(12) INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY(PCT)
`(19) World Intellectual Property
`Organization
`International Bureau
`
`CAMAAARA
`
`(43) International Publication Date
`27 April 2017 (27.04.2017)
`
`WIPOIPCT
`
`\=
`
`(10) International Publication Number
`WO 2017/070170 Al
`
`6)
`
`International Patent Classification:
`
`GOIN33/68 (2006.01)
`
`GOIN33/86 (2006.01)
`
`(21)
`
`International Application Number:
`
`PCT/US201 6/057640
`
`(22)
`
`International Filing Date:
`
`(25)
`
`Filing Language:
`
`(26)
`
`Publication Language:
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`19 October 2016 (19.10.2016)
`
`English
`
`English
`
`(30)
`
`(71)
`
`(72)
`
`(74)
`
`(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, Melrose, 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 of national protection available): AE, AG, AL, AM,
`
`AO, 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, NL NO, NZ,
`OM,PA,PF, 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, FL, FR, GB, GR, HR, HU,IE, IS, IT, LT, LU,
`LV, MC, MK, MT, NL, NO, PL, PT, RO, RS, SE, SL 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))
`
`(54) Title: IMMUNOASSAY TO DETECT CLEAVED HIGH MOLECULAR WEIGHT KININOGEN
`
`FIG. 9
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`3000
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`2500
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`2000
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`1500
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`500
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`[2-chain](ng/mL)
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`
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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
`(HMWE)with highsensitivity and specificity and isolated antibodies that specifically bind cleaved HMWK.
`
`Singapore Exhibit 2012
`Singapore Exhibit 2012
`Lassen v. Singapore et al.
`Lassen v. Singaporeet al.
`PGR2019-00053
`PGR2019-00053
`
`
`
`wo2017/070170A[IITANRITIMTUMITATANIAAMTREYAA
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`

`

`WO 2017/070170
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`PCT/US2016/057640
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`IMMUNOASSAY TO DETECT CLEAVED HIGH MOLECULAR WEIGHT KININOGEN
`
`CROSS REFERENCE TO RELATED APPLICATIONS
`
`This application claims the benefit of U.S. Provisional Application Numbers 62/243,505,
`
`filed October 19, 2015, and 62/335,311, filed May 12, 2016 under 35 U.S.C. 8119, the entire
`
`content of cach of which is herein incorporated by reference.
`
`10
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`15
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`BACKGROUNDOF PRESENT DISCLOSURE
`
`Kininogensare precursors of kinin, such as bradykinin and kallidin. There are two types
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`of human kininogens, high molecular-weight kininogen (HMWK)and low molecular-weight
`
`kininogen (LMWK&), which are splicing variants. HMWKacts mainly as a cofactor on
`
`coagulation and inflammation andis the preferred substrate for plasma kallikrein (pKal)-
`
`mediated bradykinin generation.
`
`Plasmakallikrein (pKal) is the primary bradykinin-generating enzymein the circulation.
`
`The activation of pKal occurs via the contact system which has been linked to disease pathology
`
`associated with hereditary angioedema (HAE). pKal cleaves HMWK(asingle-chain
`
`polypeptide) to produce bradykinin and a cleaved form HMWK,which contains two polypeptide
`
`chains held together by a disulfide bond. Cugnoet al., Blood (1997) 89:3213-3218.
`
`Cleaved HMWKincreased to about 47% of total kininogen during a hereditary
`
`angioedema (HAE) attack. Cugnoetal., Blood (1997) 89:3213-3218, making it a biomarker for
`
`monitoring HAEattack. It is therefore of interest to develop sensitive and reliable assays for
`
`detecting the level of cleaved HMWKin biological samples.
`
`SUMMARYOF PRESENT DISCLOSURE
`
`NoAL
`
`Someaspects of the present disclosure provide an immunoassay for detecting a cleaved
`
`high molecular weight kininogen (HMWK)with high sensitivity and specificity. The method
`
`comprises (i) providing a support memberon whicha first agent (e.g., an antibody such as
`
`559B-M004-B04)that specifically binds a cleaved HMWKis attached; (ii) contacting the
`
`support memberof(i) with a biological sample suspected of containing a cleaved HMWK;(iii)
`
`30
`
`contacting the support memberobtained in (11) with a second agent that binds HMWK,wherein
`
`the second agent is conjugated to a label; and (iv) detecting a signal released from the label of
`
`the second agent that is bound to the support member, directly or indirectly, to determine the
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`WO 2017/070170
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`PCT/US2016/037640
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`level of the cleaved HMWKin the biological sample. In some instances, step (ii) may be
`
`performed in the presence of ZnCl.
`
`In some embodiments, prior to step (ii), the support memberof(i) is incubated with a
`
`blocking buffer.
`
`In some embodiments, the second agentis a polyclonal antibody, a monoclonal
`
`antibodies, or a mixture of two or more monoclonal antibodies that bind to HMWK. The two or
`
`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),
`
`10
`
`contacting the second agent in (iii), which is immobilized on the support member, with the other
`
`memberof the receptor-ligand pair, wherein the other memberis 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
`
`15
`
`suspected of containing a cleaved HMWKwith any ofthe antibodies described herein (e.g.
`
`559B-M004-B04); (ii) measuring a complex of the cleaved HMWKand 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 (1) is performed in the presence of ZnCl2.
`
`In some
`
`embodiments, step (i) is performed using an enzyme-linked immunosorbent assay (ELISA) or an
`
`immunoblotting assay.
`
`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.
`
`In some
`
`embodiments, the method further comprises collecting the sample into an evacuated blood
`
`collection tube, which comprises one or more protease inhibitors.
`
`Any of the assay methods(e.g., immunoassays) described herein may be a ELISA assay,
`
`a Western blot assay, orlateral flow assay.
`
`In some embodiments, the biological sample is obtained from a subject (e.g., a human
`
`patient) having a disease. The assay method mayfurther comprise determining whetherthe
`
`disease is mediated by plasma kallikrein based on the level of the cleaved HMWK,a deviation
`
`30
`
`of the level of the cleaved HMWKin the sample from that of a control sample being indicative
`
`that the disease is mediated by plasma kallikrein.
`
`Anyof the assay methods described herein may further comprise identifying patients
`
`with diseases or disorders mediated by plasma kallikrein, or evaluating the efficacy of a
`
`i)
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`WO 2017/070170
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`PCT/US2016/037640
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`treatment of the disease or disorder based on the levels of cleaved HMWK.
`
`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 C1 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 paticnt who is on a treatment for the
`
`disorder, and wherein the method further comprises assessing the efficacy of the treatment based
`
`10
`
`on the level of the cleaved HMWKdeterminingin step (111), a deviation of the level of the
`
`cleaved HMWKin the sample from the subject fromthat of a control sample being indicative of
`
`the treatmentefficacy. In some embodiments, the method further comprisesidentifying 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
`
`15
`
`of the disease based on the level of the cleaved HMWK.
`
`In some embodiments, the human paticnt has a history of the discase (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 memberis a 96-well plate.
`
`In anotheraspect 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-B04 or competes against 559B-M004-B04 for
`
`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-B04. In one example, the antibodyis
`
`559B-M004-B04.
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`

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`WO 2017/070170
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`PCT/US2016/037640
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`Anyofthe antibodies specific to a cleaved HMWKasdescribed herein can be used in a
`
`method for detecting a cleaved high molecular kininogen (HMWK)ina sample. Such a method
`
`may comprise (i) contacting a sample suspected of containing a cleaved HMWKwith the
`
`antibody; (ii) measuring a complex of the cleaved HMWKandthe antibody formedin step (i);
`
`and determining the level of the cleaved HMWKin the sample based on theresult 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 result obtained from this method maybe relied on
`
`to determinethe risk of a subject from whom the sample is obtained for developing a disorder
`
`mediated by plasma kallikrein such as HAE. In someinstances, step (i) can be performed in the
`
`10
`
`presence of ZnCly.
`
`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 kininogen (HMWK)and a cleaved HMWK.
`
`15
`
`In some embodiments, the antibody that binds both intact and cleaved HMWKdoesnot
`
`bind to low molecular weight kininogen (LMWK). In some embodiments, the antibody binds
`
`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 against 559B-M0067-E02,
`
`559B-M0039-G07, 559B-M0044-E09, 559B-M0003-C08, 559B-M0039-H06, 559B-M0039-
`
`DO08, 559B-M0068-C07, 559B-M0021-G11, 559B-M0061-G06, 559B-M0036-G12, 559B-
`
`M0042-E06, 559B-M0070-H10, 559B-M0068-D01, or 559B-M0004-E08for binding to the
`
`intact HMWKand/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-
`
`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 antibodyis selected from the group consisting of 559B-M0067-
`
`30
`
`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.
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`In other embodiments, the antibody that binds both intact and cleaved HMWKalso binds
`
`LMWK.In some embodiments, the antibody binds the same epitope as 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-
`
`MO0035-F05, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B 11.
`
`559B-M0067-G11, 559B-M0064-H02, or 559B-M0065-B10.
`
`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-
`
`C06, 559B-M0003-A08, 559B-M0054-B11, 559B-M0067-G11, 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
`
`CDRsas 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-
`
`10
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`15
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`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-BO5,
`
`559B-M0048-D 12, 559B-M0053-G01, 559B-M0038-H03, 559B-M0017-H08, 559B-M0035-
`
`FO5, 559B-M0035-H09, 559B-M0043-C06, 559B-M0003-A08, 559B-M0054-B11, 559B-
`
`M0067-G11, 559B-M0064-H02, and 559B-M0065-B 10.
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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
`
`appendedclaims.
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`BRIEF DESCRIPTION OF DRAWINGS
`
`The following drawings form part of the present specification and are included to further
`
`demonstrate certain aspects of the present disclosure, which can be better understood by
`
`30
`
`reference to one or more of these drawings in combination with the detailed description of
`
`specific embodiments presented herein.
`
`FIG. 1 is a graph showing binding of 559B-M0004-B04 to intact HMWK(dark gray
`
`bars) or cleaved HMWK(light gray bars) under the indicated ELISA conditions.
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`10
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`15
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`FIG.2 presents graphs showing binding of various Fab clonesto intact 1-chain (intact)
`
`HMWK,2-chain (cleaved) HMWK,or LMWK.A: Fab clonesidentified using the phage
`
`display screening methods described herein. Intact HWMKis shownin dark gray bars, cleaved
`
`HMWEKinlight gray bars, and LMWKin medium gray bars. B: binding for several example
`
`Fab clones. LWMkKis shownin dark gray bars, intact HMWKin light gray bars, and cleaved
`
`HWMkKin medium graybars.
`
`FIG.3 is a graph showing specificity of 559B-M0004-B04 towards intact HMWK,
`
`cleaved HMWK,or LMWK. Purificd cleaved HMWKwasspiked into SBT assay buffer
`
`(circles) or HMWK-deficient plasma (squares). Purified intact HMWKwasspiked into SBT
`
`assay buffer (triangles). Purified LMWKwas spiked into SBT assay buffer (diamonds). The y-
`
`axis presents the ELISA signal in absorbance units, and the x-axis presents the concentration of
`
`kininogen in pg/mL.
`
`FIG.4 is a graph showing detection of 2-Chain HMWK(cleaved HMWK)in plasma or
`
`assay buffer. Purified cleaved HMWKwasspiked into SBT assay buffer (open circles), SBT
`
`assay buffer and analyzed in the presence of 10% plasma (squares), or HMWkK-deficient plasma
`
`and analyzed in the presence of 10% plasma(triangles). Purificd cleaved HMWKwasalso
`
`spiked into assay buffer and analyzed in the presence of 2.5% plasma (diamonds) or HMWK
`
`deficient plasma and analyzed in the presence of 2.5% plasma (closed circles). The y-axis
`
`presents the ELISA signal in absorbance units, and the x-axis presents the concentration of
`
`kininogen in ug/mL.
`
`FIG. 5 is a graph showinglevels of cleaved HMWKinthe indicated human plasma
`
`samples prior to and after contact system activation. A: prior to and after contact system
`
`activation with FXIJa or ellagic acid. B: prior to and after contact system activation with FXJa,
`
`pKal, or ellagic acid.
`
`FIG.6 is a graph showing levels of cleaved HMWK in plasma samples from 12 normal
`
`human donorsprior to andafter activation of the contact system with ellagic acid.
`
`FIG. 7 presents graphs showing levels of cleaved HMWKfollowing inhibition with a
`
`pKalinhibitor. A: inhibition with landadelumab/DX-2930 or C1-INH prior to contact system
`
`activation with ellagic acid. B: inhibition of pooled sodium citrate plasma samples with
`
`30
`
`landadelumab/DX-2930 prior to contact system activation with 10 nM FXUHa.
`
`FIG.8 is a graph showing cleaved HMWKgeneration at the indicated time points
`
`following contact system activation with FXIIa orellagic acid.
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`FIG. 9 is a graph showing levels of 2-chain HMWKin plasma samples from normal
`
`subjects and subjects having HAE.
`
`FIG.10 is photo showing results obtained from a HMWK Western blot analysis, which
`
`are consistent with the results obtained from the 2-Chain HMWKELISAassay described herein.
`
`Humancitrated plasma samples (normal plasma, FXII-deficient plasma, and prekallikrein-
`
`deficient plasma) were probed with a mouse monoclonal anti-HMWkKlight 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 FXIa, or 10% cllagic acid.
`
`FIG.11 is a graph showingthat the addition of ZnCl, to either citrated or EDTA plasma
`
`10
`
`samples increased the signal of the 2-Chain HMWKin an ELISA assay. The x-axis shows the
`
`concentration of ZnCl, in the assay well after a 40-fold dilution.
`
`FIG. 12 presents schematics of the discovery and developmentof assays using the
`
`antibodies described herein. A: schematic of the phage display methods usedto discover 2-
`
`chain HMWKbinding antibodies. B: an example sandwich ELISA assay in which the 2-chain
`
`15
`
`HMWkKspecific antibody/Fab (e.g., 559B-M0004-B04) is immobilized in 96-well plates to
`
`capture 2-chain HMWKin citrated plasma, followed by washing and detection with an anti-
`
`HMWKkKantibody conjugated to a label (anticHMWK-HRP).
`
`FIG. 13 is a graph showing results from a 2-chain HMWKsandwich ELISA standard
`
`curve, in which citrated plasma samples were spiked with 2-chain HMWK(10% finaldilution).
`
`FIG. 14 showsthe identification of 2-chain HMWkK-specific antibodies by phage display
`
`selection and screening. A: plots the ratio of the result of a 2-chain HWMKbinding assay to a
`
`LMWKbinding assay on the y-axis comparedto the ratio of the result of a 2-chain HMWK
`
`binding assay to a 1-chain HMWKbinding 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,1-chain HMWK, or LMWK,followed by streptavidin-HRP. B:
`
`shows binding to 1-chain HMWK, 2-chain HMWK, or LMWKforthe indicated isolated Fab
`
`fragments.
`
`FIG. 15 is a graph showing competition of 2-chain HMWKandkininogenpeptides
`
`(HKH20 and GCP28) for binding to 559B-M0004-B04.
`
`30
`
`FIG.16 is a graph showing a standard curve for an optimized sandwich ELISA forthe
`
`detection of 2-chain HMWKin human plasma samples.
`
`FIG. 17 presents graphs of Western blotting analyses comparing the level of 2-chain
`
`HMWkKincitrated plasma samples from healthy subjects and HAE patients. A: scatter plot
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`comparing the percent 2-chaim HMWKin samples from healthy subjects (“TV”) and HAE
`
`patients between HAEattacks (“Basal”) and during an HAEattack (“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
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`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
`
`HMWKin SCAT169 plasma samples from healthy subjects and HAEpatients. A: scatter plot
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`comparing the percent 2-chaim HMWKin samples from healthy subjects (“HV”) and HAE
`
`patients between HAEattacks (“Basal”) and during an HAEattack (“Attack”). B: ROC analysis
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`comparing the sensitivity and specificity for the detection of HAE basal samples versus samples
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`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
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`= 0.967). D: ROC analysis comparing the sensitivity and specificity for the detection of HAE
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`attack samples vcrsus HAE basal samples (AUC = 0.597).
`
`FIG. 19 presents graphs of ELISA analyses comparing the level of 2-chain HMWK in
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`citrated plasma samples from healthy subjects and HAEpatients. A: scatter plot comparing the
`
`percent 2-chaim HMWKin samples from healthy subjects (“HV”) and HAEpatients between
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`HAEattacks (“Basal”) and during an HAEattack (“Attack”). B: ROC analysis comparing the
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`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
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`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
`
`HAEbasal samples (AUC = 0.709).
`
`FIG.20 presents graphs of ELISA analyses comparing the level of 2-chain HMWKin
`
`SCAT169 samples from healthy subjects and HAEpatients. A: scatter plot comparing the
`
`percent 2-chaim HMWKinsamples from healthy subjects (“HV”) and HAEpatients between
`
`HAEattacks (“Basal”) and during an HAEattack (“Attack”). B: ROC analysis comparing the
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`30
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`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
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`HAEbasal samples (AUC = 0.8176).
`
`DETAILED DESCRIPTION OF PRESENT DISCLOSURE
`
`Plasmakallikrein (PKal) is a serine protease componentof the contact system andis the
`
`primary bradykinin-generating enzymein the circulation. The contact system is activated by
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`either factor XIa (the active form of Factor XII or FXII) upon exposure to foreign or negatively
`
`charged surfaces or on endothclial cell surfaces by prolylcarboxypcptidases (Sainz IM. ct al.,
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`Thromb Haemost 98, 77-83, 2007). Activation of the plasmakallikrein amplifies intrinsic
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`coagulationvia 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
`
`a disulfide bond (also known as 2-chain HMWK).
`
`Asthe 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 C1-inhibitor
`
`protein (C1-INH)lcads to hereditary angiocdema (HAE). Paticnts with HAE suffer from acute
`
`attacks of painful edema often precipitated by unknowntriggers (Zuraw B.L.et al., N Engl J
`
`Med359, 1027-1036, 2008). Through the use of pharmacological agents or genetic studies in
`
`animal models, the plasma kallikrein-kinin system (plasma KKS) has been implicated in various
`
`diseases.
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`The level of cleaved HMWKwas found to be elevated in HAEattack, as well as in other
`
`pKal-associated disorders. Thus, cleaved HMWKcanserve as a biomarker for monitoring
`
`disease development and/or treatment efficacy. However, the art lacks suitable agents and/or
`
`suitable assays that can effectively distinguish intact HMWKfrom its cleaved version.
`
`The present disclosure is based, at least in part, on the developmentof specific
`
`immunoassaysthat allows for detection of cleaved HMWKwith high specificity and sensitivity.
`
`It was observed that a Sandwich ELISA in which an agentthat specifically binds cleaved
`
`HMWKis immobilized on a support member(e.g., a multi-well plate) unexpectedly enhanced
`
`detection efficiency as comparedto 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,
`
`unexpectedly, that using the LowCross blocking buffer (containing casein), rather than a
`
`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, atleast in
`
`part, the isolation of antibodies that specifically bind a cleaved HMWK.
`
`Accordingly, provided herein are immunoassays for detecting the presence or measuring
`
`the level of a cleaved HMWKin a biological sample suspected of containing HMWKspecies,
`
`using an agent(e.g., an antibody) that specifically binds a cleaved HMWK(e.g., the cleaved
`
`HMWkKhaving a molecular weight of 46 kDa). Given the correlation between the level of
`
`cleaved HMWKanddisorders associated with or mediated by pKal (e.g., HAE), the
`
`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
`
`disorder.
`
`I.
`
`Immunoassaysfor Specific Detection of Cleaved HMWK
`
`Onceaspect of the present disclosure relatcs to immunoassays for detecting cleaved
`
`HMWkKwith high sensitivity and specificity. Such immunoassays may involve a Sandwich
`
`ELISA in whichan agentthat specifically binds a cleaved HMWKis immobilized on a support
`
`member, which can be a 96-well plate. The immunoassays described herein allows for selective
`
`detection of cleaved HMWKinbiological samples, e.g., serum samples or plasma samples,
`
`which may contain both intact and cleaved HMWK,as well as LMWK.
`
`(i) High Molecular-Weight Kininogen
`
`High molecular-weight kininogen (HMWK)exists in the plasmaasa single polypeptide
`
`(1-chain) multi-domain (domains 1-6) protein with a molecular weight of approximately 110
`
`kDa, referred to herein as intact HWMK. The human gene encoding HMWKis kininogen 1
`
`(KNG1). KNG1 is transcribed and alternatively spliced to form mRNAsthat encode either
`
`HMWkKorlow molecular weight kininogen (LMWK). An exemplary protein sequence of
`
`HMWkKis provided below:
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`>gi|156231037|ref|NP_001095886.1| kininogen-1 isoform 1 precursor [Homo
`sapiens]
`MKLITILFLCSRLLLSLTQESQSEEIDCNDKDLFKAVDAALKKYNSQNOSNNOFVLYRITEATKTVGSDT
`FYSFKYEIKEGDCPVQOSGKTWQODCEYKDAAKAATGECTATVGKRSSTKFSVATOTCOITPAEGPVVTAQY
`DCLGCVHPISTQOSPDLEP ILRHGIQOYFNNNTQHSSLFMLNEVKRAQROVVAGLNERITYSIVQOTNCSKEN
`FLFLTPDCKSLWNGDTGECTDNAY IDIQLRIASFSQONCDIYPGKDFVQPP TKICVGCPRDIPTNSPELEE
`TLTHTITKLNAENNATFYFKIDNVKKARVOVVAGKKYF IDFVARETTCSKESNEELTESCETKKLGOSLD
`CNAEVYVVPWEKKIYPTVNCQPLGMISLMKRPPGE'SPFRSSRIGEIKEETTVSPPHTSMAPAQDEERDSG
`KEQGHTRRHDWGHEKQRKHNLGHGHKHERDQOGHGHORGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHV
`LDHGHKHKHGHGHGKHKNKGKKNGKHNGWKTEHLASSSEDSTTPSAQTQOEKTEGPTPIPSLAKPGVTVTF
`SDFQDSDLIATMMPP ISPAP IQSDDDWIPDIQIDPNGLSFNP ISDFPDTTSPKCPGRPWKSVSEINPTTQ
`MKESYYFDLTDGLS (SEQ ID NO: 1)
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`Intact HMWK, also referred to herein as “intact kininogen,” can be assayed, for example,
`
`using coagulant or immunological methods, e.g., radioimmunoassay(see, e.g., Kerbiriou-
`
`Nabias, D.M., Br J Haematol, 1984, 56(2):2734-86). A monoclonal antibody to the light chain
`
`of human HMWKis known. See,e.g., Reddigari, S.R. & Kaplan, A.P., Blood, 1999, 74:695-
`
`702, An assay for HMWKthatrelies on a chromogenic substrate can also be used. See,e.g.,
`
`Scott, C.F. et al. Thromb Res, 1987, 48(6):685-700; Gallimore, M.J. et al. Thromb Res, 2004,
`
`114(2):91-96.
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`HMWkKis cleaved by pKal within domain 4 to release the 9 amino acid, pro-
`
`inflammatory peptide bradykinin, and a 2-chain form of HMWK,referred to herein as cleaved
`
`HMWE.The 2 chains of HMWKare the heavy chain, which contains domains 1-3, and the
`
`light chain, which contains domains 5 and 6, joined by a disulfide bond. Uponinitial cleavage
`
`of intact HMWK,the heavy and light chains have a molecular weight of approximately 65 kDa
`
`and 56 kDa, respectively. Further proteolytic processing results in generation of a 46 kDalight
`
`chain.
`
`bclow.
`
`Exemplary sequences of the heavy and light chains of cleaved kininogen are provided
`
`> cleaved kininogen-1 heavy chain
`
`QESQSEEIDCNDKDLFKAVDAALKKYNSQNOSNNOFVLYRITEATKTVGSDTFYSFKYEI
`KEGDCPVOSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTA
`QYDCLGCVHPISTQSPDLEP ILRHGIOYFNNNTOQHSSLFMLNEVKRAQROVVAGLNFRIT
`YSIVOTNCSKENFLFLTPDCKSLWNGDTGECTDNAY IDIOLRIASFSONCDIYPGKDFVO
`PPTKICVGCPRDIPTNSPELEETLTHT ITKLNAENNATFYFK IDNVKKARVOVVAGKKYF
`IDFVARETTCSKESNEELTESCETKKLGOSLDCNAEVYVVPWEKKIYPTVNCQPLGMISL
`MK (SEQ ID NO: 2)
`
`> cleaved kininogen-1 light chain
`
`SSRIGEIKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKORKHNLGHGHKHER
`DQGHGHORGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNK
`GKKNGKHNGWKTEHLASSSEDSTTPSAQTOEKTEGPTP IPSLAKPGVIVTFSDFODSDLI
`ATMMPP ISPAP IOSDDDWIPDIQIDPNGLSFENPISDFPDTTSPKCPGRPWKSVSEINPTT
`QMKESYYFDLTDGLS (SEQ ID NO: 3)
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`(ti) Antibodies Specific to Cleaved HAWK
`
`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 HMWKthatis
`
`not present on intact HMWK.In some embodiments, the cleaved HMWK-binding agentis an
`
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`antibody.
`
`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,
`
`polypeptide, etc., throughat least one antigen recognition site, located in the variable region of
`
`the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact
`
`(ie., full-length) polyclonal or monoclonal antibodies, but also antigen-binding fragments
`
`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
`
`antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any
`
`other modified configuration of the immunoglobulin molecule that compriscs an antigen
`
`recognition site of the required specificity, including glycosylation variants of antibodies, amino
`
`acid sequencevariants of antibodies, and covalently modified antibodies. An antibody includes
`
`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, immunogl