(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
`
`(19) World Intellectual Property
`Organization
`1nternational Bureau
`
`(43) International Publication Date
`27 April 2017 (27.04.2017)
`
`\9
`
`WlPOl PCT
`
`(10) International Publication Number
`
`WO 2017/070170 A1
`
`(51)
`
`International Patent Classification:
`
`G01N 33/68 (2006.01)
`
`G01N 33/86 (2006.01)
`
`(21)
`
`International Application Number:
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`PCTflJS2016/057640
`
`(22)
`
`International Filing Date:
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`(25)
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`Filing Language:
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`(26)
`
`Publication Language:
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`19 October 2016 (19.10.2016)
`
`English
`
`English
`
`(30)
`
`(71)
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`(72)
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`(74)
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`(81)
`
`Priority Data:
`62/243,505
`62/335,311
`
`19 October 2015 (19.10.2015)
`12 May 2016 (12.05.2016)
`
`US
`US
`
`Applicant: DYAX CORP. [US/US]; 55 Network Drive,
`Burlington, MA 01803-2756 (US).
`
`Inventors: SEXTON, Daniel, J.; 59 Marvin Road, Mel-
`rosc, MA 02176 (US). FAUCETTE, Ryan; 29 Birch Hill
`Road, Me1rose, MA 02176 (US). COSIC, Janja; 112 War-
`ren St, #2, Arlington, MA 02474 (US).
`
`Agent: CHEN. Yahua; Wolf, Greenfield & Sacks, P.C.,
`600 Atlantic Avenue, Boston, MA 02210-2206 (US).
`
`Designated States (unless otherwise indicated, for every
`kind ofnational protection available): AE, AG, AL, AM,
`
`A0, AT, AU, AZ, BA, BB, BG, BH, BN, BR, BW, BY,
`BZ, CA, CH, CL, CN, Co, CR, CU, Cz, DE, DJ, DK, DM,
`DO, DZ, EC, EE, EG, ES, FI, GB, GD, GE, GH, GM, GT,
`HN, HR, HU, ID, IL, IN, IR, IS, JP, KE, KG, KN, KP, KR,
`KW, KZ, LA, LC, LK, LR, LS, LU, LY, MA, MD, ME,
`MG, MK, MN, MW, MX, MY, MZ, NA, NG, NI, No, NZ,
`OM, PA, PE, PG, PH, PL, PT, QA, Ro, RS, RU, RW, SA,
`SC, SD, SE, SG, SK, SL, SM, ST, SV, SY, TH, TJ, TM,
`TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, ZA, ZM,
`ZW.
`
`(84)
`
`Designated States (unless otherwise indicated, for every
`kind of regional protection available): ARIPO (BW, GH,
`GM, KE, LR, LS, MW, MZ, NA, RW, SD, SL, ST, SZ,
`Tz, UG, ZM, ZW), Eurasian (AM, AZ, BY, KG, KZ, RU,
`TJ, TM), European (AL, AT, BE, BG, CH, CY, CZ, DE,
`DK, EE, ES, FI, FR, GB, GR, HR, HU, IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SI, SK,
`SM, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, GQ,
`GW, KM, ML, MR, NE, SN, TD, TG).
`Published:
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`with international search report (Art. 21(3))
`
`with sequence listing part ofdescription (Rule 5.2(a))
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`(54) Title: IMMUNOASSAY TO DETECT CLEAVED HIGH MOLECULAR WEIGHT KININOGEN
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`FIG. 9
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`[2-chain](ng/mL)
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`Normal
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`HAE
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`(57) Abstract: The present disclosure provides immunoassay methods of detecting a cleaved high molecular weight kininogen
`(HMWK) With high sensitivity and specificity and isolated antibodies that specifically bind cleaved HMWK.
`
`Singapore Exhibit 2012
`Singapore Exhibit 2012
`Lassen v. Singapore et al.
`Lassen v. Singapore et a1.
`PGR2019-00053
`PGR2019-00053
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`level of the cleaved HMWK in the biological sample. In some instances, step (ii) may be
`
`performed in the presence of ZnClg.
`
`In some embodiments, prior to step (ii), the support member of (i) is incubated with a
`
`blocking buffer.
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`In some embodiments, the second agent is a polyclonal antibody, a monoclonal
`
`antibodies, or a mixture of two or more monoclonal antibodies that bind to HMWK. The two or
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`more monoclonal antibodies in the mixture may bind to different epitopes in HMWK.
`
`In some
`
`embodiments, the label is a signal releasing agent. In some embodiments, the label is a member
`
`of a receptor-ligand pair. In that case, the immunoassay may further comprise, prior to step (iv),
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`contacting the second agent in (iii), which is immobilized on the support member, with the other
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`member of the receptor—ligand pair, wherein the other member is conjugated to a signal releasing
`
`agent. In one example, the receptor-ligand pair is biotin and streptavidin.
`
`Another aspect of the present disclosure provides methods for detecting a cleaved high
`
`molecular kininogen (HMWK) in a sample, the method comprising (i) contacting a sample
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`suspected of containing a cleaved HMWK with any of the antibodies described herein (e. g.
`
`559B-MOO4-BO4); (ii) measuring a complex of the cleaved HMWK and the antibody formed in
`
`step (i); and (iii) determining the level of the cleaved HMWK in the sample based on the result
`
`of step (ii).
`
`In some embodiments, step (i) is performed in the presence of Zl’lClg.
`
`In some
`
`embodiments, step (i) is performed using an enzyme-linked immunosorbent assay (ELISA) or an
`
`10
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`15
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`immunoblotting assay.
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`In any of the methods described herein, the sample may be a biological sample obtained
`
`from a subject (e.g., a human patient), such as a serum sample of a plasma sample.
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`In some
`
`embodiments, the method further comprises collecting the sample into an evacuated blood
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`collection tube, which comprises one or more protease inhibitors.
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`Any of the assay methods (e.g., immunoassays) described herein may be a ELISA assay,
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`a Western blot assay, or lateral flow assay.
`
`In some embodiments, the biological sample is obtained from a subject (e. g., a human
`
`patient) having a disease. The assay method may further comprise determining whether the
`
`disease is mediated by plasma kallikrein based on the level of the cleaved HMWK, a deviation
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`30
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`of the level of the cleaved HMWK in the sample from that of a control sample being indicative
`
`that the disease is mediated by plasma kallikrein.
`
`Any of the assay methods described herein may further comprise identifying patients
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`with diseases or disorders mediated by plasma kallikrein, or evaluating the efficacy of a
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`treatment of the disease or disorder based on the levels of cleaved HMWK.
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`In some
`
`embodiments, the method may further comprises administering to the subject an effective
`
`amount of a therapeutic agent, such as a plasma kallikrein (pKal) inhibitor, a bradykinin 2
`
`receptor (B2R) inhibitor, and/or a Cl esterase inhibitor, for treating the disorder, if the subject is
`
`identified as having the disorder.
`
`In some embodiments the pKal inhibitor is an anti-pKal
`
`antibody. In some embodiments, the therapeutic agent is lanadelumab, ecallantide, icatibant, or
`
`human plasma—derived C1 esterase inhibitor.
`
`In some embodiments, the subject is a human patient who is on a treatment for the
`
`disorder, and wherein the method further comprises assessing the efficacy of the treatment based
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`10
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`on the level of the cleaved HMWK determining in step (iii), a deviation of the level of the
`
`cleaved HMWK in the sample from the subject from that of a control sample being indicative of
`
`the treatment efficacy. In some embodiments, the method further comprises identifying a
`
`suitable treatment for the subject based on the level of the cleaved HMWK. In some
`
`embodiments, the method further comprises identifying the subject as a candidate for a treatment
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`15
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`of the disease based on the level of the cleaved HMWK.
`
`In some embodiments, the human patient has a history of the disease (e.g., HAE). In
`
`some embodiments, the method further comprises assessing the risk of disease attack in the
`
`subject based on the level of the cleaved HMWK, a deviation of the level of the cleaved HMWK
`
`in the sample from the subject from that of a control sample being indicative of the risk of
`
`disease attack.
`
`In some embodiments, the method further comprises administering a therapeutic
`
`agent to the subject, if the subject is at risk of disease attack.
`
`In another aspect, a kit is provided for detecting a cleaved high molecular weight
`
`kininogen (HMWK). the kit comprising a first agent (e.g., an antibody as described herein) that
`
`specifically binds a cleaved HMWK. In some embodiments, the kit further comprises a second
`
`agent that binds HMWK, a support member, or both, and optionally instructions for detecting
`
`the cleaved HMWK.
`
`In some examples, the support member is a 96-well plate.
`
`In another aspect of the disclosure, an isolated antibody is provided, which specifically
`
`binds a cleaved high molecular weight kininogen (HMWK). In some embodiments, the
`
`antibody binds the same epitope as 559B-M004-BO4 or competes against 559B-M004-B04 for
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`30
`
`binding to the cleaved HMWK. In some embodiments, the antibody comprises the same heavy
`
`chain and light chain complementary determining regions as 559B—M004—B04, e. g., the same
`
`heavy chain and light variable regions as 559B—M004—BO4. In one example, the antibody is
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`559B-MOO4-BO4.
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`Any of the antibodies specific to a cleaved HMWK as described herein can be used in a
`
`method for detecting a cleaved high molecular kininogen (HMWK) in a sample. Such a method
`
`may comprise (i) contacting a sample suspected of containing a cleaved HMWK with the
`
`antibody; (ii) measuring a complex of the cleaved HMWK and the antibody formed in step (i);
`
`and determining the level of the cleaved HMWK in the sample based on the result of step (ii).
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`In some embodiments, the sample is a biological sample such as a serum sample or a plasma
`
`sample obtained from a human subject. The result obtained from this method may be relied on
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`to determine the risk of a subject from whom the sample is obtained for developing a disorder
`
`mediated by plasma kallikrein such as HAE. In some instances, step (i) can be performed in the
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`10
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`presence of ZIICig.
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`Any of the immunoassay methods described herein can be in Western blot format or
`
`ELISA format.
`
`In yet another aspect, an isolated antibody is provided that binds both intact high
`
`molecular weight kinino gen (HMWK) and a cleaved HMWK.
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`15
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`In some embodiments, the antibody that binds both intact and cleaved HMWK does not
`
`bind to low molecular weight kininogen (LMWK). In some embodiments, the antibody binds
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`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-Gl 1, 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 against 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-GO6, 559B-M0036-G12, 559B-
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`M0042—E06, 559B—M0070—H10, 559B—M0068—D01, or 559B—M0004—E08 for binding to the
`
`intact HMWK and/or the cleaved HMWK.
`
`In some embodiments, the antibody comprising the same heavy chain and light chain
`
`CDRs as 559B—M0067-E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-
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`M0039-H06, 559B-M0039-D08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06,
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`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-
`
`30
`
`E02, 559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-HO6, 559B-
`
`M0039—D08, 559B—M0068—C07, 559B—M0021—G1 1, 559B—M0061—G06, 559B—M0036—G12,
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`559B-MOO42—E06, 559B—M0070-H10, 559B-M0068—D01, and 559B-M0004-E08.
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`In other embodiments, the antibody that binds both intact and cleaved HMWK also binds
`
`LMWK.
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`In some embodiments, the antibody binds the same epitope as 559B-M0069-C09,
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`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-
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`M0035-F05, 559B-M0035—H09, 559B—M0043-C06, 559B-M0003—A08, 559B—M0054-B11,
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`559B—M0067—G1 1, 559B—M0064—H02, or 559B—M0065—B 10.
`
`In some embodiments, the
`
`antibody competes against 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—
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`C06, 559B—M0003-A08, 559B-M0054-B11, 559B—M0067-G1 1, 559B-M0064-H02, or 559B-
`
`M0065-B 10 for binding to the intact HMWK, the cleaved HMWK, and/or the LMWK.
`
`In some embodiments, the antibody comprises the same heavy chain and light chain
`
`CDRs as 559B—M0069-C09, 559B-M0038—FO4, 559B-M0044—C05, 559B-M0047-H01, 559B-
`
`M0019-E12, 559B-X0004-B05, 559B-M0048—D12, 559B—M0053-G01, 559B-M0038-H03,
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`559B—M0017—H08, 559B—M0035—F05, 559B—M0035—H09, 559B—M0043—C06, 559B—M0003—
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`10
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`15
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`A08, 559B-M0054-B11, 559B-M0067-G1 1, 559B-M0064—H02, or 559B—M0065-B 10.
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`In some
`
`examples, the antibody is selected from the group consisting of 559B-M0069—C09, 559B—
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`M0038-F04, 559B-M0044—C05, 559B-M0047—H01, 559B-M0019—E12, 559B-X0004-B05,
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`559B-M0048-D12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017—H08, 559B—M0035-
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`F05, 559B-M0035-H09, 559B-M0043—C06, 559B-M0003-A08, 559B-M0054—B1 1, 559B-
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`M0067-G11, 559B-M0064-H02, and 559B-M0065—B10.
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`The details of one or more embodiments of the disclosure are set forth in the description
`
`below. Other features or advantages of the present disclosure will be apparent from the
`
`following drawings and detailed description of several embodiments, and also from the
`
`appended claims.
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`BRIEF DESCRIPTION OF DRAWINGS
`
`The following drawings form part of the present specification and are included to further
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`demonstrate certain aspects of the present disclosure, which can be better understood by
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`30
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`reference to one or more of these drawings in combination with the detailed description of
`
`specific embodiments presented herein.
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`FIG. 1 is a graph showing binding of 559B-M0004-B04 to intact HMWK (dark gray
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`bars) or cleaved HMWK (light gray bars) under the indicated ELISA conditions.
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`FIG. 2 presents graphs showing binding of various Fab clones to intact l—chain (intact)
`
`HMWK, 2—chain (cleaved) HMWK, or LMWK. A: Fab clones identified using the phage
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`display screening methods described herein. Intact HWMK is shown in dark gray bars, cleaved
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`HMWK in light gray bars, and LMWK in medium gray bars. B: binding for several example
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`Fab clones. LWMK is shown in dark gray bars, intact HMWK in light gray bars, and cleaved
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`HWMK in medium gray bars.
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`FIG. 3 is a graph showing specificity of 559B—M0004—B04 towards intact HMWK.
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`cleaved HMWK, or LMWK. Purified cleaved HMWK was spiked into SBT assay buffer
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`(circles) or HMWK-deficient plasma (squares). Purified intact HMWK was spiked into SBT
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`assay buffer (triangles). Purified LMWK was spiked into SBT assay buffer (diamonds). The y-
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`axis presents the ELISA signal in absorbance units, and the X-aXlS presents the concentration of
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`kininogen in ug/mL.
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`FIG. 4 is a graph showing detection of 2-Chain HMWK (cleaved HMWK) in plasma or
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`assay buffer. Purified cleaved HMWK was spiked into SBT assay buffer (open circles), SBT
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`assay buffer and analyzed in the presence of 10% plasma (squares), or HMWK—deficient plasma
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`and analyzed in the presence of 10% plasma (triangles). Purified cleaved HMWK was also
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`spiked into assay buffer and analyzed in the presence of 2.5% plasma (diamonds) or HMWK
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`deficient plasma and analyzed in the presence of 2.5% plasma (closed circles). The y-axis
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`presents the ELISA signal in absorbance units, and the x-axis presents the concentration of
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`kininogen in ug/mL.
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`FIG. 5 is a graph showing levels of cleaved HMWK in the indicated human plasma
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`samples prior to and after contact system activation. A: prior to and after contact system
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`activation with FXHa or ellagic acid. B: prior to and after contact system activation with FXIIa,
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`pKal, or ellagic acid.
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`FIG. 6 is a graph showing levels of cleaved HMWK in plasma samples from 12 normal
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`human donors prior to and after activation of the contact system with ellagic acid.
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`FIG. 7 presents graphs showing levels of cleaved HMWK following inhibition with a
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`pKal inhibitor. A: inhibition with landadelumab/DX—293O or Cl-INH prior to contact system
`
`activation with ellagic acid. B: inhibition of pooled sodium citrate plasma samples with
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`30
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`landadelumab/DX-2930 prior to contact system activation with 10 nM FXIIa.
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`FIG. 8 is a graph showing cleaved HMWK generation at the indicated time points
`
`following contact system activation with FXIIa or ellagic acid.
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`FIG. 9 is a graph showing levels of 2-chain HMWK in plasma samples from normal
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`subjects and subjects having HAE.
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`FIG. 10 is photo showing results obtained from a HMWK Western blot analysis, which
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`are consistent with the results obtained from the 2-Chain HMWK ELISA assay described herein.
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`Human citrated plasma samples (normal plasma, FXII—deficient plasma, and prekallikrein-
`
`deficient plasma) were probed with a mouse monoclonal anti—HMWK light chain antibody
`
`followed by a goat anti—mouse detection antibody. The analyzed plasma samples were either
`
`untreated or activated with 100 nM pKal, 10 nM FXlla, or 10% cllagic acid.
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`FIG. 11 is a graph showing that the addition of ZnClz to either citrated or EDTA plasma
`
`samples increased the signal of the 2-Chain HMWK in an ELISA assay. The x-axis shows the
`
`concentration of ZnClz in the assay well after a 40-fold dilution.
`
`FIG. 12 presents schematics of the discovery and development of assays using the
`
`antibodies described herein. A: schematic of the phage display methods used to discover 2-
`
`chain HMWK binding antibodies. B: an example sandwich ELISA assay in which the 2-chain
`
`HMWK specific antibody/Fab (e.g., 559B—M0004—B04) is immobilized in 96—well plates to
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`capture 2-chain HMWK in citrated plasma, followed by washing and detection with an anti-
`
`HMWK antibody conjugated to a label (anti-HMWK-HRP).
`
`FIG. 13 is a graph showing results from a 2-chain HMWK sandwich ELISA standard
`
`curve, in which citrated plasma samples were spiked with 2—chain HMWK (10% final dilution).
`
`FIG. 14 shows the identification of 2-chain HMWK-specific antibodies by phage display
`
`selection and screening. A: plots the ratio of the result of a 2-chain HWMK binding assay to a
`
`LMWK binding assay on the y-aXis compared to the ratio of the result of a 2-chain HMWK
`
`binding assay to a l—chain HMWK binding assay on the X—aXis for each antibody (Fab) tested.
`
`Recombinant Fab fragments were passively immobilized onto 384-well plates prior to addition
`
`of biotinylated 2—chain HMWK, l-chain HMWK, or LMWK, followed by streptavidin-HRP. B:
`
`shows binding to l-chain HMWK, 2-chain HMWK, or LMWK for the indicated isolated Fab
`
`fragments.
`
`FIG. 15 is a graph showing competition of 2-chain HMWK and kininogen peptides
`
`(HKH20 and GCP28) for binding to 559B-M0004—BO4.
`
`FIG. 16 is a graph showing a standard curve for an optimized sandwich ELISA for the
`
`detection of 2—chain HMWK in human plasma samples.
`
`FIG. 17 presents graphs of Western blotting analyses comparing the level of 2-chain
`
`HMWK in citrated plasma samples from healthy subjects and HAE patients. A: scatter plot
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`comparing the percent 2—chaim HMWK in samples from healthy subjects (“HV”) and HAE
`
`patients between HAE attacks (“Basal”) and during an HAE attack (“Attack”). B: ROC
`
`(receiver operating characteristic) analysis comparing the sensitivity and specificity for the
`
`detection of HAE basal samples versus samples from healthy subjects (AUC = 0.977). C: ROC
`
`analysis comparing the sensitivity and specificity for the detection of HAE attack samples versus
`
`samples from healthy subjects (AUC = 1). D: ROC analysis comparing the sensitivity and
`
`specificity for the detection of HAE attack samples versus HAE basal samples (AUC = 0.625).
`
`FIG. 18 presents graphs of Western blotting analyses comparing the level of 2-chain
`
`HMWK in SCAT169 plasma samples from healthy subjects and HAE patients. A: scatter plot
`
`comparing the percent 2—chaim HMWK in samples from healthy subjects (“HV”) and HAE
`
`patients between HAE attacks (“Basal”) and during an HAE attack (“Attack”). B: ROC analysis
`
`comparing the sensitivity and specificity for the detection of HAE basal samples versus samples
`
`from healthy subjects (AUC = 0.915). C: ROC analysis comparing the sensitivity and
`
`specificity for the detection of HAE attack samples versus samples from healthy subjects (AUC
`
`= 0.967). D: ROC analysis comparing the sensitivity and specificity for the detection of HAE
`
`10
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`15
`
`attack samples versus HAE basal samples (AUC = 0.597).
`
`FIG. 19 presents graphs of ELISA analyses comparing the level of 2—chain HMWK in
`
`citrated plasma samples from healthy subjects and HAE patients. A: scatter plot comparing the
`
`percent 2-chaim HMWK in samples from healthy subjects (“HV”) and HAE patients between
`
`HAE attacks (“Basal”) and during an HAE attack (“Attack”). B: ROC analysis comparing the
`
`sensitivity and specificity for the detection of HAE basal samples versus samples from healthy
`
`subjects (AUC = 0.795). C: ROC analysis comparing the sensitivity and specificity for the
`
`detection of HAE attack samples versus samples from healthy subjects (AUC = 0.866). D: ROC
`
`analysis comparing the sensitivity and specificity for the detection of HAE attack samples versus
`
`HAE basal samples (AUC = 0.709).
`
`FIG. 20 presents graphs of ELISA analyses comparing the level of 2—chain HMWK in
`
`SCAT169 samples from healthy subjects and HAE patients. A: scatter plot comparing the
`
`percent 2-chaim HMWK in samples from healthy subjects (“HV”) and HAE patients between
`
`HAE attacks (“Basal”) and during an HAE attack (“Attack”). B: ROC analysis comparing the
`
`30
`
`sensitivity and specificity for the detection of HAE basal samples versus samples from healthy
`
`subjects (AUC = 0.999). C: ROC analysis comparing the sensitivity and specificity for the
`
`detection of HAE attack samples versus samples from healthy subjects (AUC = 1). D: ROC
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`analysis comparing the sensitivity and specificity for the detection of HAE attack samples versus
`
`HAE basal samples (AUC : 0.8176).
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`DETAILED DESCRIPTION OF PRESENT DISCLOSURE
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`Plasma kallikrein (PKal) is a serine protease component of the contact system and is the
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`primary bradykinin— generating enzyme in the circulation. The contact system is activated by
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`either factor XIIa (the active form of Factor XH or FXII) upon exposure to foreign or negatively
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`charged surfaces or on endothelial cell surfaces by prolylcarboxypeptidases (Sainz I.M. et al.,
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`Thromb Haemost 98, 77-83, 2007). Activation of the plasma kallikrein amplifies intrinsic
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`coagulation via its feedback activation of factor XII and proteolytically cleaves the kininogen
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`precursor, high molecular weight kininogen (HMWK), releasing the proinflammatory
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`nonapeptide bradykinin and a cleaved HMWK, which contains two polypeptide chains linked by
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`a disulfide bond (also known as 2-chain HMWK).
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`As the primary kininogenase in the circulation, plasma kallikrein is largely responsible
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`for the generation of bradykinin in the vasculature. A genetic deficiency in the Cl—inhibitor
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`protein (C l—INH) leads to hereditary angioedema (HAE). Patients with HAE suffer from acute
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`attacks of painful edema often precipitated by unknown triggers (Zuraw B.L. et al., N Engl J
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`Med 359, 1027-l036, 2008). Through the use of pharmacological agents or genetic studies in
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`animal models, the plasma kallikrein—kinin system (plasma KKS) has been implicated in various
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`diseases.
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`The level of cleaved HMWK was found to be elevated in HAE attack, as well as in other
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`pKal-as sociated disorders. Thus, cleaved HMWK can serve as a biomarker for monitoring
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`disease development and/or treatment efficacy. However. the art lacks suitable agents and/or
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`suitable assays that can effectively distinguish intact HMWK from its cleaved version.
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`The present disclosure is based, at least in part, on the development of specific
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`immunoassays that allows for detection of cleaved HMWK with high specificity and sensitivity.
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`It was observed that a Sandwich ELISA in which an agent that specifically binds cleaved
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`HMWK is immobilized on a support member (e.g., a multi-well plate) unexpectedly enhanced
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`detection efficiency as compared to the setting of ELISA in which the antigen (in this case, the
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`cleaved HMWK) is immobilized on the support member. Further, it was observed,
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`unexpectedly, that using the LowCross blocking buffer (containing casein), rather than a
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`blocking buffer containing bovine serum album (BSA), enhanced detection specificity and
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`sensitivity during the initial screening to discover antibodies specific for cleaved HMWK.
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`Moreover, the detection specificity and sensitivity was further enhanced when a 96-well plate
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`was used, as compared with a 384-well plate. The present disclosure is also based on, at least in
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`part, the isolation of antibodies that specifically bind a cleaved HMWK.
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`Accordingly, provided herein are immunoas says for detecting the presence or measuring
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`the level of a cleaved HMWK in a biological sample suspected of containing HMWK species,
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`using an agent (e.g., an antibody) that specifically binds a cleaved HMWK (e.g., the cleaved
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`HMWK having a molecular weight of 46 kDa). Given the correlation between the level of
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`cleaved HMWK and disorders associated with or mediated by pKal (e.g., HAE), the
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`imunoassays described herein can be applied to identify patients who are at risk of such
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`diseases, to monitor disease progression, and/or to monitor efficacy of a treatment against such a
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`disorder.
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`I.
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`Immunoassays for Specific Detection of Cleaved HMWK
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`One aspect of the present disclosure relates to immunoassays for detecting cleaved
`
`HMWK with high sensitivity and specificity. Such immunoassays may involve a Sandwich
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`ELISA in which an agent that specifically binds a cleaved HMWK is immobilized on a support
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`member, which can be a 96—well plate. The immunoassays described herein allows for selective
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`detection of cleaved HMWK in biological samples, e. g., serum samples or plasma samples,
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`which may contain both intact and cleaved HMWK, as well as LMWK.
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`(1') High Molecular- Weight Kinino gen,
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`High molecular-weight kininogen (HMWK) exists in the plasma as a single polypeptide
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`(l-chain) multi—domain (domains 1—6) protein with a molecular weight of approximately 110
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`kDa, referred to herein as intact HWMK. The human gene encoding HMWK is kininogen 1
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`(KNGl). KNGl is transcribed and alternatively spliced to form mRNAs that encode either
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`HMWK or low molecular weight kininogen (LMWK). An exemplary protein sequence of
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`HMWK is provided below:
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`>gi|156231037|ref|NP_001095886.1| kininogen-l isoform 1 precursor [Homo
`sapiens]
`MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDT
`FYSFKYEIKEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTAQY
`DCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRITYSIVQTNCSKEN
`FLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQPPTKICVGCPRDIPTNSPELEE
`TLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYFIDFVARETTCSKESNEELTESCETKKLGQSLD
`CNAEVYVVPWEKKIYPTVNCQPLGMISLMKRPPGFSPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSG
`KEQGHTRRHDWGHEKQRKHNLGHGHKHERDQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHV
`LDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTF
`SDFQDSDLIATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTTQ
`MKESYYFDLTDGLS (SEQ ID NO: 1)
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`Intact HMWK, also referred to herein as “intact kininogen,” can be assayed, for example,
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`using coagulant or immunological methods, e. g., radioimmunoassay (see, e.g., Kerbiriou-
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`Nabias, D.M., Br J Haematol, 1984, 56(2):2734—86). A monoclonal antibody to the light chain
`
`of human HMWK is known. See, e.g., Reddigari, SR. & Kaplan, A.P., Blood, 1999, 74:695-
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`702. An assay for HMWK that relies on a chromogenic substrate can also be used. See, e. g.,
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`Scott, C.F. et al. Thromb Res, 1987, 48(6):685—700; Gallimore. M.J. et al. Thromb Res, 2004,
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`114(2):91—96.
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`HMWK is cleaved by pKal within domain 4 to release the 9 amino acid, pro-
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`inflammatory peptide bradykinin, and a 2-chain form of HMWK, referred to herein as cleaved
`
`HMWK. The 2 chains of HMWK are the heavy chain, which contains domains 1-3, and the
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`light chain, which contains domains 5 and 6, joined by a disulfide bond. Upon initial cleavage
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`of intact HMWK, the heavy and light chains have a molecular weight of approximately 65 kDa
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`and 56 kDa, respectively. Further proteolytic processing results in generation of a 46 kDa light
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`chain.
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`below.
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`Exemplary sequences of the heavy and light chains of cleaved kininogen are provided
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`> cleaved kininogen-l heavy chain
`
`QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEI
`KEGDCPVQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTA
`QYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRIT
`YSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQ
`PPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYF
`IDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISL
`MK (SEQ ID No: 2)
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`> cleaved kininogen-l light chain
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`S SRIGE I KEETTVSPPHTSMAPAQDEERD SGKEQGHTRRHDWGHEKQRKHNLGHGHKHER
`DQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNK
`GKKNGKHNGWKTEHLAS S SED STTP SAQTQEKTEGP TP IP SLAKPGVTVTFSDFQD SDLI
`ATMMPP I SPAP IQSDDDWIPD IQIDPNGLSFNP I SDFPDTTSPKCPGRPWKSVSEINPTT
`QMKE SYYFDLTDGLS (SEQ ID NO: 3)
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`(ii) Antibodies Specific to Clem/ed HMWK
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`The immunoassays described herein may use any agent that can specifically bind a
`
`cleaved HMWK, for example, an agent that recognizes a neoepitope on cleaved HMWK that is
`
`not present on intact HMWK. In some embodiments, the cleaved HMWK-binding agent is an
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`antibody.
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`An antibody (interchangeably used in plural form) is an immunoglobulin molecule
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`capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid,
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`polypeptide, etc., through at least one antigen recognition site, located in the variable region of
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`the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact
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`(i.e., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments
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`thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants thereof, fusion proteins
`
`comprising an antibody portion, humanized antibodies, chimeric antibodies, diabodies, linear
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`antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any
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`other modified configuration of the immunoglobulin molecule that comprises an antigen
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`recognition site of the required specificity, including glycosylation variants of antibodies, amino
`
`acid sequence variants of antibodies, and covalently modified antibodies. An antibody includes
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`an antibody of any class, such as IgD, IgE, IgG, IgA, or IgM (or sub-class thereof), and the
`
`antibody need not be of any particular class. Depending on the antibody amino acid sequence of
`
`the constant domain of its heavy chains, immunoglobulins can be assigned to different classes.
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`There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of
`
`these may be further divided into subclasses (isotypes), e.g., lgGl, IgG2, lgG3, IgG4, IgAl and
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`lgAZ. The heavy-chain constant domains that correspond to the different classes of
`
`immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit
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`structures and three-dimensional configurations of different classes of immunoglobulins are well
`
`known.
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`Any of the antibodies described herein can be either monoclonal or polyclonal. A
`
`“monoclonal antibody” refers to a homogenous antibody population and a “polyclonal antibody”
`
`refers to a heterogeneous antibody population. These two terms do not limit the source of an
`
`antibody or the manner in which it is made.
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`An antibody that “specifically binds” a cleaved HMWK or an epitope thereof is a term
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`well understood in the art, and methods to determine such specific binding are also well known
`
`in the art. A molecule is said to exhibit “specific binding” if it reacts or associates more
`
`frequently, more rapidly, with greater duration and/or with greater affinity with a particular
`
`target antigen (here a cleaved HMWK) than it does with alternative targets (e.g., intact HMWK
`
`and/or LMWK). An antibody “specifically binds” to a target antigen if it binds with greater
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`affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For
`
`example, an antibody that specifically (or preferentially) binds to cleaved HMWK or an epitope
`
`therein is an antibody that binds this target antigen with greater affinity, avidity, more readily,
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`and/or with greater duration than it binds to other antigens (e.g., intact HMWK or LMWK) or
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`other epitopes in the same antigen. It is also understood by reading this definition that, for
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`example, an antibody that specifically binds to a first target antigen may