved HMWK can serve as a biomarker for monitoring disease progression and / or therapeutic efficacy. However, the art lacks suitable reagents and / or suitable assays that can effectively distinguish intact HMWK from its cleaved form.

[0051] The present disclosure is based at least in part on the development of a specific immunoassay method that allows the detection of cleaved HMWK with high specificity and sensitivity. It was observed that compared with the ELISA setting in which the antigen (in this case, the cleaved HMWK) was fixed on the support member, the sandwich ELISA in which the reagent that specifically binds to the cleaved HMWK was fixed on the support member (e.g., a multi-well plate) unexpectedly improved the detection efficiency. In addition, it was unexpectedly observed that the use of LowCross blocking buffer (containing casein) instead of a blocking buffer containing bovine serum albumin (BSA) improved the specificity and sensitivity of the detection during the initial screening to find antibodies specific to the cleaved HMWK. In addition, compared with 384-well plates, when 96-well plates were used, the detection specificity and sensitivity were further improved. The present disclosure is also based at least in part on the separation of antibodies that specifically bind to the cleaved HMWK.

[0052] Therefore, provided herein is an immunoassay for detecting the presence of or measuring the level of cleaved HMWK in a biological sample suspected of containing HMWK species using a reagent (e.g., an antibody) that specifically binds to cleaved HMWK (e.g., a cleaved HMWK with a molecular weight of 46 kDa). Considering the correlation between the level of cleaved HMWK and pKal-related or pKal-mediated disorders (e.g., HAE), the immunoassay described herein can be used to identify patients at risk of such a disease, monitor disease progression, and / or monitor the therapeutic effect for such a disorder.

[0053] I. Immunoassay for the specific detection of cleaved HMWK

[0054] One aspect of the present disclosure relates to an immunoassay for detecting cleaved HMWK with high sensitivity and specificity. The immunoassay may involve a sandwich ELISA in which a reagent that specifically binds to cleaved HMWK is immobilized on a support member that may be a 96-well plate. The immunoassay described herein allows for the selective detection of cleaved HMWK in a biological sample (e.g., a serum sample or a plasma sample), which may contain intact HMWK and cleaved HMWK as well as LMWK.

[0055] (i) High molecular weight kininogen

[0056] High molecular weight kininogen (HMWK) is present in plasma as a single polypeptide (1-chain) multi-domain (domain 1-6) protein with a molecular weight of about 110 kDa, referred to herein as complete HWMK. The human gene encoding HMWK is kininogen 1 (KNG1). KNG1 is transcribed and selectively spliced ​​to form mRNA encoding HMWK or low molecular weight kininogen (LMWK). An exemplary protein sequence of HMWK is provided below:

[0057] >gi|156231037|ref|NP_001095886.1|Kinogen-1 isoform 1 precursor [Homo sapiens]

[0058] (SEQ ID NO:1)

[0059] For example, intact HMWK, also referred to herein as "intact kininogen", can be assayed using coagulants or immunological methods, such as radioimmunoassays (see, e.g., Kerbiriou-Nabias, DM, Br J Haematol, 1984, 56(2): 2734-86). Monoclonal antibodies to the light chain of human HMWK are known. See, e.g., Reddigari, SR & Kaplan, AP, Blood, 1999, 74: 695-702. HMWK assays that rely on chromogenic substrates can also be used. See, e.g., Scott, CF et al., Thromb Res, 1987, 48(6): 685-700; Gallimore, MJ et al. Thromb Res, 2004, 114(2): 91-96.

[0060] HMWK is cleaved by pKal in domain 4 to release the proinflammatory peptide bradykinin of 9 amino acids and the 2-chain form of HMWK, referred to herein as the cleaved HMWK. The 2 chains of HMWK are a heavy chain comprising domains 1-3, and a light chain comprising domains 5 and 6, which are connected by a disulfide bond. After the initial cleavage of complete HMWK, the heavy chain and the light chain have a molecular weight of about 65kDa and 56kDa, respectively. Further proteolytic processes cause the generation of a 46kDa light chain.

[0061] Exemplary sequences of the heavy and light chains of cleaved kininogens are provided below.

[0062] >Cleaved kininogen-1 heavy chain

[0063] QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTAQYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSI VQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISLMK(SEQ ID NO:2)

[0064] >Cleaved kininogen-1 light chain

[0065] SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNKGKKNGKHNG WKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQMKESYYFDLTDGLS(SEQ ID NO:3)

[0066] (ii) Antibodies specific for cleaved HMWK

[0067] The immunoassay methods described herein can use any reagent that can specifically bind to cleaved HMWK, such as a reagent that recognizes a neo-epitope on cleaved HMWK that is not present on intact HMWK. In some embodiments, the reagent that binds to cleaved HMWK is an antibody.

[0068] Antibodies (used interchangeably in the plural) are immunoglobulin molecules that can specifically bind to a target (e.g., carbohydrates, polynucleotides, lipids, polypeptides, etc.) through at least one antigen recognition site, which is located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes not only complete (i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chains (scFv), mutants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, double antibodies, linear antibodies, single-chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, such as IgD, IgE, IgG, IgA, or IgM (or its subclass), and antibodies do not need to be of any particular class. Depending on the antibody amino acid sequence of its heavy chain constant domain, immunoglobulins can be divided into different classes. There are five major types of immunoglobulins: IgA, IgD, IgE, IgG and IgM, several of which can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different types of immunoglobulins are well known.

[0069] Any antibody described herein can be monoclonal or polyclonal. "Monoclonal antibody" refers to a homogenous antibody population, and "polyclonal antibody" refers to a heterogenous antibody population. These two terms do not limit the source of the antibody or its production method.

[0070] "Specific binding" is a term well known in the art for the HMWK of the cracking or the antibody of its epitope, and the method for determining such specific binding is also well known in the art. If the molecule reacts or associates with a longer duration and / or higher affinity more frequently and more quickly with a specific target antigen (herein the HMWK of the cracking) than it and an alternative target (for example, complete HMWK and / or LMWK), then the molecule is considered to show "specific binding". If the antibody is bound to other substances with greater affinity, avidity, more easily and / or with a longer duration than it, the antibody "specifically binds" to the target antigen. For example, the antibody that specifically (or preferentially) binds to the HMWK of the cracking or the epitope therein is compared with being bound to other antigens (for example, complete HMWK or LMWK) or other epitopes in the same antigen with greater affinity, avidity, more easily and / or with a longer duration in conjunction with the antibody of the target antigen. It can also be understood by reading this definition that, for example, the antibody that specifically binds to the first target antigen can specifically or preferentially bind to the second target antigen or can be non-specific or preferentially bound to the second target antigen. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding. Typically, but not necessarily, reference to binding implies preferential binding.

[0071] In some embodiments, the antibodies described herein that specifically bind to cleaved HMWK (as well as other antibodies that bind both cleaved HMWK and intact HMWK and optionally LMWK) have a suitable binding affinity for cleaved HMWK (or another target antigen as described herein). As used herein, "binding affinity" refers to the apparent association constant or K A . K A is the dissociation constant (K D ). The antibodies described herein may have at least 10 -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (K D ). Increased binding affinity corresponds to a decreased K D The higher affinity of an antibody binding to a first target relative to a second target can be determined by the K for binding to the second target. A (or value K D ), for a higher K for binding to the first target A (or a smaller value K D). In this case, the antibody has specificity for a first target (e.g., a protein in a first conformational form or a mimetic thereof) relative to a second target (e.g., the same protein in a second conformational form or a mimetic thereof; or a second protein). The difference in binding affinity (e.g., for specificity or other comparisons) can be at least 1.5, 2, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1000, 10,000, or 10 5 For example, the binding affinity of an antibody that specifically binds to cleaved HMWK as described herein may be 10 times, 100 times, 10,000 times, or 10 times greater than the binding affinity of the antibody to intact HMWK and / or LMWK. 5 times.

[0072] 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 fluorimetry). Exemplary conditions for evaluating binding affinity are in HBS-P buffer (10 mM HEPES pH 7.4, 50 mM NaCl, 0.005% (v / v) surfactant P20). These techniques can be used to measure the concentration of bound binding protein as a function of target protein concentration. The concentration of bound binding protein ([bound]) is related to the concentration of free target protein ([free]) and the concentration of binding sites for binding protein on the target, as represented by the following equation, where (N) is the number of binding sites per target molecule:

[0073] [Bound] = [N][Free] / (Kd + [Free])

[0074] However, it is not always necessary to determine K precisely. A , because sometimes it is enough to obtain A A quantitative measure of affinity is proportional (e.g., determined using methods such as ELISA or FACS analysis) and can therefore be used for comparison, e.g., to determine whether a higher affinity (e.g., 2-fold higher) yields a qualitative measure of affinity or to derive an inference about affinity, e.g., by activity in a functional assay (e.g., an in vitro or in vivo assay).

[0075] In some embodiments, an antibody that specifically binds to cleaved HMWK (also referred to as an anti-cleaved HMWK antibody) binds to an epitope of the cleaved HMWK identical to 559B-M004-B04. An "epitope" refers to a site on a target antigen to which a binding protein (e.g., an antibody, such as a Fab or full-length antibody) binds. The site may be composed entirely of amino acid components, entirely of chemical modifications of the amino acids of a protein (e.g., a glycosyl moiety), or a combination thereof. An overlay epitope includes at least one common amino acid residue, glycosyl, phosphate, sulfate, or other molecular feature. In some cases, an epitope is linear; in other cases, an epitope is conformational.

[0076] A first antibody "binds to the same epitope" as a second antibody if the first antibody binds to the same site on the target antigen as the second antibody binds, or to a site that overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlaps), e.g., in terms of amino acid sequence or other molecular features (e.g., sugar, phosphate, or sulfate groups) with the site to which the second antigen binds.

[0077] In some embodiments, an antibody that specifically binds to cleaved HMWK competes with 559B-M004-B04 for binding to HMWK. If the binding of the first antibody to its epitope reduces the amount of the second antibody bound to its epitope (e.g., by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more), the first antibody "competes for binding" with the second antibody. Competition can be direct (e.g., the first antibody binds to an epitope that is identical to or overlaps with the epitope bound by the second antibody), or indirect (e.g., the binding of the first antibody to its epitope results in a steric change in the target antigen that reduces the ability of the second antibody to bind to its epitope).

[0078] In some embodiments, antibodies that specifically bind to cleaved HMWK include V H chain, the V H Chain includes corresponding V with 559B-M004-B04 H The CDRs are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to each other. H CDR1, V H CDR2 and / or V H Alternatively or additionally, an antibody that specifically binds to cleaved HMWK comprises a CDR3 corresponding to 559B-M004-B04. L The CDRs are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to each other. LCDR1, V L CDR2 and / or V L CDR3. In some embodiments, the antibody that specifically binds to cleaved HMWK has the same heavy chain complementarity determining region (CDR) and / or light chain complementarity determining region (CDR) as 559B-M004-B04.

[0079] "Complementarity Determining Region" or "CDR" as known in the art refers to a non-contiguous sequence of amino acids within an antibody variable region that confers antigen specificity and binding affinity. Typically, there are three (3) CDRs in each heavy chain variable region and three (3) CDRs in each light chain variable region. The precise amino acid sequence boundaries of a given CDR can be readily determined using any of a number of well-known schemes, including those described by Kabat et al., (1991), 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (Kabat's numbering scheme); Al-Lazikani et al., (1997) JMB 273, 927-948 (Chothia's numbering scheme); MacCallum et al., J. Mol. Biol. 262:732-745 (1996) (Contact numbering scheme); Lefranc MP et al., Dev Comp Immunol, 2003 January; 27(1):55-77 (IMGT numbering scheme); and Honegger A and Pluckthun A, J Mol Biol, 2001 Jun. 8; 309(3):657-70, (AHo numbering scheme).

[0080] The boundaries of a given CDR may vary depending on the scheme used for identification. For example, the Kabat scheme is based on structural alignments, while the Chothia scheme is based on structural information. The Contact scheme is based on analysis of complex crystal structures and is similar in many respects to the Chothia numbering scheme. Therefore, unless otherwise indicated, the term "complementarity determining region" or "CDR" for a given antibody should be understood to encompass complementarity determining regions defined by any known scheme as described above.

[0081] If an antibody has the same V as 559B-M004-B04 (and other exemplary antibodies disclosed herein) by the same numbering scheme, H CDR and / or V LCDRs, such antibodies are considered to have the same CDRs as 559B-M004-B04 (or other exemplary antibodies disclosed herein), and such antibodies are within the scope of the present disclosure. For example, such antibodies may have the same V CDRs as clone 559B-M004-B04 as determined by the Chothia numbering scheme. H CDR and / or V L In another example, an anti-cleavage HMWK antibody within the scope of the present disclosure may have the same CDR as clone 559B-M004-B04, as determined by the Kabat numbering scheme. H CDR and / or V L CDR.

[0082] Alternatively or additionally, the anti-split HMWK antibody comprises a V H The V chains are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical. H chain, and / or with 559B-M004-B04 V L The V chains are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical. L In some embodiments, the antibody is 559B-M004-B04.

[0083] The "percent identity" of two amino acid sequences is determined using an algorithm based on Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified from Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. This algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. J. Mol. Biol. 215:403-10, 1990. BLAST protein searches can be performed with the XBLAST program, score = 50, wordlength = 3, to obtain amino acid sequences homologous to the protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST as described in Altschul et al., Nucleic Acids Res. 25 (17):3389-3402, 1997 can be used. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (eg, XBLAST and NBLAST) can be used.

[0084] The sequences of the heavy chain variable region and the light chain variable region of 559B-M004-B04 are shown below. The CDR1, CDR2 and CDR3 sequences of the heavy chain and the CDR1, CDR2 and CDR3 sequences of the light chain are underlined and shown in bold (identified by a scheme as an example).

[0085] >559B-R0048-A01 (559B-M0004-B04) heavy chain amino acid sequence (SEQ ID NO: 4)

[0086]

[0087] >559B-R0048-A01 (559B-M0004-B04) light chain amino acid sequence (SEQ ID NO: 5)

[0088]

[0089] In some cases, the antibody specifically binding to the HMWK of the cracking may be included in one or more (e.g., at most 5, at most 3, or at most 1) conservative mutations in one or more heavy chain CDRs or one or more light chain CDRs of 559B-M0004-B04, such as at a position where the residue is unlikely to participate in the interaction with the HMWK of the cracking. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is performed. Variants can be prepared according to methods known to those of ordinary skill in the art for changing polypeptide sequences, such as those found in the literature of the compilation of these methods, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al., ed., 2nd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al., John Wiley & Sons, Inc., New York. Conservative substitutions of amino acids include substitutions between amino acids within the following groups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T; (f) Q, N; and (g) E, D.

[0090] Antibodies capable of binding to cleaved HMWK as described herein (as well as antibodies capable of binding to intact HMWK and / or LMWK) can be prepared by any method known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0091] In some embodiments, antibodies specific to the target antigen (cracked HMWK, complete HMWK and / or LMWK) can be prepared by conventional hybridoma technology. Full-length target antigens or fragments thereof, optionally coupled to carrier proteins (such as KLH), can be used to immunize host animals to produce antibodies that bind to the antigen. As further described herein, the immunization pathways and plans of host animals are generally consistent with established and conventional techniques for antibody stimulation and production. General techniques for producing mouse antibodies, humanized antibodies and human antibodies are known in the art and described herein. It is expected that any mammalian subject including humans or cells producing antibodies therefrom can be manipulated as the basis for producing mammalian (including human) hybridoma cell lines. Typically, a certain amount of immunogens, including those described herein, are inoculated intraperitoneally, intramuscularly, orally, subcutaneously, plantarly and / or intradermally to host animals.

[0092] Hybridomas can be prepared from lymphocytes and immortalized myeloma cells using conventional somatic cell hybridization techniques, such as Kohler, B. and Milstein, C. (1975) Nature 256: 495-497 or as modified by Buck, DW, et al., In Vitro, 18: 377-381 (1982). Available myeloma lines, including but not limited to X63-Ag8.653 and those from Salk Institute, Cell Distribution Center, San Diego, Calif., USA, can be used for hybridization. In general, the technology involves the use of a fusion agent (such as polyethylene glycol) or electrical techniques well known to those skilled in the art to fuse myeloma cells and lymphocytes. After fusion, cells are separated from the fusion culture medium and grown in a selective growth culture medium, such as hypoxanthine-aminopterin-thymidine (HAT) culture medium, to eliminate unhybridized parental cells. Any culture medium supplemented with serum or without serum as described herein can be used to culture hybridomas secreting monoclonal antibodies. As another alternative of cell fusion technology, EBV immortalized B cells can be used for producing anti-PKal monoclonal antibodies as described herein. If necessary, then amplify and subclone hybridoma, and measure the anti-immunogenic activity of supernatant by conventional immunoassay procedure (for example, radioimmunoassay, enzyme immunoassay or fluorescent immunoassay).

[0093] Hybridoma that can be used as antibody source comprises all derivatives, daughter cells of parent hybridoma that produces the monoclonal antibody that can disturb PKal activity.The hybridoma that produces such antibody can use known program growth in vitro or in vivo.If necessary, monoclonal antibody can be separated from culture medium or body fluid by conventional immunoglobulin purification procedure (such as ammonium sulfate precipitation, gel electrophoresis, dialysis, chromatography and ultrafiltration).If there is undesirable activity, can for example by making goods run on the adsorbent prepared by the immunogen attached to solid phase and from the immunogen elution desired antibody or make the desired antibody be released from the immunogen, and remove undesirable activity. Large quantities of antibodies (e.g., monoclonal antibodies) can be produced by immunizing a host animal with a target antigen or a fragment comprising a target amino acid sequence conjugated to a protein that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor, using a bifunctional reagent or a derivatizing reagent, such as maleimidobenzoyl sulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R1N=C=NR (wherein R and R1 are different alkyl groups).

[0094] If necessary, the antibody (monoclonal or polyclonal) of interest (e.g., produced by a hybridoma) can be sequenced and then the polynucleotide sequence can be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector of a host cell and the host cell can then be amplified and frozen for further use. Alternatively, the polynucleotide sequence can be used for genetic manipulation to improve the affinity (affinity maturation) of the antibody, or other features. It can be expected that the genetic manipulation antibody sequence is to obtain greater affinity and / or specificity to the target antigen. It will be apparent to those skilled in the art that one or more polynucleotide changes can be made to the antibody and still maintain its binding specificity to the target antigen.

[0095] In other embodiments, fully human antibodies can be obtained by using commercially available mice that have been engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce more desirable (e.g., fully human antibodies) or more robust immune responses can also be used to produce humanized or human antibodies. An example of such technology is the Xenomouse from Amgen, Inc. (Fremont, Calif.). RTM and HuMAb-Mouse from Medarex, Inc. (Princeton, NJ) RTM and TC Mouse TMAlternatively, antibodies can be produced recombinantly by phage display or yeast technology. See, for example, U.S. Patent Nos. 5,565,332, 5,580,717, 5,733,743, and 6,265,150; and Winter et al. (1994) Annu. Rev. Immunol. 12: 433-455. Alternatively, phage display technology (McCafferty et al. (1990) Nature 348: 552-553) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors.

[0096] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by conventional methods. For example, F(ab')2 fragments can be produced by digesting antibody molecules with pepsin, and Fab fragments can be produced by reducing the disulfide bonds of F(ab')2 fragments.

[0097] Single-chain antibodies can be prepared by connecting the nucleotide sequence encoding the heavy chain variable region and the nucleotide sequence encoding the light chain variable region through recombinant technology. Preferably, a flexible linker is incorporated between the two variable regions. Alternatively, the technology described for producing single-chain antibodies (U.S. Patents 4,946,778 and 4,704,692) can be used to produce phage or yeast scFv libraries, and scFv clones specific to PKal can be identified from the library according to the following routine procedures. Positive clones can be further screened to identify those clones of specific binding target antigens, such as cracked HMWK.

[0098] In some embodiments, antibodies specific to cleaved HMWK (or complete HMWK or LMWK) can be isolated from an antibody library, which can be a synthetic library or a natural library. A natural antibody library refers to a library derived from a natural source (e.g., a human donor) after conventional procedures. A synthetic antibody library refers to a library whose members are designed according to predetermined rules (e.g., with a complete randomized CDR region, such as CDR; or a semi-randomized CDR region, such as CDR1 or CDR2 of a heavy chain, a light chain, or both a heavy chain and a light chain).

[0099] In some cases, the antibody library is a display library (e.g., a phage display library or a yeast display library). A display library is a collection of entities; each entity includes an available polypeptide component and a recyclable component encoding or identifying the polypeptide component. The polypeptide component is diverse, showing different amino acid sequences. The polypeptide component can have any length, for example, from three amino acids to more than 300 amino acids. The display library entity can include more than one polypeptide component, such as two polypeptide chains of sFab. In an exemplary embodiment, the display library can be used to identify proteins that bind to cleaved HMWK (and other target antigens described herein). In the selection, the polypeptide component of each member of the library is probed with cleaved HMWK (or its fragment), and if the polypeptide component is bound to the cleaved HMWK, the display library member is usually identified by being retained on a carrier. Fig.12 An exemplary description of using a phage display antibody library to identify antibodies specific for cleaved HMWK is provided in .

[0100] The retained display library members are recovered from the support and analyzed. The analysis can include amplification and subsequent selection under similar or dissimilar conditions. For example, positive selection and negative selection can be performed alternately. The analysis can also include determining the amino acid sequence of the polypeptide component and purifying the polypeptide component for detailed characterization.

[0101] Antibodies obtained according to methods known in the art and described herein can be characterized using methods well known in the art. For example, one method is to identify the epitope to which the antigen binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes on proteins, including solving crystal structures of antibody-antigen complexes, competition assays, gene fragment expression assays, and synthetic peptide-based assays, such as described in Chapter 11 of Harlow and Lane's Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In another example, an epitope is a protein that is bound by an antigen. PlottingCan be used to determine the sequence to which the antibody is bound. The epitope can be a linear epitope, that is, contained in a single amino acid segment, or a conformational epitope formed by the three-dimensional interaction of amino acids, which may not necessarily be contained in a single segment (primary structure linear sequence). Peptides of different lengths (e.g., at least 4-6 amino acids long) can be separated or synthesized (e.g., recombined) and used for binding tests with antibodies. In another example, the epitope to which the antibody is bound can be determined in a system screening by using overlapping peptides derived from the target antigen sequence and binding by antibody determination. According to the gene fragment expression test, the open reading frame encoding the target antigen is randomly or fragmented by specific gene construction, and the reactivity of the expressed fragment of the antigen with the antibody to be tested is determined. Gene fragments can be produced, for example, by PCR, and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. The combination of the antibody and the radiolabeled antigen fragment is then determined by immunoprecipitation and gel electrophoresis.

[0102] Certain epitopes can also be identified by using a large library (phage library) of random peptide sequences displayed on the surface of phage particles. Alternatively, a library of defined overlapping peptide fragments can be tested in a simple binding assay for the combination of test antibodies. In another example, mutagenesis, domain exchange experiments and alanine scanning mutagenesis of the antigen binding domain can be performed to identify residues required, sufficient and / or necessary for epitope binding. For example, domain exchange experiments can be performed using mutants of target antigens in which various fragments of the HMWK polypeptide have been replaced (exchanged) with sequences from closely associated but antigenically different proteins. By assessing the combination of antibodies with mutant HMWK, the importance of specific antigen fragments to antibody binding can be assessed.

[0103] Alternatively, competition assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibodies. Competition assays are well known to those skilled in the art.

[0104] Any anti-cleaved HMWK antibody is also within the scope of the present disclosure.

[0105] (iii) Immunoassay method

[0106] Provided herein is an immunoassay for detecting cracked HMWK. As used herein, the term "immunoassay" may be interchangeably referred to as an immunoassay based on immunity or an immunological assay based on immunology. Typically, an immunoassay uses a reagent (e.g., antibody) that is bound to a molecule to detect the presence and / or concentration (level) of a molecule (e.g., HMWK) in a sample. Examples of immunoassays include Western blots, enzyme-linked immunosorbent assays (ELISA), lateral flow assays, radioimmunoassays, electrochemiluminescence-based detection assays, magnetic immunoassays, and related techniques. In some embodiments, the immunoassay is an ELISA assay. In some embodiments, the immunoassay is a sandwich ELISA assay. In some embodiments, the immunoassay is a lateral flow assay.

[0107] ELISA is known in the art (see, e.g., Crowther, John R (2009). "The ELISA Guidebook." 2nd ed. Humana Press and Lequin R (2005). "Enzyme immunoassay (EIA) / enzyme-linked immunosorbent assay (ELISA)". Clin. Chem. 51 (12): 2415-8), and exemplary ELISAs are described herein. Kits for performing ELISA are also known in the art and commercially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).

[0108] In order to perform the immunoassay method described herein, a sample can be obtained from a subject. As used herein, "sample" refers to a composition comprising a tissue (e.g., blood, plasma, or protein) from a subject. The sample includes an initial untreated sample taken from a subject and a subsequently treated sample, such as a partially purified or preserved form. Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is a body fluid sample, such as a serum or plasma sample. The sample analyzed by the immunoassay method described herein can be an initial untreated sample taken from a subject or a subsequently treated sample, such as a partially purified or preserved form. In some embodiments, multiple (e.g., at least 2, 3, 4, 5 or more) samples can be collected from a subject over time or at specific time intervals, for example, to assess the progression of a disease or disorder or to evaluate the efficacy of a treatment. Multiple samples can be obtained before and after treatment or during treatment.

[0109] The sample can be obtained from the subject using any method known in the art. In some embodiments, the sample is obtained from the subject by collecting the sample (e.g., blood sample) into an evacuated collection tube (e.g., a vacuum blood collection tube). In some embodiments, the vacuum collection tube contains one or more protease inhibitors, for example to reduce or prevent in vitro activation of the contact system during sample collection. Such protease inhibitors may be included in a liquid formulation. In some embodiments, the protease inhibitor includes at least one serine protease inhibitor and at least one cysteine ​​protease inhibitor. Such vacuum collection tubes are known in the art. See, for example, PCT Application No. US2016 / 046681. Optionally, the vacuum blood collection tube may further include one or more anticoagulants.

[0110] A "patient," "subject," or "host" to be treated by the disclosed methods (these terms are used interchangeably) may refer to a human or non-human animal. In some embodiments, the subject is suspected of having or is at risk of or has a disorder mediated by kallikrein, such as a disorder mediated by bradykinin, such as hereditary angioedema (HAE), non-histamine-dependent idiopathic angioedema, rheumatoid arthritis, Crohn's disease, lupus, Alzheimer's disease, septic shock, burns, cerebral ischemic / reperfusion injury, cerebral edema, diabetic retinopathy, diabetic nephropathy, macular edema, vasculitis, arterial or venous thrombosis, thrombosis associated with ventricular assist devices or stents, heparin-induced thrombocytopenia with thrombosis, thromboembolic disease, and coronary heart disease with unstable angina, edema, eye disease, gout , intestinal diseases, oral mucositis, neuropathy pain, inflammatory pain, spinal stenosis degenerative spondylosis, postoperative ileus, aortic aneurysm, osteoarthritis, hereditary angioedema, pulmonary embolism, stroke, head trauma or peritumoral brain 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 related to protein misfolding, diseases related to angiogenesis, hypertensive nephropathy and diabetic nephropathy, allergic and respiratory diseases (e.g., allergies, asthma, chronic obstructive pulmonary disease, acute respiratory distress syndrome, cystic fibrosis, persistent rhinitis) and tissue injury (e.g., burns or chemical injury).

[0111] Alternatively or additionally, the subject requiring analysis as described herein may be a patient with the disease or disorder. Such a subject may be currently under an attack of a disease (e.g., HAE), or may have suffered from a disease in the past (e.g., currently during a period of disease quiescence). In some instances, the subject is a human patient who may be treating a disease, such as a C1 esterase inhibitor (C1-INH), a plasma kallikrein inhibitor, or a bradykinin inhibitor. In other cases, such a human patient may not have such treatment.

[0112] The samples described herein can be analyzed using reagents that specifically bind to the cleaved HMWK to determine the level of cleaved HMWK in the sample. In some embodiments, the immunoassay method described herein can be in the form of a sandwich ELISA, in which a first reagent (e.g., an antibody described herein) that specifically binds to the cleaved HMWK is fixed to a support member. The support member can then be cultured with the sample described herein for a suitable period of time under conditions that allow the formation of a cleaved HMWK / first reagent (e.g., antibody) complex. This complex can then be detected using a second reagent that binds to HMWK. The second reagent can be conjugated to a marker that can release a signal directly or indirectly. The intensity of the signal represents the level of cleaved HMWK in the sample.

[0113] Any support member known in the art can be used in the method, including but not limited to membranes, beads, slides or multiwell plates. The selection of a suitable support member for an immunoassay method will depend on various factors, such as sample quantity and the method of detecting the signal released from the label conjugated to the second reagent.

[0114] In some embodiments, the support member is a membrane, such as a nitrocellulose membrane, a polyvinylidene fluoride (PVDF) membrane, or a cellulose acetate membrane. In some examples, the immunoassay method can be a Western blot assay format or a lateral flow assay format.

[0115] In some embodiments, the support member is a multi-well plate, such as an ELISA plate. In some embodiments, the immunoassay method described herein can be performed on a high-throughput platform. In some embodiments, multi-well plates, such as plates with 24 wells, 48 ​​wells, 96 wells, 384 wells or more wells, can be used for high-throughput immunoassay methods. A separate immunoassay method can be performed in parallel in each well. Therefore, it is generally desirable to use a plate reader to measure multiple wells in parallel to increase the assay throughput. In some embodiments, a plate reader capable of imaging multiple wells (e.g., 4 wells, 16 wells, 24 wells, 48 ​​wells, 96 wells, 384 wells or more wells) in parallel can be used for the platform. For example, a commercially available plate reader (e.g., a plate::visual system available from Perkin Elmer, Waltham, MA) can be used. The plate reader is capable of performing kinetic-based fluorescence analysis. The plate::visual system has optical devices with high collection efficiency and has optical devices specifically designed for analyzing 96 wells in parallel. Other suitable parallel plate readers include, but are not limited to, SAFIRE (Tecan, San Jose, CA), (Molecular Devices, Union City, CA), FDSS7000 (Hamamatsu, Bridgewater, NJ), and CellLux (Perkin Elmer, Waltham, MA).

[0116] As described in Example 1, it was unexpectedly discovered that the surface area and / or volume of the wells of a multi-well plate can affect the results of an immunoassay. In some embodiments, the immunoassay is performed in a 96-well plate, such as a 96-well ELISA plate.

[0117] In other embodiments, the high throughput screening immunoassay methods of the present disclosure can be automated (eg, adapted for robotic assays).

[0118] In some embodiments, immunoassay methods can be performed on low-throughput platforms, including single immunoassay formats. For example, low-throughput platforms can be used to measure the presence and amount of HMWKs that are cleaved in biological samples (e.g., biological tissues, tissue extracts) for diagnostic methods, monitoring disease and / or treatment progress, and / or predicting whether a disease or disorder may benefit from a specific treatment.

[0119] Any method known in the art can be used, as described herein, to fix a reagent (e.g., an antibody described herein) that specifically binds to the cleaved HMWK to a support member. In some embodiments, fixation includes binding a reagent (e.g., an antibody) to a support member. In other embodiments, fixation includes adsorbing the antibody to the support member. This adsorption method can be performed, for example, by culturing the antibody in a buffer in the wells of a multi-well plate. In some embodiments, reagents such as antibodies are provided in a coating buffer and cultured in the wells of a multi-well plate. Coating buffers are obvious to those skilled in the art and can be prepared or obtained from commercial sources. Non-limiting examples of coating buffers include 50mM sodium bicarbonate, pH 9.6; 0.2M sodium bicarbonate, pH 9.4; phosphate buffer solution (50mM phosphate, pH8.0, 0.15M NaCl); carbonate-bicarbonate solution; and TBS (50mM TRIS, pH 8.0, 0.15M NaCl).

[0120] In some embodiments, the first reagent is immobilized on the support member by hydrophobic interactions between the first reagent and the support member. In some embodiments, the first reagent is immobilized on the support member using electrophoretic transfer.

[0121] Whether before or after fixing, or before and after fixing, the support member can be cultivated with blocking buffer. Generally, blocking buffer is used for any exposed surface of the blocking support film (for example, the position not occupied by the first reagent on the support film). Using blocking buffer can reduce the detected baseline signal (i.e. "background interference") and / or improve the sensitivity of immunoassay and / or reduce the non-specific binding of sample components to the support film. As described in Example 1, the selection of blocking buffer affects the result of immunoassay. In some embodiments, the blocking buffer contains serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the blocking buffer is a BSA buffer (for example, 2% BSA in a PBS buffer). In some embodiments, the blocking buffer does not contain serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the blocking buffer comprises casein fragments, and optional NaCl and tween (Tween), and can have pH 7.0-7.4. In some embodiments, the casein fragments are high-purity casein fragments. Such blocking buffer can be prepared or obtained from commercial sources (eg, Blocking Solution Low Cross from CANDOR Bioscience).

[0122] A support member to which a reagent specific for the cracked HMWK is attached can be contacted (cultured) with a sample suspected of containing the cracked HMWK described herein. In general, the term "contact" refers to exposing the support member to a biological sample or reagent for a suitable period of time, sufficient to form a complex between the reagent, such as an antibody and the cracked HMWK in the sample, if any. Afterwards, the sample can be removed from the support member and then washed multiple times to remove any unbound cracked HMWK. In some embodiments, the contact is performed by capillary action, wherein the biological sample or reagent moves on the surface of the support membrane.

[0123] The support member may then be incubated with the second agent that binds to the HMWK for a suitable period of time to allow the second agent to bind to the HMWK attached to the support member.

[0124] The second reagent can be any reagent capable of binding to HMWK, such as an antibody capable of binding to HMWK (specific for HMWK in cleaved form or cross-reacting with both cleaved HMWK and intact HMWK). In some embodiments, the second reagent comprises one or more antibodies that bind to HWMK (cleaved and / or intact). In some embodiments, the antibody is a mouse monoclonal antibody or a monoclonal sheep antibody. It is conjugated to a marker that is a compound capable of directly or indirectly releasing a signal (e.g., by interacting with one or more additional compounds).

[0125] In some embodiments, the marker is a signal releasing agent, which is an agent that releases a signal directly (e.g., a dye or fluorophore) or an agent that releases a signal upon interaction with a substrate (e.g., such as HRP or β-galactosidase, which can convert a colorless substrate into a colored product). As used herein, the term "fluorophore" (also called "fluorescent marker" or "fluorescent dye") refers to a portion that absorbs light energy at a defined excitation wavelength and emits light energy at a different wavelength.

[0126] In other embodiments, the marker can be a member of a receptor-ligand pair. As used herein, "ligand-receptor pair" refers to a pair of molecules (e.g., biomolecules) having specific affinity to each other, such as biotin-streptavidin. In this case, the support member carrying the HMWK-second reagent of the first reagent-cracking can also be cultivated with another member of the ligand-receptor pair for a suitable time period so that two members of the receptor-ligand pair interact. As described herein, another member of the receptor-ligand pair is conjugated with a signal release agent. In an example, the second reagent is conjugated with biotin and detected using the streptavidin conjugated with HRP.

[0127] After washing away any unbound conjugate, a substrate solution may be added to aid detection. For example, after a set interval, the reaction may be stopped (e.g., by adding 1N NaOH) and the concentration of the colored product formed may be measured in a spectrophotometer. The intensity of the color is proportional to the concentration of bound antigen.

[0128] Next, the signal released from the marker as described herein can be detected / measured by conventional methods, which depend on the specific form of the immunoassay method and the signal releasing agent used therein. As used herein, the term "measuring" or "measurement" or alternatively "detecting" or "detection" means assessing the presence, absence, quantity or amount (which may be an effective amount) of a substance in a sample, including deriving a qualitative or quantitative concentration level of such a substance, or otherwise assessing a value or classification of a subject.

[0129] Assays, such as Western blot assays, can further involve the use of a quantitative imaging system, such as the commercially available LICOR imaging technology (see, e.g., LICOR Imaging from LI-COR Biosciences). CLx infrared imaging system). In some embodiments, an electrochemiluminescence detection assay or an assay relying on a combination of electrochemiluminescence and patterned array technology (e.g., ECL or MULTI-ARRAY technology assays from Meso Scale Discovery (MSD)) is used.

[0130] Any immunoassay method described herein, such as one or more steps of the immunoassay method, can be carried out in a suitable assay buffer, which is obvious to those skilled in the art. In some embodiments, the assay buffer contains or has been supplemented with ZnCl2. In some embodiments, the assay buffer contains at least about 10 μM, 20 μM, 30 μM, 40 μM, 50 μM, 60 μM, 70 μM, 80 μM, 90 μM, 100 μM, 150 μM, 200 μM, 250 μM, 300 μM, 350 μM, 400 μM, 450 μM, 500 μM or more ZnCl2. In some embodiments, such an assay buffer containing ZnCl2 is used in such a step: wherein a reagent specific to the HMWK of cleavage (e.g., an antibody specific to the HMWK of cleavage) is combined with the HMWK of cleavage. ZnCl2 enhances the binding activity of reagents (e.g., antibodies) to the HMWK of cleavage.

[0131] In some embodiments, the assay buffer contains serum albumin, such as bovine serum albumin or human serum albumin. In some embodiments, the assay buffer contains at least about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.12%, 0.014%, 0.16%, 0.18%, 0.2%, 0.25%, 0.3%, 0.4% or more BSA. In some embodiments, the assay buffer contains a surfactant, such as Tween-20. In some embodiments, the assay buffer contains 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1% or more surfactant. In one example, the assay buffer contains 0.1% BSA and 0.05% Tween-20 in PBS.

[0132] (iv) Diagnostic and prognostic applications

[0133] Given the correlation between the level of cleaved HMWK and such a disease or disorder (e.g., as a biomarker), the assays and kits described herein can be used to assess a disease or disorder associated with plasma kallikrein, such as those described herein (e.g., HAE). Alternatively or additionally, the assays and kits described herein can be used to monitor the progression of such a disease, assess the efficacy of a disease treatment, identify patients suitable for a particular treatment, and / or predict a disease state (e.g., onset versus quiescence) in a subject.

[0134] In some embodiments, the level of cleaved HMWK determined by the immunoassay method described herein can be relied upon to assess whether the subject (e.g., a human patient) from which the biological sample is obtained has a disease or disorder associated with plasma kallikrein (e.g., HAE or an autoimmune disease, such as RA, UC, and Crohn's disease), or is at risk of the disease or disorder. The level of cleaved kininogen can then be compared with the total amount of intact kininogen or kininogen in the sample to determine the value (e.g., percentage) of cleaved kininogen, the value of intact kininogen, or both in the sample. The value of cleaved kininogen and / or intact kininogen can be compared with a reference value to determine whether the subject has a pKal-mediated disorder, such as HAE or an autoimmune disease, such as RA, UC, and Crohn's disease, or is at risk of the disease. For example, if the percentage of cleaved kininogen is or is higher than a reference number, the subject can be identified as having a pKal-mediated disorder, such as HAE, RA, UC, and Crohn's disease, or is at risk of the disease. Alternatively, if the percentage of intact kininogen is at or below a reference number, the subject can be identified as having, or being at risk for, a pKal-mediated disorder, such as HAE, RA, UC, and Crohn's disease.

[0135] In some embodiments, the samples for analysis of the methods described herein are derived from human subjects with hereditary angioedema (HAE) or at risk of HAE. HAE is also known as "Quincke edema", C1 esterase inhibitor deficiency, C1 inhibitor deficiency, and hereditary angioneurotic edema (HANE). HAE is characterized by recurrent attacks of severe swelling (angioedema), which can affect, for example, the limbs, face, genitals, gastrointestinal tract, and trachea. Symptoms of HAE include, for example, swelling of the arms, legs, lips, eyes, tongue, and / or throat; tracheal obstruction that may involve swelling of the throat and sudden hoarseness; repeated attacks of abdominal cramps without obvious cause; and / or swelling of the intestines, which may be severe and can lead to abdominal cramps, vomiting, dehydration, diarrhea, pain, and / or shock. About one-third of individuals with this HAE develop a non-itching rash called erythema marginale during an attack.

[0136] Swelling of the trachea can be life-threatening and causes death in some patients. The estimated mortality rate is 15-33%. HAE results in approximately 15,000-30,000 emergency room visits each year.

[0137] Trauma or stress, for example, dental surgery, illness (e.g., viral illnesses, such as colds and flu), menstruation, and surgery can trigger an attack of angioedema. To prevent acute attacks of HAE, patients can try to avoid specific stimuli that previously caused an attack. However, in many cases, attacks occur without a known trigger. Typically, HAE symptoms first appear in childhood and worsen during adolescence. On average, untreated individuals have attacks every 1 to 2 weeks, and most attacks last about 3 to 4 days (ghr.nlm.nih.gov / condition / hereditary-angioedema). The frequency and duration of attacks vary greatly among people with hereditary angioedema, even among people in the same family.

[0138] There are three types of HAE, called types I, II, and III. It is estimated that HAE affects 1 in 50,000 people, with type I accounting for about 85% of cases, type II accounting for about 15% of cases, and type III being very rare. Type III is the most recently described form and was originally thought to occur only in women, but families with affected males have been identified.

[0139] HAE is inherited in an autosomal dominant pattern, so that an affected person can inherit the mutation from an affected parent. New mutations in the gene can also occur, so HAE can also occur in people with no history of the disorder in their family. It is estimated that 20-25% of cases result from new spontaneous mutations.

[0140] Mutations in the SERPING1 gene cause hereditary angioedema types I and II. The SERPING1 gene provides instructions for making the C1 inhibitor protein, which is important for controlling inflammation. C1 inhibitor blocks the activity of certain proteins that promote inflammation. Mutations that cause hereditary angioedema type I result in decreased levels of C1 inhibitor in the blood. In contrast, mutations that cause type II result in the production of dysfunctional C1 inhibitor. In the absence of appropriate levels of functional C1 inhibitor, excess bradykinin is produced. Bradykinin promotes inflammation by increasing the leakage of fluid through blood vessel walls into body tissues. Excessive accumulation of fluid in body tissues causes episodes of swelling seen in individuals with hereditary angioedema types I and II.

[0141] Mutations in the F12 gene are associated with some cases of type III hereditary angioedema. The F12 gene provides guidance for the production of coagulation factor XII. In addition to playing a key role in blood clotting (coagulation), factor XII is also an important stimulant 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 leaky, which causes episodes of swelling. The causes of other cases of type III hereditary angioedema are still unknown. Mutations in one or more genes that have not yet been identified may cause the disorders in these cases.

[0142] HAE can present similarly to other forms of angioedema from allergies or other medical conditions, but the causes and treatments are significantly different. When HAE is misdiagnosed as an allergy, it is most often treated with antihistamines, steroids, and / or epinephrine, which are generally ineffective against HAE, although epinephrine can be used for life-threatening reactions. Misdiagnosis has also led to unnecessary exploratory surgeries for patients with abdominal swelling, and abdominal pain has been mistakenly diagnosed as a psychosomatic disorder in some HAE patients.

[0143] C1 inhibitor therapy, as well as other therapies for HAE, are described in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925.

[0144] Emergency treatment of HAE attacks is provided to stop the development of edema as quickly as possible. C1 inhibitor concentrate from donor blood administered intravenously is an emergency treatment; however, this treatment is not available in many countries. In emergency situations where C1 inhibitor concentrate is not available, fresh frozen plasma (FFP) can be used as a substitute because it also contains C1 inhibitor.

[0145] Purified C1 inhibitor derived from human blood has been used in Europe since 1979. Several C1 inhibitor treatments are now available in the United States and two C1 inhibitor products are now available in Canada. Pasteurized Berinert P (CSLBehring) was approved by the FDA in 2009 for acute attacks. Nanofiltered It was approved by the FDA for prevention in 2008. Rhucin / Ruconest (Pharming) is a recombinant C1 inhibitor in development that does not carry the risk of infectious disease transmission due to human blood-borne pathogens.

[0146] Treatment of an acute HAE attack may also include medications and / or IV fluids for pain relief.

[0147] Other treatment modalities stimulate the synthesis of C1 inhibitor or reduce C1 inhibitor consumption. Androgenic drugs, such as danazol, can reduce the frequency and severity of attacks by stimulating the production of C1 inhibitor.

[0148] Helicobacter pylori can trigger abdominal attacks. Antibiotics that treat Helicobacter pylori will reduce abdominal attacks.

[0149] Newer treatments attack the contact cascade. Ecallantide Inhibits plasma kallikrein and is already approved in the United States. Shire) inhibits the bradykinin B2 receptor and is already approved in Europe and the United States.

[0150] The diagnosis of HAE can rely on, for example, family history and / or blood tests. Laboratory findings associated with type I, type II, and type III HAE are described, for example, in Kaplan, AP, J Allergy Clin Immunol, 2010, 126(5):918-925. In type I HAE, levels of C1 inhibitor, like levels of C4, decrease, while levels of C1q are normal. In type II HAE, levels of C1 inhibitor are normal or increased; however, C1 inhibitor function is abnormal. C4 levels decrease and C1q levels are normal. In type III, levels of C1 inhibitor, C4, and C1q can all be normal. The present disclosure is based, at least in part, on the identification of additional proteins that have different levels in samples from HAE patients compared to healthy individuals (Table 1). Measuring the level or presence of 2-HMWK can be used to identify whether a subject has a disease, such as HAE. In some embodiments, these methods can be used to determine whether a patient has had an episode of HAE or is having an episode of HAE.

[0151] Symptoms of HAE can be assessed, for example, using a questionnaire, for example, one typically completed by the patient, clinician, or family member. Such questionnaires are known in the art and include, for example, visual analog scales. See, for example, McMillan, CV et al. Patient. 2012; 5(2): 113-26.

[0152] The values ​​of cleaved kininogen and / or intact kininogen detected in a sample from a subject can be compared to reference values ​​to determine whether the subject has a pKal-mediated disorder (e.g., HAE), or is at risk for the disease. Alternatively or additionally, the values ​​of cleaved kininogen and / or intact kininogen detected in a sample from a subject can be compared to reference values ​​to assess the efficacy of a disorder treatment, the prognosis or severity of the disorder, and / or to identify the subject as a candidate for treatment.

[0153] Reference value can be the control level of the kininogen percentage of cracking.In some embodiments, control level is the percentage of the kininogen of cracking in the sample (such as blood or plasma sample) obtained from control sample, for example, healthy subject or healthy subject colony, and the healthy subject is preferably the species identical with candidate subject.As used herein, healthy subject is to measure cracking and / or complete kininogen level and obviously does not contain target disease (for example, PKal mediation disorder, such as HAE or autoimmune disease, such as RA, US and Crohn's disease) or the subject without disease history.

[0154] The control level can also be a predetermined level or threshold. Such a predetermined level can represent the percentage of cleaved kininogen in a subject population without the target disease or without the risk of the target disease. It can also represent the percentage of cleaved kininogen in a subject population with the target disease.

[0155] The predetermined level can be in various forms. For example, it can be a single cut-off value, such as a median or mean value. In some embodiments, such a predetermined level can be determined based on a comparison group, such as one of the defined groups is known to have a target disease and another defined group is known to not have the target disease. Alternatively, the predetermined level can be a range, such as a range representing the percentage of kininogen cleaved in a control population within a predetermined percentage.

[0156] The control level as described herein can be determined by conventional techniques. In some embodiments, the control level can be obtained by performing conventional methods on a control sample as described herein (e.g., the same assay to obtain the level of cleaved and / or intact kininogen in a test sample as described herein). In other examples, the level of cleaved and / or intact kininogen can be obtained from members of a control population and the results can be analyzed, for example, by a computer program to obtain a control level (predetermined level) representing the level of cleaved and / or intact kininogen in the control population.

[0157] By comparing the percentage of cleaved kininogen in the sample obtained from the candidate subject with the reference value as described herein, it can be determined whether the candidate subject has a pKal-mediated disease (e.g., HAE or autoimmune diseases, such as RA, UC and Crohn's disease) or is at risk of the disease. For example, if the percentage of cleaved kininogen in the sample of the candidate subject deviates from the reference value (e.g., increases compared to the reference value or decreases compared to the reference value), the candidate subject may be identified as having the disease or being at risk of the disease. When the reference value represents the percentage range of cleaved kininogen in the subject population with the target disease, the percentage of cleaved kininogen in the candidate sample falling within the range indicates that the candidate subject has the target disease or is at risk of the target disease. In some cases, the reference value can represent a background level, which indicates that there is no cleaved kininogen. The presence of cleaved kininogen is considered to be a deviation from this background reference value. As used herein, "deviation" from a control sample or reference value covers the level of cleaved HMWK and the presence or absence of cleaved HMWK in the sample.

[0158] As used herein, "elevated levels or levels above a reference value" means that the level / percentage of cleaved kininogen is above a reference value, such as a predetermined threshold value of the level / percentage of cleaved kininogen in a control sample. Control levels are described in detail herein.

[0159] The percentage of increased cleaved kininogen includes, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more of the percentage of cleaved kininogen compared to the reference value. The percentage of increased cleaved kininogen also includes an increase in the phenomenon from a zero state (e.g., no or undetectable cleaved kininogen and / or intact kininogen bound to the capture agent in the sample) to a non-zero state (e.g., some or detectable cleaved kininogen and / or intact kininogen).

[0160] As used herein, "reduced percentage / level, or percentage / level below a reference value" means that the percentage / level of cleavage is less than a reference value, such as a predetermined threshold of cleaved kininogen in a control sample. Control levels are described in detail herein.

[0161] The reduced level of cleaved kininogen includes, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, 300%, 400%, 500% or more cleaved kininogen below the reference value. The reduced level of cleaved kininogen bound to the capture agent also includes reducing the phenomenon from a non-zero state (e.g., some or detectable cleaved kininogen in the sample) to a zero state (e.g., no or undetectable cleaved kininogen in the sample).

[0162] In some embodiments, the candidate subject is a human patient with symptoms of a pKal-mediated disorder, such as HAE or an autoimmune disease, such as RA, UC, and Crohn's disease. For example, the subject has edema, swelling, wherein the swelling is entirely or primarily peripheral; urticaria; redness, pain, and swelling without signs of infection; non-histamine-mediated edema, recurrent episodes of swelling, or a combination thereof. In other embodiments, the subject has no symptoms of a pKal-mediated disorder, no history of symptoms of a pKal-mediated disorder, or no history of a pKal-mediated disorder, such as HAE, at the time the sample is collected. In other embodiments, the subject is resistant to antihistamine therapy, corticosteroid therapy, or both antihistamine therapy and corticosteroid therapy.

[0163] Subjects identified in the methods described herein may be subjected to appropriate treatment.

[0164] Taking into account the correlation between the level of cleaved HMWK and this disease, the assay method and kit described herein can be used to evaluate the therapeutic efficacy of diseases related to plasma kallikrein, such as those described herein. For example, before and after treatment or during the course of treatment, multiple biological samples (e.g., blood or plasma samples) can be collected from the subject being treated. The level of cleaved and / or intact kininogen can be measured by any assay method as described herein, and the value (e.g., percentage) of cleaved and / or intact kininogen can therefore be determined. If the percentage of cleaved kininogen decreases after treatment or during treatment (compared to the sample collected earlier, the percentage of cleaved kininogen in the sample collected later) or the percentage of intact kininogen increases after treatment or during treatment, it indicates that treatment is effective. In some instances, treatment relates to therapeutic agents, such as kallikrein inhibitors, bradykinin B2 receptor antagonists or C1-INH replacement agents. Examples of therapeutic agents include, but are not limited to, landarumab (DX-2930), ecallantide (DX88), icatibant, and human plasma-derived C1-INH.

[0165] If the subject is identified as not responding to treatment, a higher dose and / or a higher dosing frequency of the therapeutic agent is administered to the identified subject. In some embodiments, the dosage or dosing frequency of the therapeutic agent is maintained, reduced or terminated in a subject identified as responding to treatment or not requiring further treatment. Alternatively, a different treatment may be applied to a subject that is found not to respond to the first treatment.

[0166] In other embodiments, the value of cleaved kininogen, either alone or in combination with intact kininogen, can also be relied upon to identify disorders that can be treated by pKal inhibitors. To implement the method, the level of cleaved kininogen and / or the level of intact kininogen in a sample (e.g., a blood sample or a plasma sample) collected from a subject with a target disease can be measured by an appropriate assay, such as those described herein, such as a Western blot assay or an ELISA assay. The value of cleaved and / or intact kininogen, such as a percentage, can be determined as described herein. The value of cleaved kininogen and / or intact kininogen can be compared with a reference value as described herein. If the value of cleaved kininogen / intact kininogen deviates from the reference value (e.g., increases or decreases), it indicates that the pKal inhibitor can effectively treat the disease. For example, if the percentage of cleaved kininogen decreases after treatment or during treatment, the treatment can be identified as effective. Alternatively, if the percentage of intact kininogen increases after treatment or during treatment, the treatment is identified as effective.

[0167] If the disease is identified as being sensitive to (treatable with) a pKal inhibitor, the method may further comprise administering to the subject having the disease an effective amount of a pKal inhibitor, such as ecallantide (DX-88), EPIKAL-2, or landarumab (DX-2930).

[0168] Also within the scope of the present disclosure is a method for assessing the severity of a disease or disorder associated with plasma kallikrein or a disease state. For example, as described herein, HAE may be in a resting state (basal state), during which the subject does not experience disease symptoms. HAE attacks are typically recurrent events in which the subject may experience pain and swelling, such as in the hands, feet, face, gastrointestinal tract, genitals, and pharynx (throat) for 2-5 days. In some embodiments, the level of 2-HMWK indicates whether the subject will experience, is experiencing, or will soon experience an HAE attack. In some embodiments, the method includes comparing the level of 2-HMWK in a sample obtained from a subject with HAE with a sample from the same subject (e.g., a sample obtained from the same subject in a basal state or a sample obtained from the same subject during an HAE attack) .

[0169] (v) Non-clinical applications

[0170] In addition, the assays described herein for detecting cleaved 2-HMWK levels can be used for research purposes. Although many diseases and disorders associated with or mediated by plasma kallikrein have been identified, other diseases may be mediated by similar mechanisms or involve similar components. In some embodiments, the methods described herein can be used to identify diseases associated with or mediated by plasma kallikrein, or associated with or mediated by components of the contact activation system. In some embodiments, the methods described herein can be used to study disease mechanisms (e.g., discovering new biological pathways or processes involved in disease development) or disease progression.

[0171] In some embodiments, the level of cleaved 2-HMWK measured using the assay described herein can be relied upon in developing new therapeutic agents for diseases associated with the contact activation system. For example, the level of cleaved 2-HMWK can be measured in samples obtained from subjects to whom a new therapy (e.g., a clinical trial) has been administered. In some embodiments, the level of cleaved 2-HMWK can indicate the efficacy of a new therapeutic agent or the progression of a disease in a subject before, during, or after a new therapy.

[0172] II. Treatment of diseases associated with plasma kallikrein

[0173] A subject at risk for or suffering from a disease associated with plasma kallikrein as identified using the methods and assays described herein can be treated with any appropriate therapeutic agent. In some embodiments, provided methods include selecting a treatment for a subject based on an output of the methods, such as measuring the level of cleaved 2-HMWK.

[0174] In some embodiments, the methods include selecting or administering a therapeutic agent (e.g., a kallikrein inhibitor, a bradykinin B2 receptor inhibitor, and / or a C1 esterase inhibitor) or selecting and administering the therapeutic agent to a subject based on the output of an assay (e.g., a 2-HMWK assay).

[0175] In some embodiments, the therapeutic agent is administered to the subject one or more times. In some embodiments, a plasma kallikrein inhibitor is administered to the subject. In some embodiments, the kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein antibody, or a fragment thereof. In some embodiments, an antagonist of the bradykinin B2 receptor is administered to the subject. In some embodiments, C1-INH is administered to the subject.

[0176] Therapeutic agents, for example, kallikrein inhibitors, bradykinin B2 receptor inhibitors and / or C1-INH, can be used together with another therapy as a part of a combination therapy for the treatment of a disease or condition involving a contact activation system. Combination therapy can be provided in a variety of different ways, for example, replacing one or more of a reagent with a kallikrein inhibitor, bradykinin B2 receptor antagonist or C1-INH, for example, replacing one or more of a reagent with a kallikrein inhibitor, bradykinin B2 receptor antagonist or C1-INH and another therapy. The first reagent can be used before or after another therapy is used. In some cases, the first reagent and another therapy (for example, therapeutic agent) are used simultaneously or very close in time (for example, a short time interval between injections, such as between the same treatment cycle). The first reagent and other therapies can also be used at larger time intervals.

[0177] Plasma kallikrein binding agents (e.g., binding proteins, e.g., polypeptides, e.g., inhibitory polypeptides, e.g., antibodies, e.g., inhibitory antibodies, or other binding agents, e.g., small molecules) are useful therapeutic agents for various diseases and conditions, e.g., diseases and conditions involving plasma kallikrein activity. For example, in some embodiments, the disease or condition involving plasma kallikrein activity is hereditary angioedema (HAE). In some embodiments, a plasma kallikrein binding agent, e.g., a plasma kallikrein inhibitor, is administered to a subject at risk of or suffering from a disease associated with a contact activation system.

[0178] Many useful protein inhibitors of kallikreins (tissue kallikrein and / or plasma kallikrein) include Kunitz domains. As used herein, a "Kunitz domain" is a polypeptide domain having at least 51 amino acids and comprising at least two, preferably three, disulfide bonds. The domain is folded so that the first and sixth cysteines, the second and fourth cysteines, and the third and fifth cysteines form a disulfide bond (e.g., in a Kunitz domain having 58 amino acids, cysteines may appear at positions corresponding to amino acids 5, 14, 30, 38, 51, and 55 (according to the numbering of the BPTI homologous sequence provided below), and disulfide bonds may be formed between the cysteines at positions 5 and 55, 14 and 38, and 30 and 51), or, if two disulfide bonds are present, they may be formed between a subset of their corresponding cysteines. According to the numbering of the BPTI sequence provided below, the spacing between individual cysteines can be within 7, 5, 4, 3, 2, 1 or 0 amino acids corresponding to the spacing between positions 5 to 55, 14 to 38 and 30 to 51 described below. The BPTI sequence can be used as a reference to indicate specific positions in any general Kunitz domain. Comparison of a Kunitz domain of interest to BPTI can be performed by identifying the best fit alignment in which the number of aligned cysteines is maximized.

[0179] The 3D structure of the Kunitz domain of BPTI is known (at high resolution). An X-ray structure is deposited in the Brookhaven Protein Data Bank as "6PTI". The 3D structures of some BPTI homologs (Eigenbrot et al., Protein Engineering (1990) 3(7):591-598; Hynes et al., Biochemistry (1990) 29:10018-10022) are known. At least 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., J. Biol. Chem. (1988) 263(13):6001-6004; Girard et al., Nature (1989) 338:518-20; Novotny et al., J. Biol. Chem. (1989) 264(31):18832-18837), the two Kunitz domains of the inter-α-trypsin inhibitor APP-I (Kido et al., J. Biol. Chem. (1988) 263(34):18104-18107), the Kunitz domain from collagen, the three Kunitz domains of TFPI-2 (Sprecher et al., PNAS USA (1994) 91: 3353-3357), Kunitz domain of hepatocyte growth factor type 1 activator inhibitor, Kunitz domain of hepatocyte growth factor type 2 activator inhibitor, Kunitz domain described in U.S. Patent Publication No. 2004-0152633. LACI is a human serum phosphoglycoprotein with a molecular weight of 39 kDa (amino acid sequence in Table 1) and contains three Kunitz domains.

[0180] Table 1: Exemplary native Kunitz domains

[0181]

[0182] The above Kunitz domains are called LACI-K1 (residues 50 to 107), LACI-K2 (residues 121 to 178) and LACI-K3 (213 to 270). The cDNA sequence of LACI is reported in Wun et al. (J. Biol. Chem. (1988) 263 (13): 6001-6004). Girard et al. (Nature (1989) 338: 518-20) reported mutation studies in which the P1 residue in each of the three Kunitz domains was altered. When factor VIIa is complexed with tissue factor and LACI-K2 inhibits factor Xa, it is LACI-K1 that inhibits factor VIIa (F.VIIa).

[0183] 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 from GenBank sequence databases ((National Center for Biotechnology Information, National Institutes of Health), Bethesda MD), for example, using BLAST search; from the Pfam database (Hidden Markov Models) of HMM (e.g., using the default parameters of Pfam search); from the SMART database search; or from the ProDom database search. For example, Pfam version 9 of Pfam accession number PF00014 provides many Kunitz domains and HMMs for identifying Kunitz domains. A description of the Pfam database can be found in Sonhammer et al. Proteins (1997) 28(3):405-420 and a detailed description of HMMs can be found, for example, in Gribskov et al. Meth. Enzymol. (1990) 183:146-159; Gribskov et al. Proc. Natl. Acad. Sci. USA (1987) 84:4355-4358; Krogh et al. J. Mol. Biol. (1994) 235:1501-1531; and Stultz et al. Protein Sci. (1993) 2:305-314. The SMART database of HMMs (Simple Modular Architecture Research Tool, EMBL, Heidelberg, DE) is described in Schultz et al. Proc. Natl. Acad. Sci. USA (1998) 95:5857 and Schultz et al. Nucl. Acids Res (2000) 28:231. The SMART database contains domains identified by profiling using a hidden Markov model 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. Nucl. Acids Res. (1999) 27:263-267). The current version of ProDom was constructed using a recursive PSI-BLAST search of the SWISS-PROT 38 and TREMBL protein databases (Altschul et al. Nucleic Acids Res. (1997) 25:3389-3402; Gouzy et al. Computers and Chemistry (1999) 23:333-340). The database automatically generates a consensus sequence for each domain. Prosite lists Kunitz domains as motifs and identifies proteins that include Kunitz domains. See, e.g., Falquet et al. Nucleic Acids Res. (2002) 30:235-238.

[0184] Kunitz domains primarily use amino acids in two loop regions ("binding loops") to interact with target proteases. The first loop region is between residues corresponding approximately to amino acids 13-20 of BPTI. The second loop region is between residues corresponding approximately to amino acids 31-39 of BPTI. An exemplary library of Kunitz domains differs in one or more amino acid positions in the first and / or second loop regions. When screening for Kunitz domains that interact with kallikrein or when selecting improved affinity variants, particularly useful positions to be altered include: positions 13, 15, 16, 17, 18, 19, 31, 32, 34, and 39 relative to the BPTI sequence. At least some of these positions are expected to be in close contact with the target protease. It is also useful to alter other positions, such as positions adjacent to the above positions in the three-dimensional structure.

[0185] The "framework region" of a Kunitz domain is defined as being part of the Kunitz domain, but specifically excludes those residues that are in the first and second binding loop regions, i.e., residues corresponding approximately to amino acids 13-20 of BPTI and amino acids 31-39 of BPTI. In contrast, residues that are not in the binding loops can tolerate a wider range of amino acid substitutions (e.g., conservative and / or non-conservative substitutions).

[0186] In one embodiment, these Kunitz domains are variant forms with a ring structure, including Kunitz domain 1 of the aggregation inhibitor (LACI) protein associated with human lipoprotein. LACI contains three clearly defined peptide ring structures inside, which is a typical Kunitz domain (Girard, T. et al., Nature (1989) 338: 518-520). Variants of Kunitz domain 1 of LACI described herein have been screened and isolated, which bind to kallikrein with enhanced affinity and specificity (see, e.g., U.S. Patent Nos. 5,795,865 and 6,057,287). These methods can also be used for other Kunitz domain frameworks to obtain other Kunitz domains that interact with kallikrein, such as plasma kallikrein. Useful modulators of kallikrein function generally bind and / or inhibit kallikrein, as determined using kallikrein binding and inhibition assays.

[0187] In some aspects, the plasma kallikrein inhibitor is bound to the plasma kallikrein in active form. In some embodiments, the plasma kallikrein inhibitor binds to and inhibits plasma kallikrein, such as human plasma kallikrein and / or mouse kallikrein. Exemplary polypeptide plasma kallikrein reagents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No. 7,628,983 and U.S. Patent No. 8,283,321, U.S. Patent No. 7,064,107, U.S. Patent No. 7,276,480, U.S. Patent No. 7,851,442, U.S. Patent No. 8,124,586, U.S. Patent No. 7,811,991 and U.S. Publication No. 20110086801, the entire contents of each of which are incorporated herein by reference. In some embodiments, the plasma kallikrein inhibitor is an inhibitory polypeptide or inhibitory peptide. In some embodiments, the inhibitory peptide is ecallantide (also known as DX-88 or SEQ ID NO:80). In some embodiments, the kallikrein inhibitor comprises or consists of about 58 amino acid sequence of amino acids 3-60 of SEQ ID NO:80 or a DX-88 polypeptide having 60 amino acid sequence of SEQ ID NO:80.

[0188] Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys ArgAla Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe IleTyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys LysMet Cys Thr Arg Asp (SEQ ID NO:80).

[0189] The plasma kallikrein inhibitor can be a full-length antibody (e.g., IgG (e.g., IgG1, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgA1, IgA2), IgD, and IgE) or can include only an antigen binding fragment (e.g., a Fab, F(ab′)2, or scFv fragment). The binding protein can include two heavy chain immunoglobulins and two light chain immunoglobulins, or can be a single chain antibody. The plasma kallikrein inhibitor can be a recombinant protein, such as a humanized, CDR-grafted, chimeric, deimmunized, or in vitro generated antibody, and can optionally include a constant region derived from a human germ line immunoglobulin sequence. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.

[0190] Exemplary plasma kallikrein binding proteins are disclosed in U.S. Publication No. 20120201756, the entire contents of which are incorporated herein by reference. In some embodiments, the kallikrein binding protein is an antibody (e.g., a human antibody) having a light chain and / or a heavy chain selected from the group consisting of: M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also known as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03 , X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01 (also referred to herein as DX-2930 or landarumab), X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein competes with or binds to the same epitope as M162-A04, M160-G12, M142-H08, X63-G06, X101-A01 (also referred to herein as DX-2922), X81-B01, X67-D03, X67-G04, X81-B01, X67-D03, X67-G04, X115-B07, X115-D05, X115-E09, X115-H06, X115-A03, X115-D01, X115-F02, X124-G01, X115-G04, M29-D09, M145-D11, M06-D09, and M35-G04. In some embodiments, the plasma kallikrein binding protein is landarumab. See US 20110200611 and US20120201756, which are incorporated herein by reference.

[0191] An example of a plasma kallikrein inhibitory antibody is landarumab. The amino acid sequences of the heavy and light chain variable regions of landarumab are provided below, with the CDR regions indicated in bold and underlined.

[0192] Heavy chain variable region sequence of landarumab (SEQ ID NO: 81)

[0193] EVQLLESGGG LVQPGGSLRL SCAASGFTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVYADSVKGRFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD IWGQGTMVTV SS

[0194] Light chain variable region sequence of landarumab (SEQ ID NO: 82)

[0195] DIQMTQSPS TLSASVGDRV TITCRASQSI SSWLAWYQQK PGKAPKLLIY KASTLESGVPSRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI

[0196] In some embodiments, the plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein. In some embodiments, the plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein in the HC and / or LC framework regions (e.g., HC and / or LC FR 1, 2, 3 and / or 4). In some embodiments, the plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein in the HC and / or LC CDRs (e.g., HC and / or LC CDR1, 2, and / or 3). In some embodiments, the plasma kallikrein inhibitor may have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity with the plasma kallikrein inhibitor described herein in the constant region (e.g., CH1, CH2, CH3 and / or CL1).

[0197] In some aspects, the small molecule binds to and inhibits the active form of plasma kallikrein.

[0198] Bradykinin B2 receptor inhibitors

[0199] In some embodiments, a bradykinin B2 receptor inhibitor (eg, antagonist) is administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant It is a peptidomimetic drug consisting of 10 amino acids that blocks the binding of natural bradykinin to the bradykinin B2 receptor.

[0200] C1-INH replacement reagent

[0201] In some embodiments, a C1 esterase inhibitor (C1-INH), such as a C1-INH replacement agent, is administered to a subject. Exemplary C1-INH replacement agents are publicly available and include, for example, human plasma-derived C1-INH, such as and

[0202] III. Kit for detecting cleaved HMWK

[0203] The present disclosure also provides a kit for evaluating the cleaved HMWK in a sample suspected of containing cleaved HMWK, such as a biological sample from a human patient. Such a kit may include a first reagent that specifically binds to the cleaved HMWK compared to an intact HMWK or LMWK. In some embodiments, the first reagent is an antibody, such as any antibody that specifically binds to the cleaved HMWK (e.g., 559B-M004 or a functional variant thereof described herein). In some embodiments, the kit also includes a second reagent (e.g., an antibody that binds to HMWK) for detecting the binding of the first reagent to the cleaved HMWK. The second reagent may be conjugated to a marker. In some embodiments, the second reagent is an antibody that specifically binds to the cleaved HMWK. In other embodiments, the second reagent is an antibody that cross-reacts with both the cleaved HMWK and the intact HMWK.

[0204] The kit may further include a support member for implementing the immunoassay method and fixing the first reagent. In some embodiments, the support member is a 96-well plate, such as a 96-well ELISA plate. The kit may also include one or more buffers described herein, but not limited to a coating buffer; an assay buffer, such as an assay buffer containing ZnCl2; a blocking buffer; a washing buffer; and / or a stop buffer.

[0205] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include a description of how to use the components contained in the kit to measure the level of cleaved and / or intact HMWK in a sample, which may be a biological sample collected from a human patient. Alternatively or additionally, the kit may include a description of how to use the components contained in the kit to measure the level of LMWK.

[0206] Instructions for use of the kit generally include information about the amount of each component and suitable conditions for implementing the assay methods described herein. The components in the kit may be unit doses, large packages (e.g., multi-dose packages), or subunit doses. The instructions provided in the kit of the present disclosure are generally written instructions on a label or package insert (e.g., a sheet of paper included in the kit), but machine-readable instructions (e.g., instructions carried on a disk or CD) are also acceptable.

[0207] The label or package insert indicates that the kit is used to assess the level of cleaved and / or intact HMWK. In some embodiments, the kit is used to assess the level of LWMK. Instructions for practicing any of the methods described herein may be provided.

[0208] The kit of the present disclosure is in appropriate packaging. Suitable packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed polyester film or plastic bags), etc. It is also contemplated that packaging used in conjunction with a dedicated device, such as an inhaler, a nasal application device (e.g., a nebulizer), or an injection device, such as a miniature pump. The kit may have a sterile inlet (e.g., the container may be an intravenous solution bag or vial with an obstruction that can be punctured by a hypodermic injection needle). The container may also have a sterile inlet (e.g., the container may be an intravenous solution bag or vial with an obstruction that can be punctured by a hypodermic injection needle).

[0209] The kit may optionally provide additional components, such as explanatory information, such as controls and / or standards or reference samples. Typically, the kit includes a container and a label or package insert (one or more) on or associated with the container. In some embodiments, the present disclosure provides an article of manufacture comprising the contents of the above-mentioned kit.

[0210] IV. Other Antibodies Binding to Cleaved HMWK

[0211] Also provided herein are isolated antibodies that bind to both cleaved HMWK and intact HMWK. In some embodiments, such antibodies do not bind to LMWK or bind to LMWK with low affinity. In other embodiments, such antibodies also bind to LMWK.

[0212] In some embodiments, antibodies that specifically bind to cleaved HMWK and intact HMWK (or additional LMWK) described herein have suitable binding affinity for one or more target antigens. -5 , 10 -6 , 10 -7 , 10 -8 , 10 -9 , 10 -10 M or lower binding affinity (KD).

[0213] Examples of such antibodies and their binding specificities are provided in Table 2 below in Example 2. The amino acid sequences of the heavy and light chain variable regions are provided below, with the CDR regions (determined by one approach as an example) marked in bold and underlined:

[0214] >559B-R0049-A01 (559B-M0067-E02) heavy chain amino acid sequence (SEQ ID NO: 6)

[0215]

[0216] >559B-R0049-A01 (559B-M0067-E02) light chain amino acid sequence (SEQ ID NO: 7)

[0217]

[0218] >559B-R0049-G05 (559B-M0039-G07) heavy chain amino acid sequence (SEQ ID NO: 8)

[0219]

[0220] >559B-R0049-G05 (559B-M0039-G07) light chain amino acid sequence (SEQ ID NO: 9)

[0221]

[0222] >559B-R0048-A09 (559B-M0044-E09) heavy chain amino acid sequence (SEQ ID NO: 10)

[0223]

[0224] >559B-R0048-A09 (559B-M0044-E09) light chain amino acid sequence (SEQ ID NO: 11)

[0225]

[0226] >559B-R0048-E01 (559B-M0003-C08) heavy chain amino acid sequence (SEQ ID NO: 12)

[0227]

[0228] >559B-R0048-E01 (559B-M0003-C08) light chain amino acid sequence (SEQ ID NO: 13)

[0229]

[0230] >559B-R0049-G01 (559B-M0039-H06) heavy chain amino acid sequence (SEQ ID NO: 14)

[0231]

[0232] >559B-R0049-G01 (559B-M0039-H06) light chain amino acid sequence (SEQ ID NO: 15)

[0233]

[0234] >559B-R0049-E05 (559B-M0039-D08) heavy chain amino acid sequence (SEQ ID NO: 16)

[0235]

[0236] >559B-R0049-E05 (559B-M0039-D08) light chain amino acid sequence (SEQ ID NO: 17)

[0237]

[0238] >559B-R0048-A11 (559B-M0068-C07) heavy chain amino acid sequence (SEQ ID NO: 18)

[0239]

[0240] >559B-R0048-A11 (559B-M0068-C07) light chain amino acid sequence (SEQ ID NO: 19)

[0241]

[0242] >559B-R0048-A03 (559B-M0021-G11) heavy chain amino acid sequence (SEQ ID NO: 20)

[0243]

[0244] >559B-R0048-A03 (559B-M0021-G11) light chain amino acid sequence (SEQ ID NO: 21)

[0245]

[0246] >559B-R0048-C05 (559B-M0061-G06) heavy chain amino acid sequence (SEQ ID NO: 22)

[0247]

[0248] >559B-R0048-C05 (559B-M0061-G06) light chain amino acid sequence (SEQ ID NO: 23)

[0249]

[0250] >559B-R0049-A03 (559B-M0036-G12) heavy chain amino acid sequence (SEQ ID NO: 24)

[0251]

[0252] >559B-R0049-A03 (559B-M0036-G12) light chain amino acid sequence (SEQ ID NO: 25)

[0253]

[0254] >559B-R0048-C09 (559B-M0042-E06) heavy chain amino acid sequence (SEQ ID NO: 26)

[0255]

[0256] >559B-R0048-C09 (559B-M0042-E06) light chain amino acid sequence (SEQ ID NO: 27)

[0257]

[0258] >559B-R0048-E09 (559B-M0070-H10) heavy chain amino acid sequence (SEQ ID NO: 28)

[0259]

[0260] >559B-R0048-E09 (559B-M0070-H10) light chain amino acid sequence (SEQ ID NO: 29)

[0261]

[0262] >559B-R0048-E05 (559B-M0068-D01) heavy chain amino acid sequence (SEQ ID NO: 30)

[0263]

[0264] >559B-R0048-E05 (559B-M0068-D01) light chain amino acid sequence (SEQ ID NO: 31)

[0265]

[0266] >559B-R0048-C01 (559B-M0004-E08) heavy chain amino acid sequence (SEQ ID NO: 32)

[0267]

[0268] >559B-R0048-C01 (559B-M0004-E08) light chain amino acid sequence (SEQ ID NO: 33)

[0269]

[0270] >559B-R0049-C01 (559B-M0069-C09) heavy chain amino acid sequence (SEQ ID NO: 34)

[0271]

[0272] >559B-R0049-C01 (559B-M0069-C09) light chain amino acid sequence (SEQ ID NO: 35)

[0273]

[0274] >559B-R0049-A05 (559B-M0038-F04) heavy chain amino acid sequence (SEQ ID NO: 36)

[0275]

[0276] >559B-R0049-A05 (559B-M0038-F04) light chain amino acid sequence (SEQ ID NO: 37)

[0277]

[0278] >559B-R0048-G05 (559B-M0044-C05) heavy chain amino acid sequence (SEQ ID NO: 38)

[0279]

[0280] >559B-R0048-G05 (559B-M0044-C05) light chain amino acid sequence (SEQ ID NO: 39)

[0281]

[0282] >559B-R0048-C11 (559B-M0047-H01) heavy chain amino acid sequence (SEQ ID NO: 40)

[0283]

[0284] >559B-R0048-C11 (559B-M0047-H01) light chain amino acid sequence (SEQ ID NO: 41)

[0285]

[0286] >559B-R0048-C03 (559B-M0019-E12) heavy chain amino acid sequence (SEQ ID NO: 42)

[0287]

[0288] >559B-R0048-C03 (559B-M0019-E12) light chain amino acid sequence (SEQ ID NO: 43)

[0289]

[0290] >559B-R0048-A05 (559B-X0004-B05) heavy chain amino acid sequence (SEQ ID NO: 44)

[0291]

[0292] >559B-R0048-A05 (559B-X0004-B05) light chain amino acid sequence (SEQ ID NO: 45)

[0293]

[0294] >559B-R0048-E11 (559B-M0048-D12) heavy chain amino acid sequence (SEQ ID NO: 46)

[0295]

[0296] >559B-R0048-E11 (559B-M0048-D12) light chain amino acid sequence (SEQ ID NO: 47)

[0297]

[0298] >559B-R0048-G11 (559B-M0053-G01) heavy chain amino acid sequence (SEQ ID NO: 48)

[0299]

[0300] >559B-R0048-G11 (559B-M0053-G01) light chain amino acid sequence (SEQ ID NO: 49)

[0301]

[0302] >559B-R0049-C05 (559B-M0038-H03) heavy chain amino acid sequence (SEQ ID NO: 50)

[0303]

[0304] >559B-R0049-C05 (559B-M0038-H03) light chain amino acid sequence (SEQ ID NO: 51)

[0305]

[0306] >559B-R0048-E03 (559B-M0017-H08) heavy chain amino acid sequence (SEQ ID NO: 52)

[0307]

[0308] >559B-R0048-E03 (559B-M0017-H08) light chain amino acid sequence (SEQ ID NO: 53)

[0309]

[0310] >559B-R0049-E03 (559B-M0035-F05) heavy chain amino acid sequence (SEQ ID NO: 54)

[0311]

[0312] >559B-R0049-E03 (559B-M0035-F05) light chain amino acid sequence (SEQ ID NO: 55)

[0313]

[0314] >559B-R0049-G03 (559B-M0035-H09) heavy chain amino acid sequence (SEQ ID NO: 56)

[0315]

[0316] >559B-R0049-G03 (559B-M0035-H09) light chain amino acid sequence (SEQ ID NO: 57)

[0317]

[0318] >559B-R0048-A07 (559B-M0043-C06) heavy chain amino acid sequence (SEQ ID NO: 58)

[0319]

[0320] >559B-R0048-A07 (559B-M0043-C06) light chain amino acid sequence (SEQ ID NO: 59)

[0321]

[0322] >559B-R0048-G01 (559B-M0003-A08) heavy chain amino acid sequence (SEQ ID NO: 60)

[0323]

[0324] >559B-R0048-G01 (559B-M0003-A08) light chain amino acid sequence (SEQ ID NO: 61)

[0325]

[0326] >559B-R0048-G09 (559B-M0054-B11) heavy chain amino acid sequence (SEQ ID NO: 62)

[0327]

[0328] >559B-R0048-G09 (559B-M0054-B11) light chain amino acid sequence (SEQ ID NO: 63)

[0329]

[0330] >559B-R0048-E07 (559B-M0067-G11) heavy chain amino acid sequence (SEQ ID NO: 64)

[0331]

[0332] >559B-R0048-E07 (559B-M0067-G11) light chain amino acid sequence (SEQ ID NO: 65)

[0333]

[0334] >559B-R0048-C07 (559B-M0065-B10) heavy chain amino acid sequence (SEQ ID NO: 66)

[0335]

[0336] >559B-R0048-C07 (559B-M0065-B10) light chain amino acid sequence (SEQ ID NO: 67)

[0337]

[0338] >559B-R0049-C03 (559B-M0037-E08) heavy chain amino acid sequence (SEQ ID NO: 68)

[0339]

[0340] >559B-R0049-C03 (559B-M0037-E08) light chain amino acid sequence (SEQ ID NO: 69)

[0341]

[0342] >559B-R0049-E01 (559B-M0035-A01) heavy chain amino acid sequence (SEQ ID NO: 70)

[0343]

[0344] >559B-R0049-E01 (559B-M0035-A01) light chain amino acid sequence (SEQ ID NO: 71)

[0345]

[0346] >559B-R0048-G03 (559B-M0003-E08) heavy chain amino acid sequence (SEQ ID NO: 72)

[0347]

[0348] >559B-R0048-G03 (559B-M0003-E08) light chain amino acid sequence (SEQ ID NO: 73)

[0349]

[0350] >559B-R0048-G07 (559B-M0052-E02) heavy chain amino acid sequence (SEQ ID NO: 74)

[0351]

[0352] >559B-R0048-G07 (559B-M0052-E02) light chain amino acid sequence (SEQ ID NO: 75)

[0353]

[0354] >559B-M0064-H02 heavy chain amino acid sequence (SEQ ID NO: 76)

[0355]

[0356] >559B-M0064-H02 light chain amino acid sequence (SEQ ID NO: 77)

[0357]

[0358] Also within the scope of the present disclosure are functional equivalents of any of the exemplary antibodies listed above. Such functional equivalents can bind to the same epitope of HMWK and / or intact HMWK or LMWK of the same cracking as one of the exemplary antibodies listed above. In some embodiments, the functional equivalent competes with one of the exemplary antibodies listed above for binding to the target antigen.

[0359] In some embodiments, functional equivalents include V H chain, the V H The chain includes a V corresponding to one of the exemplary antibodies listed above. H The CDRs are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to each other. H CDR1, V HCDR2 and / or V H Alternatively or additionally, functional equivalents include VDR3 that is at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%) identical to the exemplary antibodies listed above. L CDR1, V L CDR2 and / or V L CDR3. In some embodiments, the functional equivalent has the same heavy chain complementarity determining region and / or light chain complementarity determining region (CDR) as one of the exemplary antibodies listed above.

[0360] Alternatively or additionally, functional equivalents include V H The V sequences are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the V sequences of the H Chain and / or V with exemplary antibodies L The V sequences are at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the V sequences of the L chain.

[0361] In some cases, a functional equivalent may comprise one or more (e.g., up to 5, up to 3, or up to 1) conservative mutations in one or more heavy chain CDRs or one or more light chain CDRs in an exemplary antibody, for example at positions where the residue is unlikely to be involved in interaction with the target antigen.

[0362] Without further explanation, it is believed that those skilled in the art can fully use the present disclosure based on the above description. Therefore, the following specific embodiments are only interpreted as illustrative and do not limit the other parts of the present disclosure in any way. All publications cited herein are incorporated herein by reference for the purpose of the themes mentioned in the text.

[0363] Example

[0364] Example 1: Development of an immunoassay for specific detection of cleaved HMWK

[0365] Initially, ELISA-based immunoassay screening was developed to identify Fab fragments in phage display libraries that bind to cleaved or intact HMWK. Typically, assay conditions rely on biotinylated intact or cleaved HMWK immobilized on 384-well assay plates coated with streptavidin, blocked with bovine serum albumin (BSA) blocking buffer, and contacting the immobilized HMWK with Fab displayed on phage from overnight cultures in E. coli (detected with anti-M13-HRP antibody).

[0366] like Fig.12 As shown in panel A of FIG. 1 , the biotinylated 1-strand HMWK-immobilized streptavidin-coated magnetic beads (Dynabeads M280, Thermo Fisher) were preformed with approximately 1×10 12 The negative selection of the library of phage is selected for obtaining the antibody specific for 2-chain HMWK.Then the exhausted library is contacted with the biotinylated 2-chain HMWK fixed on the magnetic beads coated with streptavidin.The beads are fully washed with PBS buffer and used to infect Escherichia coli for phage output amplification to complete a round of selection.Three rounds of selection are carried out before screening single phage clones using the biotinylated 1-chain and 2-chain HMWK fixed on the plate coated with streptavidin by ELISA, and then the anti-M13 antibody conjugated with horseradish peroxidase (HRP) is detected and the absorbance detection caused by the substrate hydrolysis of 3,3',5,5'-tetramethylbenzidine (TMB) is carried out.Recombinant Fab fragments are expressed in Escherichia coli and purified by protein A agarose chromatography (Wassaf et al. Anal. Biochem. (2006) 351:241-253). The specificity of each purified Fab was determined by coating 384-well plates and measuring binding to biotinylated 1-chain HMWK, biotinylated 2-chain HMWK, or biotinylated LMWK, followed by detection with streptavidin conjugated to HRP and TMB detection. These assay conditions resulted in the identification of the 559B-M004-B04 isolate, which specifically binds to cleaved HMWK ( Figure 1 ).

[0367] Immobilized HMWK was also contacted with a crude (unpurified) 559B-M004-B04 Fab preparation from an overnight culture in E. coli. Fab bound to HMWK was detected using an anti-human Fab-HRP antibody, but no specific binding to cleaved HMWK was resulted ( Figure 1 ).

[0368] The immunoassay configuration was reversed by inertly immobilizing the purified Fab fragment of 559B-M004-B04 on a polystyrene 384-well assay plate. The Fab was contacted with biotinylated HMWK and bound HMWK was detected with streptavidin-HRP. Figure 1 ).

[0369] Unexpectedly, when the BSA blocking buffer was replaced with a commercially available blocking buffer (LowCross Blocking Solution from Candor Biosciences) during the initial screening assays, the specificity of the 559B-M004-B04 Fab for cleaved HMWK was enhanced ( Figure 1). In addition, the use of 96-well assay plates instead of 384-well plates for the immunoassay further increased the observed specificity of 559B-M004-B04 for cleaved HMWK ( Figure 1 ).

[0370] The results obtained with the 559B-M004-B04 isolate led to the development of an immunoassay (ELISA) for the detection of 2-HMWK in samples ( Fig.12 , Block B). This assay can also be used to further evaluate the binding characteristics of other Fab fragments and antibodies. In short, Fab is coated on a multi-well plate overnight. The flat plate is washed the next day and then blocked with BSA buffer. After washing, samples, standards and QC diluted in LowCross buffer are added to the flat plate, and after subsequent culture and then washing, any combined 2-chain HMWK is detected by adding HRP-labeled sheep anti-HMWK polyclonal detection antibodies. After culturing with the detection antibody, the flat plate is washed and TMB substrate is added to the flat plate. After a short culture, the reaction is terminated with phosphoric acid. The optical density is then measured at 450nm-630nm.

[0371] Example 2: Evaluation of binding specificity of Fab clones using the immunoassay method described herein

[0372] The immunoassay method described in Example 1 was used to evaluate the binding of 36 purified Fab clones (see Table 2 below) to cleaved HWMK, intact HMWK and LWMK. Specifically, each purified Fab clone was fixed on a 96-well assay plate with a total volume of 100 μL in PBS at a concentration of 1 μg / L and cultured overnight at 2-8°C. The assay plate was blocked with LowCross blocking buffer. Biotinylated intact HMWK, biotinylated cleaved HMWK or biotinylated LMWK (each 1 μg / L) were added to each well with a total volume of 100 μL and cultured for 2 hours, then washed with wash buffer. HRP-labeled streptavidin was added to each well at a concentration of 100 ng / mL, and the signal was developed using Ultra TMB substrate. The signal-to-noise ratio was calculated using the signal observed when the biotinylated protein was added to the uncoated well. ( Figure 2 , Panels A and B). Based on the ELISA results, the antibodies could be divided into five categories (Table 2).

[0373] Table 2: Binding characteristics of Fab fragments

[0374]

[0375]

[0376] Several antibodies that bind to both 1-chain HMWK, 2-chain HMWK and LMWK, such as 559B-M0064-H02, have been obtained. These antibodies may bind to epitopes in domains 1 to 4 shared between HMWK and LMWK. M070-H10 is an example of an antibody that is thought to bind to an epitope shared between 1-chain and 2-chain HMWK but not on LMWK. LMWK is a kininogen splice variant that results in a truncated protein including domains 1 to 4 and a portion of domain 5 (Colman et al. Blood (1997) 90: 3819-3843). Therefore, antibodies such as M070-H10 may bind to domain 5 or domain 6.

[0377] like Fig.14 As shown in panel A of FIG, 559B-M0004-B04 showed selectivity for 2-chain over 1-chain HMWK and LMWK and was selected for further assay optimization. A sandwich ELISA was developed to detect cleaved HMWK in human plasma samples, in which 559B-M0004-B04 (100 μL, 2 μg / mL) was inertly immobilized on a 96-well plate (Nunc Maxisorp plate) ( Fig.12 , Panel B). The next day, the plates were washed and then blocked with 2% BSA (protease / IgG-free) in PBS buffer. After washing, the samples contained cleaved HMWK in 0.1% BSA buffer (2-chain HMWK assay buffer) in PBS containing 0.05% Tween-20. Purified protein standards (e.g., 2-chain HMWK, intact HMWK, or LMWK) were spiked into HNKW HMWK-deficient plasma and diluted 1:320 in 2-chain HMWK assay buffer. After washing the plates with PBST, a mixture of 2 mouse monoclonal antibodies (11H05 and 13B12) at 1 μg / mL in 2-chain HMWK assay buffer was added at room temperature for 1 hour. Unbound detection antibodies were washed and a 1:2000 dilution of goat anti-mouse secondary antibody conjugated to horseradish peroxidase (HRP) was added. The samples containing the secondary antibody were incubated for 1 hour at room temperature and the unbound secondary antibody was removed by washing with 2-chain HMWK assay buffer. The signal was detected by adding 3,3',5,5'-tetramethylbenzidine (TMB), a HRP substrate. The reaction was terminated with phosphoric acid. The hydrolysis of TMB substrate was detected at 450nm-630nm using a microplate reader ( Figure 3 In addition, ELISA assays were performed using samples containing HMWK cleaved in 2-chain HMWK assay buffer or HMWK-deficient plasma and analyzed in the presence of 2.5% or 10% plasma, resulting in similar binding ( Figure 4Using these immunoassay conditions, specific binding to cleaved HMWK was detected. The assay resulted in comparable performance when HMWK was provided in 2-chain HMWK assay buffer or HMWK-deficient plasma ( Figure 3 and Figure 4 ). In addition, there was no binding of 559B-M0004-B04 to LMWK.

[0378] Evaluation of an ELISA assay for the detection of cleaved HMWK ( Figure 5 A and Figure 5 B). The amount of cleaved HMWK in normal human plasma was measured in the absence or presence of catalytic amounts of FXIIa, pKal or ellagic acid, which causes autoactivation of FXII to FXIIa and thus produces cleaved HMWK ( Figure 5 , Panels A and B). Consistent with the role of plasma kallikrein as the major plasma enzyme required for the production of 2-chain HMWK, neither ellagic acid nor FXIIa addition resulted in the production of cleaved HMWK in prekallikrein-deficient plasma. The contact system in FXI-deficient plasma was similarly activated using FXIIa, pKal, or ellagic acid; this result is consistent with the understanding that FXIa is produced by FXIIa and does not produce 2-chain HMWK.

[0379] The results from the 2-chain HMWK ELISA were confirmed by Western blot analysis using mouse monoclonal antibody 11H05 by detecting cleaved HMWK generated after contact activation in human plasma ( Fig.10 ). The 11H05 antibody specifically binds to the light chain of HMWK and elucidates both a 56 kDa light chain and a further proteolytically cleaved 46 kDa light chain, which is subsequently generated by the proteolytic activity of plasma kallikrein at a site near the N-terminus of the HMWK light chain (Colman et al. Blood (1997) 90:3819-3843).

[0380] The ability of the ELISA assay to detect cleaved HMWK produced in plasma from 12 normal donors was also evaluated ( Figure 6 After activation with ellagic acid in the contact activation system, cleaved HMWK was detected in each of the 12 samples. After inhibition of the contact activation system in normal plasma using various concentrations of landarumab (DX-2930; a specific inhibitor of plasma kallikrein) or the serine protease inhibitor C1-INH and then activation with ellagic acid ( Figure 7, Panels A and B), the amount of cleaved HMWK was also measured. Landalumab (DX-2930) is a potent fully human antibody (Ki = 0.12 nM) and a specific inhibitor of plasma kallikrein (Chyung et al. Ann. Allergy Asthma Immunol. (2014) 113: 460-466; Kenniston et al. J. Biol. Chem. (1994) 289: 23596-23608) discovered using phage and is being clinically developed for the preventive treatment of HAE-C1INH attacks. When landalumab was spiked into citrated plasma at different concentrations, it effectively inhibited the production of 2-chain HMWK induced by FXIIa as shown by Western blots and sandwich ELISA ( Figure 7 B) IC of Landalumab for inhibition of 2-chain HMWK production 50 The concentration of M004-B04 was 212±28 nM, which is consistent with the expected value for activation of all prekallikreins in pure plasma (approximately 500 nM). The complete inhibition of the signal by landarumab in plasma treated with a contact system activator confirmed that M004-B04 is specific for the double-chain HMWK generated by plasma kallikreins.

[0381] In this preliminary assay, using M004-B04 as the capture antibody and HRP-conjugated sheep polyclonal anti-kininogen as the detection antibody, activation of the contact system in kininogen-deficient plasma did not result in an increase in the ELISA signal (data not shown).

[0382] from Fig.10 It can also be seen that plasma collected from healthy subjects using EDTA as an anticoagulant is activated similarly to citrated plasma; supporting the argument that metal ions are not required for contact system activation (Colman et al. Blood (1997) 90:3819-3843). However, no 2-chain HMWK was detected by ELISA in EDTA plasma ( Figure 5 B), indicating that the binding of M004-B04 antibody to 2-chain HMWK depends on metal ions. The zinc binding site on HMWK in light chain domain 5 (amino acids 479-498) was previously identified and shown to mediate kininogen interactions with endothelial cell surface receptors gC1qR, cytokeratin 1, and urokinase plasminogen activator receptors, thereby enhancing contact system activation (Kaplan et al. Adv. Immunol. (2014) 121: 41-89; Bjorkqvist et al. Biol. Chem. (2013) 394: 1195-1204). Adding ZnCl2 to the assay buffer was tested at various concentrations and found to enhance the binding of the antibody to cleaved HMWK ( Fig.11The effect of increasing concentrations of ZnCl2 on the ELISA signal observed with ellagic acid activated citrate and EDTA plasma was investigated. The ELISA signal in EDTA plasma increased to an apparent maximum at ZnCl2 concentrations above 400 μM (well concentration).

[0383] Electron microscopy was previously used to show that the binding of 1-chain HMWK to zinc promotes a more compact and globular quaternary structure (Herwald et al. Eur J. Biochem. (2001) 268: 396-404). Electron microscopy also showed that in a buffer containing EDTA, 2-chain HMWK adopts a longer, less globular quaternary structure than 1-chain HMWK (Herwald et al. Eur J. Biochem. (2001) 268: 396-404). Although the effect of zinc on the structure of 2-chain HMWK has not been previously reported, the apparent zinc-dependent binding of M004-B04 described herein indicates that 2-chain HMWK exists in a unique conformation in the presence of zinc.

[0384] The EDTA concentration in plasma collected in commercial spray-coated K2EDTA tubes was approximately 4 mM, which translated to an in-well concentration of approximately 200 μM after a 1:20 dilution and is consistent with the restoration of zinc-dependent binding after the addition of sufficient ZnCl2 to overwhelm the chelating capacity of EDTA. In contrast, the ELISA signal for citrated plasma activated with ellagic acid did not increase in the presence of 25 μM or 50 μM ZnCl2 (well concentrations), but increased at concentrations above 100 μM ZnCl2, with the ELISA signal increasing to a maximum above 200 μM ZnCl2. ( Fig.11 ) The normal concentration of zinc in plasma from healthy volunteers is 10-17 μM (Wessells et al. J. Nutr. (2014) 144: 1204-1210). Since the ELISA signal observed in activated citrated plasma only increased when the ZnCl2 concentration in the well was >50 μM, which is equivalent to the concentration in plasma >1 mM, it seems that ELISA is not sensitive to physiological fluctuations in zinc concentration in plasma. Therefore, ZnCl2 was not added to the assay buffer in subsequent experiments.

[0385] As described above, the binding of 559B-M004-B04 to 2-chain HMWK is enhanced by supraphysiological concentrations of ZnCl2 and is inhibited by high concentrations of EDTA through metal chelation. It has been described that the zinc binding site is in the domain of 2-chain HWMK, and a synthetic peptide (HKH20, HKHGHGHGKHKNKGKKNGKH (SEQ ID NO: 83)) comprising this site was shown to inhibit contact system activation by attenuating cell surface association (Nakazawa et al. Int. Immunopharmacol. (2002) 2: 1875-1885). Therefore, the ability of the HKH20 peptide and the GCP28 peptide corresponding to the sequence in domain 3 to inhibit the binding of 2-chain HMWK to 559B-M004- was tested by ELISA. Fig.15 As shown, HKH20 peptide, but not GCP28 peptide, inhibited the binding of 2-chain HMWK to M004-B04, suggesting that the M004-B04 epitope may be located within domain 5 near the zinc binding site. To perform this assay, the kininogen peptide was diluted to 250 μg / mL and pre-incubated on the assay plate, and then purified 2-chain HMWK from deficient human plasma was diluted 160 and added to the plate.

[0386] The time dependence of cleaved HMWK production in normal citrated human plasma was assessed at different time points after activation of the contact activation system with ellagic acid or FXIIa ( Figure 8 Finally, an ELISA assay was used to assess the presence and amount of cleaved HMWK in plasma samples from patients with hereditary angioedema (HAE) compared to citrated plasma samples from normal patients (without HAE). It was found that samples from HAE patients contained elevated levels of 2-chain HMWK (1423 ± 603 ng / mL) relative to samples from normal donors (432.4 ± 186 ng / mL) ( Fig. 9 ), which was statistically significant by one-way ANOVA analysis (P=0.017).

[0387] Having determined that M004-B04 specifically binds to a novel epitope on 2-chain HMWK that is not present on 1-chain HMWK or LMWK and demonstrating that antibody binding is dependent on plasma kallikrein activity, the assay also used a pair of mouse monoclonal antibodies (11H05 and 13B12) for detection ( Fig.16 ). Antibody 13B12 appears to bind to the heavy chain of HMWK, and 11H05 appears to bind to the light chain of HMWK, the combination of the two antibodies used for detection resulted in an enhanced signal, which may be due to their non-overlapping binding epitopes in the antigen.

[0388] The importance of plasma collection for contact system assessment has been described previously (Suffritti et al. Clin. Exp. Allergy (2014) 44: 1503-1514). It is well known that contact of plasma with glass or other polar surfaces results in extensive ex vivo contact system activation, which may mask accurate determination of endogenous contact system activation (Colman et al., Blood (1997) 90: 3819-3843). The ability of the optimized sandwich ELISA to detect 2-chain HMWK was compared in different plasma types, including a custom plasma containing a cocktail of protease inhibitors in acid citrate dextrose in a vacuum plastic blood collection tube, called SCAT169 (HTI, Essex VT). Figure 6 As shown, the standard curve prepared in SCAT169 plasma is less sensitive than that prepared in citrated plasma, which may be due to the inclusion of 2mM EDTA in the collected plasma. In the plasma dilution used in this assay (1:320), this concentration of EDTA (3.1μM) does not significantly interfere with 2-chain HMWK and may help stabilize the plasma from proteolytic degradation caused by metalloproteinases.

[0389] Citrated plasma and SCAT169 plasma from healthy volunteers were compared with samples from HAE patients by Western blot and sandwich ELISA assays. Fig.17 , Panels AC, Western blot assay for detection of 2-chain HMWK (i.e., cleaved kininogen) in citrated plasma was able to differentiate samples from HAE patients from samples from healthy volunteers (HV), as shown by receiver operator characteristic (ROC) analysis with an area under the curve (AUC) value of 0.977 for basal versus HV comparisons or 1.0 for flare versus HV comparisons. Citrated plasma samples from HAE patients at rest (basal) were differentiated from flare samples with an AUC of 0.625 ( Fig.17 , Panel D).

[0390] like Fig.18 As shown in panels AC, Western blots for detection of 2-chain HMWK in SCAT169 plasma were able to differentiate samples from HAE patients from samples from healthy volunteers (HV), as shown in ROC analysis, with an AUC value of 0.915 for the comparison of basal to HV, or 0.967 for the comparison of exacerbation to HV. SCAT169 samples from HAE patients at rest (basal) were differentiated from exacerbation samples with an AUC of 0.597 ( Fig.18 , Panel D).

[0391] exist Fig.19In panels AC, the 2-chain ELISA method for detecting 2-chain HMWK in citrated plasma was able to distinguish samples from HAE patients from samples from healthy volunteers, as shown by ROC analysis, with an AUC value of 0.915 for the comparison of basal to HV, or 0.866 for the comparison of flare to HV. Citrated plasma samples from HAE patients at rest (basal) were distinguished from flare samples with an AUC of 0.709 ( Fig.19 , Panel D).

[0392] like Fig. 20 As shown in panels AC, the 2-chain ELISA method for detecting 2-chain HMWK in SCAT169 samples was able to distinguish samples from HAE patients from samples from healthy volunteers, as shown by ROC analysis, with an AUC value of 0.999 for the comparison of basal to HV, or 1.0 for the comparison of flare to HV. Citrated plasma samples from HAE patients at rest (basal) were distinguished from flare samples with an AUC of 0.8176 ( Fig. 20 , Panel D).

[0393] For the above ROC analysis, both the 2-chain HMWK Western blot and the 2-chain HMWK ELISA demonstrated herein can be used to differentiate patients with or at risk of HAE from healthy volunteers based on the levels of cleaved kininogen in plasma. The presence of protease inhibitors in SCAT169 plasma reduces ex vivo plasma activation during blood collection.

[0394] Other Implementations

[0395] All features disclosed in this specification can be combined in any combination. Each feature disclosed in this specification can be used for alternative feature replacement of the same, equivalent or similar purpose. Therefore, unless otherwise clearly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0396] From the above description, those skilled in the art can easily determine the essential characteristics of the present disclosure, and without departing from its spirit and scope, various changes and modifications can be made to the present disclosure to adapt it to various applications and conditions. Therefore, other embodiments are also within the scope of the claims of the present disclosure.

[0397] Principle and scope of equivalents

[0398] Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation many equivalents to the specific embodiments of the disclosure described herein.The scope of the present disclosure is not intended to be limited to the above description, but rather is set forth as in the appended claims.

[0399] In the claims, articles such as "a," "an," and "the" may mean one or more, unless indicated otherwise or otherwise obvious from the context. If one, more than one, or all of the members of a group appear in, apply to, or otherwise relate to a given product or method, the claim or specification is considered to satisfy the inclusion of "or" between one or more members of the group, unless indicated otherwise or otherwise obvious from the context. The present disclosure includes embodiments in which exactly one member of the group appears in, applies to, or otherwise relates to a given product or method. The present disclosure includes embodiments in which more than one or all of the members of the group appear in, apply to, or otherwise relate to a given product or method.

[0400] In addition, the present disclosure encompasses all such variations, combinations and arrangements: one or more limitations, elements, clauses and descriptive terms from one or more listed claims are introduced into another claim. For example, any claim that is subordinate to another claim may be modified to include one or more limitations that appear in any other claim that is subordinate to the same basic claim. In the case where the elements appear as a list, for example, in the form of Markush groups, each subgroup of the elements is also disclosed, and any element (one or more) can be removed from the group. In general, it should be understood that in the case where the present disclosure or aspects of the present disclosure are referred to as including specific elements and / or features, certain embodiments of the present disclosure or aspects of the present disclosure are composed of or substantially composed of such elements and / or features. For the purpose of simplification, those embodiments are not explicitly explained herein. It is also noted that the terms "include" and "comprising" are intended to be open-ended and allow other elements or steps to be included. In the case of a given range, endpoints are included. In addition, unless otherwise noted or otherwise apparent from the context, it is understood by those of ordinary skill in the art that the values ​​expressed as ranges can be considered to be any specific value or subrange within the narrative range in different embodiments of the present disclosure, to one tenth of the unit of the lower limit of the range, unless the context clearly indicates otherwise.

[0401] This application cites various published patents, published patent applications, journal articles and other publications, all of which are incorporated herein by reference. If there is a conflict between any incorporated reference and this description, this description shall prevail. In addition, any specific embodiment of the present disclosure that falls within the prior art may be explicitly excluded from any one or more of the claims. Because such embodiments are considered to be known to those of ordinary skill in the art, they may be excluded, even if the exclusion is not explicitly explained herein. Any specific embodiment of the present disclosure may be excluded from any claim for any reason, whether or not it is related to the existence of the prior art.

[0402] Using only routine experimentation, those skilled in the art will recognize or be able to ascertain 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 as 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 present disclosure as defined in the following claims. Sequence Listing <110> Takeda Pharmaceutical Company Limited <120> Immunoassay for the detection of cleaved high molecular weight kininogen <130> FPAP-1800263 <140> Not yet assigned <141> at the same time <150> US 62 / 335,311 <151> 2016-05-12 <150> US 62 / 243,505 <151> 2015-10-19 <160> 83 <170> PatentIn Version 3.5 <210> 1 <211> 644 <212> PRT <213> Homo sapiens <400> 1 Met Lys Leu Ile Thr Ile Leu Phe Leu Cys Ser Arg Leu Leu Leu Ser 1 5 10 15 Leu Thr Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp 20 25 30 Leu Phe Lys Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn 35 40 45 Gln Ser Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys 50 55 60 Thr Val Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu 65 70 75 80 Gly Asp Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr 85 90 95 Lys Asp Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly 100 105 110 Lys Arg Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile 115 120 125 Thr Pro Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly 130 135 140 Cys Val His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu 145 150 155 160 Arg His Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu 165 170 175 Phe Met Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly 180 185 190 Leu Asn Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys 195 200 205 Glu Asn Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly 210 215 220 Asp Thr Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg 225 230 235 240 Ile Ala Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe 245 250 255 Val Gln Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro 260 265 270 Thr Asn Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys 275 280 285 Leu Asn Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val 290 295 300 Lys Lys Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp 305 310 315 320 Phe Val Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu 325 330 335 Thr Glu Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn 340 345 350 Ala Glu Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val 355 360 365 Asn Cys Gln Pro Leu Gly Met Ile Ser Leu Met Lys Arg Pro Pro Gly 370 375 380 Phe Ser Pro Phe Arg Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr 385 390 395 400 Thr Val Ser Pro Pro His Thr Ser Met Ala Pro Ala Gln Asp Glu Glu 405 410 415 Arg Asp Ser Gly Lys Glu Gln Gly His Thr Arg Arg His Asp Trp Gly 420 425 430 His Glu Lys Gln Arg Lys His Asn Leu Gly His Gly His Lys His Glu 435 440 445 Arg Asp Gln Gly His Gly His Gln Arg Gly His Gly Leu Gly His Gly 450 455 460 His Glu Gln Gln His Gly Leu Gly His Gly His Lys Phe Lys Leu Asp 465 470 475 480 Asp Asp Leu Glu His Gln Gly Gly His Val Leu Asp His Gly His Lys 485 490 495 His Lys His Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 500 505 510 Asn Gly Lys His Asn Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser 515 520 525 Glu Asp Ser Thr Thr Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly 530 535 540 Pro Thr Pro Ile Pro Ser Leu Ala Lys Pro Gly Val Thr Val Thr Phe 545 550 555 560 Ser Asp Phe Gln Asp Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile 565 570 575 Ser Pro Ala Pro Ile Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln 580 585 590 Ile Asp Pro Asn Gly Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp 595 600 605 Thr Thr Ser Pro Lys Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu 610 615 620 Ile Asn Pro Thr Thr Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr 625 630 635 640 Asp Gly Leu Ser <210> 2 <211> 362 <212> PRT <213> Homo sapiens <400> 2 Gln Glu Ser Gln Ser Glu Glu Ile Asp Cys Asn Asp Lys Asp Leu Phe 1 5 10 15 Lys Ala Val Asp Ala Ala Leu Lys Lys Tyr Asn Ser Gln Asn Gln Ser 20 25 30 Asn Asn Gln Phe Val Leu Tyr Arg Ile Thr Glu Ala Thr Lys Thr Val 35 40 45 Gly Ser Asp Thr Phe Tyr Ser Phe Lys Tyr Glu Ile Lys Glu Gly Asp 50 55 60 Cys Pro Val Gln Ser Gly Lys Thr Trp Gln Asp Cys Glu Tyr Lys Asp 65 70 75 80 Ala Ala Lys Ala Ala Thr Gly Glu Cys Thr Ala Thr Val Gly Lys Arg 85 90 95 Ser Ser Thr Lys Phe Ser Val Ala Thr Gln Thr Cys Gln Ile Thr Pro 100 105 110 Ala Glu Gly Pro Val Val Thr Ala Gln Tyr Asp Cys Leu Gly Cys Val 115 120 125 His Pro Ile Ser Thr Gln Ser Pro Asp Leu Glu Pro Ile Leu Arg His 130 135 140 Gly Ile Gln Tyr Phe Asn Asn Asn Thr Gln His Ser Ser Leu Phe Met 145 150 155 160 Leu Asn Glu Val Lys Arg Ala Gln Arg Gln Val Val Ala Gly Leu Asn 165 170 175 Phe Arg Ile Thr Tyr Ser Ile Val Gln Thr Asn Cys Ser Lys Glu Asn 180 185 190 Phe Leu Phe Leu Thr Pro Asp Cys Lys Ser Leu Trp Asn Gly Asp Thr 195 200 205 Gly Glu Cys Thr Asp Asn Ala Tyr Ile Asp Ile Gln Leu Arg Ile Ala 210 215 220 Ser Phe Ser Gln Asn Cys Asp Ile Tyr Pro Gly Lys Asp Phe Val Gln 225 230 235 240 Pro Pro Thr Lys Ile Cys Val Gly Cys Pro Arg Asp Ile Pro Thr Asn 245 250 255 Ser Pro Glu Leu Glu Glu Thr Leu Thr His Thr Ile Thr Lys Leu Asn 260 265 270 Ala Glu Asn Asn Ala Thr Phe Tyr Phe Lys Ile Asp Asn Val Lys Lys 275 280 285 Ala Arg Val Gln Val Val Ala Gly Lys Lys Tyr Phe Ile Asp Phe Val 290 295 300 Ala Arg Glu Thr Thr Cys Ser Lys Glu Ser Asn Glu Glu Leu Thr Glu 305 310 315 320 Ser Cys Glu Thr Lys Lys Leu Gly Gln Ser Leu Asp Cys Asn Ala Glu 325 330 335 Val Tyr Val Val Pro Trp Glu Lys Lys Ile Tyr Pro Thr Val Asn Cys 340 345 350 Gln Pro Leu Gly Met Ile Ser Leu Met Lys 355 360 <210> 3 <211> 255 <212> PRT <213> Homo sapiens <400> 3 Ser Ser Arg Ile Gly Glu Ile Lys Glu Glu Thr Thr Val Ser Pro Pro 1 5 10 15 His Thr Ser Met Ala Pro Ala Gln Asp Glu Glu Arg Asp Ser Gly Lys 20 25 30 Glu Gln Gly His Thr Arg Arg His Asp Trp Gly His Glu Lys Gln Arg 35 40 45 Lys His Asn Leu Gly His Gly His Lys His Glu Arg Asp Gln Gly His 50 55 60 Gly His Gln Arg Gly His Gly Leu Gly His Gly His Glu Gln Gln His 65 70 75 80 Gly Leu Gly His Gly His Lys Phe Lys Leu Asp Asp Asp Leu Glu His 85 90 95 Gln Gly Gly His Val Leu Asp His Gly His Lys His Lys His Gly His 100 105 110 Gly His Gly Lys His Lys Asn Lys Gly Lys Lys Asn Gly Lys His Asn 115 120 125 Gly Trp Lys Thr Glu His Leu Ala Ser Ser Ser Glu Asp Ser Thr Thr 130 135 140 Pro Ser Ala Gln Thr Gln Glu Lys Thr Glu Gly Pro Thr Pro Ile Pro 145 150 155 160 Ser Leu Ala Lys Pro Gly Val Thr Val Thr Phe Ser Asp Phe Gln Asp 165 170 175 Ser Asp Leu Ile Ala Thr Met Met Pro Pro Ile Ser Pro Ala Pro Ile 180 185 190 Gln Ser Asp Asp Asp Trp Ile Pro Asp Ile Gln Ile Asp Pro Asn Gly 195 200 205 Leu Ser Phe Asn Pro Ile Ser Asp Phe Pro Asp Thr Thr Ser Pro Lys 210 215 220 Cys Pro Gly Arg Pro Trp Lys Ser Val Ser Glu Ile Asn Pro Thr Thr 225 230 235 240 Gln Met Lys Glu Ser Tyr Tyr Phe Asp Leu Thr Asp Gly Leu Ser 245 250 255 <210> 4 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 4 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Val Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Ser Gly Gly Asn Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Lys Leu Phe Tyr Tyr Asp Asp Thr Lys Gly Tyr Phe Asp Phe 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 5 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 5 Gln Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Leu Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Arg Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 6 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 6 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Leu Tyr 20 25 30 Pro Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Pro Ser Gly Gly Phe Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Ser Arg Tyr Tyr Tyr Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 7 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 7 Gln Tyr Glu Leu Thr Gln Pro Pro Ser Met Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Glu 20 25 30 Tyr Val Tyr Trp Phe Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asp Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Thr Asp Tyr Tyr Cys Ser Thr Trp Asp Asp Thr Leu 85 90 95 Arg Thr Gly Val Phe Gly Gly Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 8 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 8 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Arg Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Ser Gly Gly Trp Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Thr Asp Asn Gly Asp Tyr Ala Leu Ala His Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 9 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 9 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Arg Ile Ile Asn 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Ala Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Arg Val Glu Ile Lys 100 105 <210> 10 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 10 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30 Ser Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Ser Ser Gly Gly Ser Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Thr Tyr Tyr Cys 85 90 95 Ala Arg Thr Arg Arg Gly Trp Phe Gly Glu Asp Tyr Tyr Tyr Tyr Met 100 105 110 Asp Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 11 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 11 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Ile Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn 20 25 30 Asp Val Gly Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Gln Arg Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln His Asn Ser Tyr Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 12 <211> 126 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 12 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 Met Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gly Lys Thr Trp Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Gly Gly Ser Ser Ser Tyr Tyr Tyr Tyr Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 13 <211> 111 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 13 Gln Ser Ala Leu Thr Gln Ser Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Gly Asn 20 25 30 Thr Val Asn Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ser Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Ile Tyr Tyr Cys Ala Ser Trp Asp Asp Arg Leu 85 90 95 Asn Gly His Trp Val Phe Gly Gly Gly Thr Arg Leu Thr Val Leu 100 105 110 <210> 14 <211> 123 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 14 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Trp Pro Ser Gly Gly Gly Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Asp Tyr Asp Tyr Gly Asp Phe Thr Asp Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 15 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 15 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Ser Tyr 20 25 30 Asn Leu Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Gly Ser Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Ile Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Cys Ser Tyr Ala Gly Ser 85 90 95 Tyr Ser Tyr Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu 100 105 110 <210> 16 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 16 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Gln Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Tyr Ser Ser Gly Gly Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Leu Pro Gly Gln Pro Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 17 <211> 110 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 17 Gln Ser Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Tyr Val Tyr Trp Tyr Gln Gln Phe Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asp Arg Leu 85 90 95 Ser Gly Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 18 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 18 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gln Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Ser Gly Gly Ser Thr Pro Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly His His Gly Met Asp Val Trp Gly Gln Gly Thr Thr Val 100 105 110 Thr Val Ser Ser 115 <210> 19 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 19 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser 20 25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Asn Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Lys Tyr Asn Ile Ala Pro 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 20 <211> 120 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 20 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 Pro Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Ser Ser Gly Gly Phe Thr Pro Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Met Val Arg Gly Val Ile Lys Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 21 <211> 110 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 21 Gln Tyr Glu Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser His 20 25 30 Tyr Val Phe Trp Tyr Gln Gln Leu Pro Gly Ala Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Arg Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Thr Trp Asp Asn Ser Leu 85 90 95 Ser Ala Trp Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 22 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 22 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Thr Met Trp Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Ser Ser Gly Gly Lys Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Ala Asn Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met 100 105 110 Val Thr Val Ser Ser 115 <210> 23 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 23 Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Ala Leu Ser Val Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30 Asp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile His Gly Ala Ser Thr Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Arg Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Asn Asp Trp Pro 85 90 95 Pro Leu Phe Gly Pro Gly Thr Lys Val Asn Ile Lys 100 105 <210> 24 <211> 125 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 24 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Tyr Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Val Pro Ser Gly Gly Gln Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Thr Arg Arg Gly Trp Phe Gly Glu Asp Tyr Tyr Tyr Tyr Met 100 105 110 Asp Val Trp Gly Lys Gly Thr Leu Val Thr Val Ser Ser 115 120 125 <210> 25 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 25 Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Val Ser Cys Arg Ala Ser Gln Ser Val Gly Ser 20 25 30 Thr Tyr Leu Ala Trp Tyr Gln His Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Glu Pro Glu Asp Phe Ala Ile Tyr Tyr Cys Gln His Phe His Thr Ser 85 90 95 Pro Pro Gly Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 110 <210> 26 <211> 118 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 26 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Lys Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Thr Arg Thr Ser Gly Leu Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 27 <211> 110 <212> PRT <213> artificial sequence <220> <223> Synthetic polypeptide <400> 27 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Lys Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Val Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Ser 85 90 95 Thr Thr Val Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 28 <211> 119 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 28 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Thr Tyr 20 25 30 Gly Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Ser Gly Gly Lys Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Arg Pro Asp Tyr Tyr Ala Met Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 29 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 29 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gly Ala Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Ser Asn 20 25 30 Thr Val Asn Trp Tyr Gln Lys Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Tyr Asn Asp Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu Gln 65 70 75 80 Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Ser Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 30 <211> 126 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 30 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Pro Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Ser Gly Gly Lys Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Gln Gly Arg Ala Val Arg Gly Lys Leu Tyr Tyr Tyr Gly 100 105 110 Met Asp Val Trp Gly Gln Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 31 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 31 Gln Ser Ala Leu Thr Gln Pro Pro Ser Ala Ser Gln Thr Pro Gly Gln 1 5 10 15 Thr Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Thr Asn 20 25 30 Asn Val Asn Trp Tyr Gln Gln Leu Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Ser Ser His His Arg Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Ala Ser Lys Ser Gly Thr Ser Ala Ser Leu Ala Ile Ser Gly Leu Gln 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asp Ser Leu 85 90 95 Asn Gly Pro Val Phe Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 110 <210> 32 <211> 117 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 32 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 His Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Ser Ser Gly Gly Ser Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Val Arg Tyr Gly Met Asp Val Trp Gly Gln Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 33 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 33 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Val Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser 20 25 30 Trp Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ala Asn Ser Phe Pro 85 90 95 Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 34 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 34 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Met Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Gly Gly Tyr Thr Gln Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Arg Gly Leu Ile Ala Ala Ala Gly Gly Phe Asp Pro Trp 100 105 110 Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 35 <211> 108 <212> PRT <213> Artificial sequence <220> <223> synthetic peptide <400> 35 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Gly Ile 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Thr 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Arg Thr Tyr Gly Arg Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 36 <211> 121 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 36 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Glu Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Pro Ser Gly Gly Tyr Thr Met Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg His Arg Ser Lys Trp Asn Asp Ala Pro Phe Asp Ser Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 37 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 37 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Ala Ile Thr Cys Arg Ala Ser Gln Ser Ile Asp Thr 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Lys Leu Glu Asp Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Thr Gly Thr Asp Phe Thr Leu Thr Ile Arg Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Ser Tyr Phe Cys Gln Gln Ser Tyr Ser Ser Pro 85 90 95 Gly Ile Thr Phe Gly Pro Gly Thr Lys Val Glu Ile Lys 100 105 <210> 38 <211> 123 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 38 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Gln Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Tyr Ser Ser Gly Gly Arg Thr Phe Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Arg Gly Ser Trp Tyr Val Gly Gly Asn Glu Tyr Phe Gln His 100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 39 <211> 106 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 39 Gln Ser Val Leu Thr Gln Ser Pro Ser Leu Ser Leu Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Pro Cys Ser Gly Asp Thr Leu Gly Asn Lys Phe Val 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Thr Lys Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Thr Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Val Trp Asp Ser Asn Ser Tyr Ala 85 90 95 Phe Gly Pro Gly Thr Lys Val Thr Val Leu 100 105 <210> 40 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 40 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Phe Tyr 20 25 30 Met Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Ser Ser Gly Gly Phe Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Val Arg Gly Leu Ala Val Ala Ala Pro Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 41 <211> 110 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 41 Gln Ser Glu Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Ile Gly Thr Ser Ser Asp Ile Gly Thr Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Asp Val Asn Thr Arg Pro Ser Gly Val Ser Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Thr Ser 85 90 95 Val Thr Trp Val Phe Gly Gly Gly Thr Thr Leu Thr Val Leu 100 105 110 <210> 42 <211> 115 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 42 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gly Tyr 20 25 30 Asn Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Pro Ser Gly Gly Trp Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Thr Arg Gly Gln Trp Met Asp Trp Trp Gly Gln Gly Thr Met Val Thr 100 105 110 Val Ser Ser 115 <210> 43 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 43 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Ile Ile Thr Cys Arg Ala Ser Gln Asn Ile Thr Gly 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Asn Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Arg Met Asn Thr Gly Val Pro Ser Arg Phe Arg 50 55 60 Gly Ser Gly Ser Gly Thr Asp Tyr Ile Leu Thr Ile Tyr Lys Leu Glu 65 70 75 80 Pro Glu Asp Ile Gly Thr Tyr Phe Cys Gln His Thr Asp Asp Phe Ser 85 90 95 Val Thr Phe Gly Gly Gly Thr Lys Val Asp Leu Lys 100 105 <210> 44 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 44 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe His Tyr Arg 20 25 30 Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val Ser 35 40 45 Tyr Ile Ser Ser Ser Gly Gly Tyr Thr Ala Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Ala Lys Arg Asn Arg Ala Phe Asp Ile Trp Gly Gln Gly Thr Met Val 100 105 110 Thr Val Ser Ser 115 <210> 45 <211> 114 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 45 Gln Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ala Val Ser Leu 1 5 10 15 Gly Glu Arg Ala Thr Ile Asn Cys Lys Ser Ser Gln Ser Val Leu Tyr 20 25 30 Ser Ser Asn Asn Lys Asn Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly 35 40 45 Gln Pro Pro Lys Leu Leu Ile Tyr Trp Ala Ser Thr Arg Glu Ser Gly 50 55 60 Val Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu 65 70 75 80 Thr Ile Ser Ser Leu Gln Ala Glu Asp Val Ala Val Tyr Tyr Cys Gln 85 90 95 Gln Tyr Tyr Ser Thr Pro Leu Gly Phe Gly Gln Gly Thr Lys Leu Glu 100 105 110 Ile Lys <210> 46 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 46 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Gln Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Phe Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Leu Pro Ala Asn Phe Tyr Tyr Tyr Met Asp Val Trp Gly Lys 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 47 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 47 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asn Ile Tyr Ser 20 25 30 Phe Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Thr Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Ser Tyr Tyr Cys Gln Gln Asn Tyr Asn Ile Pro 85 90 95 Trp Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 48 <211> 123 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 48 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Met Met Lys Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Val Pro Ser Gly Gly Trp Thr Thr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Thr Glu Gly Asn Leu Trp Phe Gly Glu Gly Arg Ala Phe Asp Ile 100 105 110 Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 49 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 49 Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu 65 70 75 80 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp 85 90 95 Pro Pro Ser Phe Gly Gln Gly Thr Arg Leu Asp Ile Lys 100 105 <210> 50 <211> 126 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 50 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Asp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Ser Ser Ser Gly Gly Lys Thr Glu Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Tyr Arg Tyr Cys Thr Ala Asn Thr Cys Ser Leu Tyr Gly 100 105 110 Met Asp Val Trp Gly Arg Gly Thr Thr Val Thr Val Ser Ser 115 120 125 <210> 51 <211> 108 <212> PRT <213> Artificial sequence <220> <223> Synthetic polypeptide <400> 51 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Ala Ile Thr Cys Arg Thr Ser Gln Gly Val Arg Ser 20 25 30 Asp Phe Ala Trp Tyr Gln Gln Thr Pro Gly Lys Ala Pro Arg Arg Leu 35 40 45 Ile Tyr Ala Ala Phe Ile Leu Asp Asn Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Met Lys 100 105 <210> 52 <211> 120 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 52 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 Trp Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Ser Gly Gly Gly Thr Gly Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Ser Arg Gly Ser Gly Ser His Glu Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 53 <211> 109 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 53 Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Asn Arg Gly Thr Gly Ile Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Ser Glu Asp Phe Ala Val Tyr Phe Cys Gln Gln Tyr Lys Asn Trp Pro 85 90 95 Asn Leu Thr Phe Gly Gly Gly Thr Lys Val Asp Ile Lys 100 105 <210> 54 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 54 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Pro Met Ala Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Val Ser Ser Gly Gly Arg Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Pro Tyr Asp Phe Trp Ser Glu Gly Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 55 <211> 111 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 55 Gln Ser Val Leu Thr Gln Pro Pro Ser Ala Ser Gly Thr Pro Gly Gln 1 5 10 15 Arg Val Thr Ile Ser Cys Ser Gly Ser Ser Ser Asn Ile Gly Asn Asn 20 25 30 Phe Val Tyr Trp Tyr His Gln Val Pro Gly Thr Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Lys Asn Asn Gln Arg Pro Ser Gly Val Pro Asp Arg Phe Ser 50 55 60 Gly Ser Lys Ser Ala Ala Ser Ala Ser Leu Ala Ile Ser Gly Leu Arg 65 70 75 80 Ser Glu Asp Glu Ala Asp Tyr Tyr Cys Ala Ala Trp Asp Asn Ser Leu 85 90 95 Ser Gly Phe Tyr Val Phe Gly Ala Gly Thr Lys Val Thr Val Leu 100 105 110 <210> 56 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 56 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Arg Ile Gly Pro Ser Gly Gly Pro Thr Ser Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Tyr Tyr Gly Thr Gly Arg Tyr Phe Gln His Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 57 <211> 109 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 57 Gln Asp Ile Gln Met Thr Gln Ser Pro Asp Ser Leu Ser Leu Ser Pro 1 5 10 15 Gly Asp Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser 20 25 30 Asp Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Glu Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp 85 90 95 Pro Pro Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 58 <211> 116 <212> PRT <213> Artificial sequence <220> <223> synthetic peptide <400> 58 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Ala Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Ser Ser Ser Gly Gly Glu Thr Met Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Gly Tyr Gly Arg Ile Asp Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 59 <211> 110 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 59 Gln Ser Val Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Ile Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Ala Pro Lys Leu 35 40 45 Met Ile Tyr Glu Val Ser Asn Arg Pro Ser Gly Val Ser Asn Arg Phe 50 55 60 Ser Gly Ser Lys Ser Gly Asn Thr Ala Ser Leu Thr Ile Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Ser Ser Tyr Thr Ser Gly 85 90 95 Ser Thr Arg Val Phe Gly Thr Gly Thr Arg Val Thr Val Leu 100 105 110 <210> 60 <211> 121 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 60 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Val Met Arg Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Gly Pro Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Gly Gly Ser Gly Ser Ser His Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 61 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 61 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Ser Gly Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Phe Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 62 <211> 122 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 62 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Tyr Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Pro Ser Gly Gly Leu Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Thr Gly Phe Ala Val Gln His Gly Gly Gly Ala Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 63 <211> 108 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 63 Gln Asp Ile Gln Met Thr Gln Ser Pro Ala Thr Leu Ser Met Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Thr Thr 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Asp Ala Ser Ile Arg Ala Thr Gly Val Pro Ala Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Thr Ile Trp Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 64 <211> 119 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 64 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Pro Tyr 20 25 30 Glu Met Val Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Val Pro Ser Gly Gly Trp Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Ser Pro Ser Gly Arg Gly Leu Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 65 <211> 109 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 65 Gln Asp Ile Gln Met Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro 1 5 10 15 Gly Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30 Ser Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu 35 40 45 Leu Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu 65 70 75 80 Gln Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Leu Gln Gln Lys Ser Tyr 85 90 95 Pro Tyr Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 66 <211> 119 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 66 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Lys Tyr 20 25 30 Phe Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Trp Ile Ser Ser Ser Gly Gly Tyr Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Gly Ala Tyr Tyr Tyr Asp Ala Phe Asp Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115 <210> 67 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 67 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ala Ile 20 25 30 Phe Leu Asn Trp Tyr Gln Gln Thr Pro Gly Lys Pro Pro Lys Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Ala Asp Phe Thr Leu Thr Ile Ser Asn Leu Gln 65 70 75 80 Leu Glu Asp Phe Thr Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr Leu 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 68 <211> 117 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 68 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Ser Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Ser Ser Gly Gly Met Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Asp Tyr Tyr Gly Asn Met Asp Val Trp Gly Lys Gly Thr Thr 100 105 110 Val Thr Val Ser Ser 115 <210> 69 <211> 108 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 69 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Thr Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Thr Ser Gln Asp Ile Ser Gly 20 25 30 Ala Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Arg Leu Leu 35 40 45 Ile Phe Gly Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Lys Tyr Pro 85 90 95 Leu Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 70 <211> 116 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 70 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Trp Tyr 20 25 30 Thr Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Tyr Ile Tyr Pro Ser Gly Gly Tyr Thr Met Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Asn Pro Tyr Ser Ser Gly Gly Tyr Trp Gly Gln Gly Thr Leu Val 100 105 110 Thr Val Ser Ser 115 <210> 71 <211> 113 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 71 Gln Asp Ile Gln Met Thr Gln Ser Pro Leu Ser Leu Pro Val Thr Pro 1 5 10 15 Gly Glu Pro Ala Ser Ile Ser Cys Arg Ser Ser Gln Ser Leu Leu Asp 20 25 30 Ser Asn Gly Tyr Asn Tyr Leu Asp Trp Phe Leu Gln Lys Pro Gly Gln 35 40 45 Ser Pro Gln Leu Leu Ile Tyr Leu Gly Phe Asn Arg Ala Ser Gly Val 50 55 60 Pro Asp Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Lys 65 70 75 80 Ile Ser Arg Val Glu Ala Glu Asp Val Gly Val Tyr Tyr Cys Met Gln 85 90 95 Ala Leu Gln Thr Pro Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile 100 105 110 Thr <210> 72 <211> 120 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 72 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ala Tyr 20 25 30 Leu Met Thr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Ser Gly Gly Ile Thr Lys Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Ile Pro Asn Trp Ile Tyr Gly Met Asp Val Trp Gly Gln 100 105 110 Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 73 <211> 105 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 73 Gln Ser Ala Leu Thr Gln Pro Pro Ser Val Ser Val Ser Pro Gly Gln 1 5 10 15 Thr Ala Ser Ile Thr Cys Ser Gly Asp Lys Leu Gly Asn Lys Tyr Ala 20 25 30 Ser Trp Tyr Gln Gln Lys Pro Gly Gln Ser Pro Val Leu Val Ile Tyr 35 40 45 Gln Asp Arg Arg Arg Pro Ser Gly Ile Pro Glu Arg Phe Ser Gly Ser 50 55 60 Asn Ser Gly Asn Thr Ala Thr Leu Thr Ile Ser Gly Thr Gln Ala Met 65 70 75 80 Asp Glu Ala Asp Tyr Tyr Cys Gln Ala Trp Asp Ser Gly Val Val Phe 85 90 95 Gly Gly Gly Thr Lys Leu Thr Val Leu 100 105 <210> 74 <211> 124 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 74 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Leu Met Leu Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Ser Pro Ser Gly Gly Gly Thr Ala Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Met Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ala Tyr Ser Gly Ser Tyr Tyr Tyr Tyr Tyr Tyr Met Asp 100 105 110 Val Trp Gly Lys Gly Thr Thr Val Thr Val Ser Ser 115 120 <210> 75 <211> 109 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 75 Gln Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val 1 5 10 15 Gly Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser 20 25 30 Tyr Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu 35 40 45 Ile Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln 65 70 75 80 Pro Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Ser Tyr Ser Thr His 85 90 95 Ser Ile Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 76 <211> 137 <212> PRT <213> Artificial Sequence <220> <223> Synthetic Polypeptide <400> 76 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Gln Tyr 20 25 30 Ile Met Gly Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Gly Ser Ser Gly Val Thr Val Tyr Ala Asp Ser Val Lys 50 55 60 Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr Leu 65 70 75 80 Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95 Arg Gly Gly Gly Val Thr Val Leu His Ala Phe Asp Ile Trp Gly Gln 100 105 110 Gly Thr Met Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser 130 135 <210> 77 <211> 118 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 77 Gln Ser Ala Leu Thr Gln Pro Ala Ser Val Ser Gly Ser Pro Gly Gln 1 5 10 15 Ser Ile Thr Ile Ser Cys Thr Gly Thr Ser Ser Asp Val Gly Gly Tyr 20 25 30 Asn Tyr Val Ser Trp Tyr Gln Gln His Pro Gly Lys Val Pro Lys Leu 35 40 45 Ile Ile Tyr Glu Gly Asn Lys Arg Pro Ser Gly Val Pro Asp Arg Phe 50 55 60 Ser Gly Ser Lys Ala Gly Asn Thr Ala Ser Leu Thr Val Ser Gly Leu 65 70 75 80 Gln Ala Glu Asp Glu Ala Asp Tyr Tyr Cys Thr Ala Tyr Gly Gly His 85 90 95 Ser Arg Phe Tyr Val Phe Gly Thr Gly Thr Lys Val Thr Val Leu Gly 100 105 110 Gln Pro Lys Ala Asn Pro 115 <210> 78 <211> 304 <212> PRT <213> Homo sapiens <400> 78 Met Ile Tyr Thr Met Lys Lys Val His Ala Leu Trp Ala Ser Val Cys 1 5 10 15 Leu Leu Leu Asn Leu Ala Pro Ala Pro Leu Asn Ala Asp Ser Glu Glu 20 25 30 Asp Glu Glu His Thr Ile Ile Thr Asp Thr Glu Leu Pro Pro Leu Lys 35 40 45 Leu Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Lys 50 55 60 Ala Ile Met Lys Arg Phe Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu 65 70 75 80 Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu Ser 85 90 95 Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp Asn Ala Asn Arg Ile 100 105 110 Ile Lys Thr Thr Leu Gln Gln Glu Lys Pro Asp Phe Cys Phe Leu Glu 115 120 125 Glu Asp Pro Gly Ile Cys Arg Gly Tyr Ile Thr Arg Tyr Phe Tyr Asn 130 135 140 Asn Gln Thr Lys Gln Cys Glu Arg Phe Lys Tyr Gly Gly Cys Leu Gly 145 150 155 160 Asn Met Asn Asn Phe Glu Thr Leu Glu Glu Cys Lys Asn Ile Cys Glu 165 170 175 Asp Gly Pro Asn Gly Phe Gln Val Asp Asn Tyr Gly Thr Gln Leu Asn 180 185 190 Ala Val Asn Asn Ser Leu Thr Pro Gln Ser Thr Lys Val Pro Ser Leu 195 200 205 Phe Glu Phe His Gly Pro Ser Trp Cys Leu Thr Pro Ala Asp Arg Gly 210 215 220 Leu Cys Arg Ala Asn Glu Asn Arg Phe Tyr Tyr Asn Ser Val Ile Gly 225 230 235 240 Lys Cys Arg Pro Phe Lys Tyr Ser Gly Cys Gly Gly Asn Glu Asn Asn 245 250 255 Phe Thr Ser Lys Gln Glu Cys Leu Arg Ala Cys Lys Lys Gly Phe Ile 260 265 270 Gln Arg Ile Ser Lys Gly Gly Leu Ile Lys Thr Lys Arg Lys Arg Lys 275 280 285 Lys Gln Arg Val Lys Ile Ala Tyr Glu Glu Ile Phe Val Lys Asn Met 290 295 300 <210> 79 <211> 58 <212> PRT <213> Homo sapiens <400> 79 Arg Pro Asp Phe Cys Leu Glu Pro Pro Tyr Thr Gly Pro Cys Lys Ala 1 5 10 15 Arg Ile Ile Arg Tyr Phe Tyr Asn Ala Lys Ala Gly Leu Cys Gln Thr 20 25 30 Phe Val Tyr Gly Gly Cys Arg Ala Lys Arg Asn Asn Phe Lys Ser Ala 35 40 45 Glu Asp Cys Met Arg Thr Cys Gly Gly Ala 50 55 <210> 80 <211> 60 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 80 Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys 1 5 10 15 Arg Ala Ala His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys 20 25 30 Glu Glu Phe Ile Tyr Gly Gly Cys Glu Gly Asn Gln Asn Arg Phe Glu 35 40 45 Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp 50 55 60 <210> 81 <211> 122 <212> PRT <213> Artificial sequence <220> <223> Synthetic peptides <400> 81 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser His Tyr 20 25 30 Ile Met Met Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Gly Ile Tyr Ser Ser Gly Gly Ile Thr Val Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Tyr Arg Arg Ile Gly Val Pro Arg Arg Asp Glu Phe Asp Ile Trp 100 105 110 Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 82 <211> 105 <212> PRT <213> artificial sequence <220> <223> synthetic peptide <400> 82 Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Thr Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Thr Tyr Trp Thr 85 90 95 Phe Gly Gln Gly Thr Lys Val Glu Ile 100 105 <210> 83 <211> 20 <212> PRT <213> Artificial Sequence <220> <223> Synthetic polypeptide <400> 83 His Lys His Gly His Gly His Gly Lys His Lys Asn Lys Gly Lys Lys 1 5 10 15 Asn Gly Lys His 20

Claims

1. Use of a first reagent that specifically binds to cleaved high molecular weight kininogen (HMWK) and a second reagent that binds to HMWK in the manufacture of an immunoassay reagent for determining the efficacy of a treatment for a disease associated with plasma kallikrein (pKal), the determination comprising: (i) providing a supporting member on which a first reagent that specifically binds to the cleaved high molecular weight kininogen (HMWK) is immobilized; (ii) contacting the support member of (i) with a biological sample obtained from a subject before, after and / or during treatment; (iii) contacting the support member obtained in (ii) with a second reagent that binds to HMWK, wherein the second reagent is conjugated to a label; and (iv) directly or indirectly detecting a signal released from the label of the second reagent bound to the support member to determine the level of the cleaved HMWK in each of the biological samples; wherein a decreased level of cleaved HMWK in the biological sample after or during treatment compared to a biological sample obtained before treatment indicates that the treatment is effective; The first reagent is an antibody, wherein the antibody comprises a heavy chain complementary determining region (CDR) 1 sequence FSFYVMV, a heavy chain CDR2 sequence GISPSGGNTAYADSVK, and a heavy chain CDR3 sequence KLFYYDDTKGYFDF; and a light chain CDR1 sequence SGSSSNIGSNYVY, a light chain CDR2 sequence RNNQRPS, and a light chain CDR3 sequence AWDDSLNGRV; and Among the diseases associated with pKal are hereditary angioedema (HAE), rheumatoid arthritis, ulcerative colitis or Crohn's disease.

2. The use according to claim 1, wherein the antibody comprises a heavy chain variable region sequence shown in SEQ ID NO: 4 and a light chain variable region sequence shown in SEQ ID NO:

5.

3. The use according to claim 1, wherein the treatment comprises a plasma kallikrein (pKal) inhibitor.

4. The use according to claim 3, wherein the pKal inhibitor is ecallantide (DX-88), EPIKAL-2 or landarumab (DX-2930).

5. The use according to any one of claims 1 to 4, wherein the support member is a 96-well plate.

6. The use according to any one of claims 1 to 4, wherein prior to step (ii), the support member of (i) is incubated with a blocking buffer.

7. The use according to any one of claims 1 to 4, wherein the second agent is a polyclonal antibody or a monoclonal antibody.

8. The use according to any one of claims 1 to 4, wherein the marker is a signal releasing agent.

9. The use according to any one of claims 1 to 4, wherein the label is one member of a receptor-ligand pair, and the immunoassay further comprises, before step (iv), contacting the second reagent in (iii) bound to the support member with another member of the receptor-ligand pair, wherein the other member is conjugated to a signal releasing agent.

10. The use according to claim 9, wherein the receptor-ligand pair is biotin and streptavidin.

11. The use according to any one of claims 1 to 4, wherein the detection is performed using a Western blot assay, an enzyme-linked immunosorbent assay (ELISA) or a lateral flow assay.

12. Use according to any one of claims 1 to 4, wherein step (ii) is carried out in the presence of ZnCl2.

13. The use according to any one of claims 1 to 4, wherein the biological sample is obtained from a human subject.

14. The use according to claim 13, wherein each of the plurality of biological samples is a serum sample or a plasma sample processed from a blood sample collected in a vacuum blood collection tube comprising one or more protease inhibitors.

15. The use according to claim 13, wherein the human subject has a history of a disease associated with pKa.

16. The use according to claim 14, wherein the human subject has a history of a disease associated with pKa.

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