NZ741448B2 - Immunoassay for the detection of cleaved high molecular weight kininogen - Google Patents
Immunoassay for the detection of cleaved high molecular weight kininogen Download PDFInfo
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- NZ741448B2 NZ741448B2 NZ741448A NZ74144816A NZ741448B2 NZ 741448 B2 NZ741448 B2 NZ 741448B2 NZ 741448 A NZ741448 A NZ 741448A NZ 74144816 A NZ74144816 A NZ 74144816A NZ 741448 B2 NZ741448 B2 NZ 741448B2
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- New Zealand
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- hmwk
- cleaved
- antibody
- sequence
- light chain
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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.
Description
IMMUNOASSAY FOR THE ION OF CLEAVED HIGH MOLECULAR
WEIGHT KININOGEN
CROSS REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Provisional Application s 62/243,505,
filed October 19, 2015, and 62/335,311, filed May 12, 2016 under 35 U.S.C. §119, the entire
content of each of which is herein incorporated by reference.
BACKGROUND OF PRESENT DISCLOSURE
Kininogens are precursors of kinin, such as bradykinin and kallidin. There are two types
of human kininogens, high molecular-weight kininogen (HMWK) and low molecular-weight
kininogen (LMWK), which are splicing variants. HMWK acts mainly as a cofactor on
coagulation and mation and is the preferred substrate for plasma kallikrein (pKal)-
mediated bradykinin generation.
Plasma kallikrein (pKal) is the primary bradykinin-generating enzyme in 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 (a single-chain
polypeptide) to produce bradykinin and a cleaved form HMWK, which contains two polypeptide
chains held er by a disulfide bond. Cugno et al., Blood (1997) 3-3218.
Cleaved HMWK increased to about 47% of total kininogen during a hereditary
angioedema (HAE) attack. Cugno et al., Blood (1997) 89:3213-3218, making it a biomarker for
monitoring HAE attack. It is therefore of interest to develop sensitive and reliable assays for
detecting the level of cleaved HMWK in biological samples.
SUMMARY OF PRESENT DISCLOSURE
In a first aspect, the present invention provides an immunoassay method for detecting a
d high molecular weight kininogen , the method comprising: (i) providing a
support member, on which a first agent that specifically binds a d HMWK is immobilized;
(ii) contacting the support member of (i) with a biological sample suspected of containing a
cleaved HMWK; (iii) ting the support member ed in (ii) 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 , directly or
indirectly, to determine the level of the cleaved HMWK in the biological sample; wherein the
first agent is an antibody comprising a heavy chain complementarity 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
IGSNYVY, a light chain CDR2 sequence RNNQRPS, and a light chain CDR3
ce AWDDSLNGRV.
In a second aspect, the present invention es an isolated antibody, which
specifically binds a cleaved high molecular weight kininogen (HMWK);
n the antibody comprises a heavy chain complementarity ining 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.
In a third aspect, the present invention provides a kit for detecting a cleaved high
lar weight kininogen (HMWK), the kit comprising a first agent that ically binds a
cleaved HMWK; wherein the first agent is an antibody of the second aspect.
In a fourth aspect, the present invention es an isolated antibody that binds both
intact high molecular weight gen (HMWK) and a cleaved HMWK and does not bind to
low molecular weight kininogen (LMWK); wherein the antibody comprises (i) a heavy chain
complementarity ining region (CDR) 1 sequence LYPMV, a heavy chain CDR2 sequence
SIYPSGGFTTYADSVKG, a heavy chain CDR3 sequence SSRYYYYGMDV, a light chain
CDR1 sequence SGSSSNIGSEYVY, a light chain CDR2 ce RNDQRPS, and a light
chain CDR3 sequence STWDDTLRTGV; (ii) a heavy chain CDR1 sequence RYRMR, a heavy
chain CDR2 sequence GISPSGGWTYYADSVKG, a heavy chain CDR3 sequence
DNGDYALAH, a light chain CDR1 sequence RASQRIINYLN, a light chain CDR2 sequence
AASSLQS, and a light chain CDR3 sequence QQSYSAPLT; (iii) a heavy chain CDR1
sequence QYSMG, a heavy chain CDR2 sequence SIYSSGGSTQYADSVKG, a heavy chain
CDR3 sequence TRRGWFGEDYYYYMDV, a light chain CDR1 sequence RASQGIRNDVG, a
light chain CDR2 sequence AASSLQS, and a light chain CDR3 sequence QHNSYPLT; (iv) a
heavy chain CDR1 sequence PYMMY, a heavy chain CDR2 sequence
SISPSGGKTWYADSVKG, a heavy chain CDR3 sequence LGGSSSYYYYYYYGMDV, a
light chain CDR1 sequence SGSSSNIGGNTVN, a light chain CDR2 sequence S, and
a light chain CDR3 sequence ASWDDRLNGHWV; (v) a heavy chain CDR1 sequence
AYDMH, a heavy chain CDR2 sequence SIWPSGGGTYYADSVKG, a heavy chain CDR3
sequence GDYDYGDFTDAFDI, a light chain CDR1 ce TGTSSDVGSYNLVS, a light
chain CDR2 sequence EGSKRPS, and a light chain CDR3 sequence YSYV; (vi) a
heavy chain CDR1 sequence NYAMQ, a heavy chain CDR2 sequence
WIYSSGGPTYYADSVKG, a heavy chain CDR3 sequence GLPGQPFDY, a light chain CDR1
sequence IGNNYVY, a light chain CDR2 sequence RNNQRPS, and a light chain
CDR3 sequence ATWDDRLSGWV; (vii) a heavy chain CDR1 sequence SYQMH, a heavy
chain CDR2 sequence GIYSSGGSTPYADSVKG, a heavy chain CDR3 sequence V,
a light chain CDR1 sequence SSWLA, a light chain CDR2 sequence S, and a
light chain CDR3 sequence QKYNIAPYT; (viii) a heavy chain CDR1 sequence PYPMT, a
heavy chain CDR2 sequence GISSSGGFTPYADSVKG, a heavy chain CDR3 sequence
MVRGVIKAFDI, a light chain CDR1 sequence SGSSSNIGSHYVF, a light chain CDR2
sequence RNNQRPS, and a light chain CDR3 sequence ATWDNSLSAWV; (ix) a heavy chain
CDR1 sequence KYTMW, a heavy chain CDR2 sequence VISSSGGKTYYADSVKG, a heavy
chain CDR3 sequence TANRAFDI, a light chain CDR1 sequence RASQSVSSDLA, a light
chain CDR2 sequence GASTRAT, and a light chain CDR3 sequence QQYNDWPPL;
(x) a heavy chain CDR1 sequence RYYMA, a heavy chain CDR2 sequence
GIVPSGGQTGYADSVKG, a heavy chain CDR3 sequence TRRGWFGEDYYYYMDV, a light
chain CDR1 sequence RASQSVGSTYLA, a light chain CDR2 sequence GASSRAT, and a light
chain CDR3 sequence QHFHTSPPGIT; (xi) a heavy chain CDR1 sequence MYKMS, a heavy
chain CDR2 ce VISPSGGRTYYADSVKG, a heavy chain CDR3 sequence
GTRTSGLDY, a light chain CDR1 sequence TGTSSDVGGYKYVS, a light chain CDR2
sequence EVSNRPS, and a light chain CDR3 sequence TTVV; (xii) a heavy chain
CDR1 sequence TYGMR, a heavy chain CDR2 sequence VISPSGGKTNYADSVKG, a heavy
chain CDR3 sequence AMDV, a light chain CDR1 sequence SGSSSNIGSNTVN, a
light chain CDR2 sequence YNDRRPS, and a light chain CDR3 sequence AAWDDSLSGPV;
(xiii) a heavy chain CDR1 sequence IYPMS, a heavy chain CDR2 sequence
GISPSGGKTAYADSVKG, a heavy chain CDR3 sequence GQGRAVRGKLYYYGMDV, a
light chain CDR1 sequence SGSSSNIGTNNVN, a light chain CDR2 sequence SHHRRPS, and
a light chain CDR3 sequence AAWDDSLNGPV; or (xiv) a heavy chain CDR1 sequence
MYHMN, a heavy chain CDR2 sequence GSTRYADSVKG, a heavy chain CDR3
sequence GVRYGMDV, a light chain CDR1 sequence RASQGISSWLA, a light chain CDR2
sequence AASSLQS, and a light chain CDR3 sequence QQANSFPIT.
In a fifth aspect, the present invention provides an isolated antibody that binds an intact
high molecular weight kininogen (HMWK) and a cleaved HMWK, and additionally binds a low
molecular weight kininogen (LMWK), wherein the antibody comprises: (i) a heavy chain
complementarity determining region (CDR) 1 sequence MYDMH, a heavy chain CDR2
sequence SISSSGGYTQYADSVKG, a heavy chain CDR3 sequence DRGLIAAAGGFDP, a
light chain CDR1 sequence RASQSIGIYLN, a light chain CDR2 sequence AASSLQS, and a
light chain CDR3 sequence QRTYGRPLT; (ii) a heavy chain CDR1 sequence KYEMM, a
heavy chain CDR2 ce SISPSGGYTMYADSVKG, a heavy chain CDR3 sequence
HRSKWNDAPFDS, a light chain CDR1 sequence RASQSIDTYLN, a light chain CDR2
ce AASKLED, and a light chain CDR3 sequence QQSYSSPGIT; (iii) a heavy chain
CDR1 sequence IYQMY, a heavy chain CDR2 sequence GRTFYADSVKG, a heavy
chain CDR3 sequence RGSWYVGGNEYFQH, a light chain CDR1 sequence
SGDTLGNKFVS, a light chain CDR2 ce QDTKRPS, and a light chain CDR3 sequence
QVWDSNSYA; (iv) a heavy chain CDR1 sequence FYMMY, a heavy chain CDR2 sequence
SISSSGGFTRYADSVKG, a heavy chain CDR3 sequence VRGLAVAAPDY, a light chain
CDR1 sequence IGTSSDIGTYNYVSW, a light chain CDR2 sequence S, and a light
chain CDR3 sequence SSYTTSVTWV; (v) a heavy chain CDR1 sequence GYNMY, a heavy
chain CDR2 sequence GWTSYADSVKG, a heavy chain CDR3 sequence GQWMDW,
a light chain CDR1 sequence RASQNITGYLN, a light chain CDR2 sequence DASRMNT, and
a light chain CDR3 sequence QHTDDFSVT; (vi) a heavy chain CDR1 sequence YRMMW, a
heavy chain CDR2 ce ISSSGGYTAYADSVKG, a heavy chain CDR3 sequence
RNRAFDI, a light chain CDR1 sequence LYSSNNKNYL, a light chain CDR2
sequence WASTRES, and a light chain CDR3 sequence QQYYSTPLG; (vii) a heavy chain
CDR1 sequence RYQMT, a heavy chain CDR2 sequence IGSSGGFTNYADSVKG, a heavy
chain CDR3 sequence LPANFYYYMDV, a light chain CDR1 sequence RASQNIYSFLN, a
light chain CDR2 sequence ATSSLQS, and a light chain CDR3 sequence PWT; (viii)
a heavy chain CDR1 sequence WYMMK, a heavy chain CDR2 sequence
SIVPSGGWTTYADSVKG, a heavy chain CDR3 sequence EGNLWFGEGRAFDI, a light
chain CDR1 sequence SSSYLA, a light chain CDR2 sequence GASSRAT, and a light
chain CDR3 sequence QQRSNWPPS; (ix) a heavy chain CDR1 sequence KYDMH, a heavy
chain CDR2 sequence GKTEYADSVKG, a heavy chain CDR3 sequence
EYRYCTANTCSLYGMDV, a light chain CDR1 sequence RTSQGVRSDFA, a light chain
CDR2 sequence AAFILDN, and a light chain CDR3 sequence QQSYSTPLT; (x) a heavy chain
CDR1 sequence PYWMH, a heavy chain CDR2 sequence VISPSGGGTGYADSVKG, a heavy
chain CDR3 sequence ESRGSGSHEDY, a light chain CDR1 sequence RASQSVSSYLA, a
light chain CDR2 sequence GASNRGT, and a light chain CDR3 sequence QQYKNWPNLT;
(xi) a heavy chain CDR1 sequence HYPMA, a heavy chain CDR2 sequence
GIVSSGGRTVYADSVKG, a heavy chain CDR3 sequence SEGAFDI, a light chain
CDR1 sequence SGSSSNIGNNFVY, a light chain CDR2 sequence KNNQRPS, and a light
chain CDR3 sequence AAWDNSLSGFYV; (xii) a heavy chain CDR1 sequence WYGMH, a
heavy chain CDR2 sequence RIGPSGGPTSYADSVKG, a heavy chain CDR3 sequence
GYYGTGRYFQH, a light chain CDR1 sequence RASQSVGSDYLA, a light chain CDR2
sequence DASNRA, and a light chain CDR3 sequence QQRSNWPPT; (xiii) a heavy chain
CDR1 sequence AYAMR, a heavy chain CDR2 ce YISSSGGETMYADSVKG, a heavy
chain CDR3 sequence GYGRIDY, a light chain CDR1 sequence TGTSSDIGGYNYVS, a light
chain CDR2 sequence S, and a light chain CDR3 sequence SSYTSGSTRV; (xiv) a
heavy chain CDR1 sequence AYVMR, a heavy chain CDR2 ce
SIGSSGGPTYYADSVKG, a heavy chain CDR3 sequence RGGSGSSHAFDI, a light chain
CDR1 sequence SSYLN, a light chain CDR2 sequence AASSLQS, and a light chain
CDR3 ce QQYNSFPLT; (xv) a heavy chain CDR1 sequence YYGMN, a heavy chain
CDR2 sequence VISPSGGLTVYADSVKG, a heavy chain CDR3 sequence
GFAVQHGGGAFDI, a light chain CDR1 ce RASQSVTTYLA, a light chain CDR2
sequence DASIRAT, and a light chain CDR3 sequence PLT; (xvi) a heavy chain
CDR1 sequence PYEMV, a heavy chain CDR2 sequence SIVPSGGWTVYADSVKG, a heavy
chain CDR3 sequence PSGRGLAFDI, a light chain CDR1 sequence RASQSISSSYLA, a light
chain CDR2 sequence GASSRAT, and a light chain CDR3 sequence LQQKSYPYT; (xvii) a
heavy chain CDR1 sequence KYFMT, a heavy chain CDR2 ce
WISSSGGYTNYADSVKG, a heavy chain CDR3 sequence GAYYYDAFDI, a light chain
CDR1 sequence RASQSIAIFLN, a light chain CDR2 sequence GASTLQS, and a light chain
CDR3 sequence QQSYSTLYT; or (xviii) a heavy chain CDR1 sequence QYIMG, a heavy chain
CDR2 ce SIGSSGVTVYADSVKG, a heavy chain CDR3 sequence GGGVTVLHAFDI,
a light chain CDR1 sequence TGTSSDVGGYNYVS, a light chain CDR2 sequence EGNKRPS,
and a light chain CDR3 sequence TAYGGHSRFYV.
In a sixth aspect, the present invention provides a method for ing a cleaved high
molecular kininogen (HMWK) in a sample, the method comprising: contacting a sample
suspected of containing a cleaved HMWK with an antibody of the second aspect; measuring a
x 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).
Some aspects of the t 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 member on which a first agent (e.g., an antibody such as
559B-M004-B04) that specifically binds a cleaved HMWK is attached; (ii) ting the
support member of (i) with a biological sample suspected of ning a cleaved HMWK; (iii)
contacting the support member obtained in (ii) 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
[TEXT CONTINUES ON PAGE 2]
level of the d HMWK in the biological sample. In some ces, step (ii) may be
performed in the presence of ZnClz.
In some embodiments, prior to step (ii), the support member of (i) is incubated with a
blocking buffer.
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
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),
ting the second agent in (iii), which is immobilized on the support member, with the other
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 s for detecting a cleaved high
molecular kininogen (HMWK) in a sample, the method comprising (i) contacting a sample
suspected of containing a cleaved HMWK with any of the antibodies described herein (6.g.
559B—M004—B04); (ii) measuring a x 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 ZnClz. 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
ments, the method further comprises ting the sample into an evacuated blood
collection tube, which comprises one or more se inhibitors.
Any of the assay s (e.g., immunoassays) described herein may be a ELISA assay,
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
of the level of the cleaved HMWK in the sample from that of a l sample being tive
that the disease is mediated by plasma kallikrein.
Any of the assay methods described herein may further comprise identifying patients
with diseases or disorders mediated by plasma kallikrein, or ting the efficacy of a
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 (BZR) inhibitor, and/or a Cl esterase inhibitor, for treating the disorder, if the t is
identified as having the disorder. In some embodiments the pKal inhibitor is an anti—pKal
antibody. In some embodiments, the eutic agent is lanadelumab, ecallantide, icatibant, or
human plasma—derived Cl esterase tor.
In some ments, 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
on the level of the cleaved HMWK ining in step (iii), a deviation of the level of the
cleaved HMWK in the sample from the t from that of a control sample being indicative of
the treatment efficacy. In some embodiments, the method further ses identifying a
suitable treatment for the subject based on the level of the cleaved HMWK. In some
embodiments, the method further ses identifying the subject as a candidate for a treatment
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 e 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 . In some embodiments, the method further comprises administering a eutic
agent to the subject, if the subject is at risk of disease attack.
In another , 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 t 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 lar weight kininogen (HMWK). In some embodiments, the
antibody binds the same epitope as 559B B04 or competes against 559B—M004—B04 for
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 antibody is
559B—M004-BO4.
Any of the antibodies specific to a cleaved HMWK as described herein can be used in a
method for detecting a cleaved high molecular gen (HMWK) in a sample. Such a method
may comprise (i) ting 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).
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
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
presence of ZnClz.
Any of the immunoassay methods described herein can be in Western blot format or
ELISA format.
In yet another aspect, an isolated dy is provided that binds both intact high
molecular weight kininogen (HMWK) and a d HMWK.
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
the same epitope as 559B—M0067—E02, 559B-M0039—GO7, 559B—M0044—E09, 559B—M0003—
C08, 559B—M0039—H06, 559B—M0039—D08, 559B—M0068—CO7, 559B—M0021—Gll, 559B—
M006l—G06, 559B—M0036—G12, 559B—M0042—E06, 0070—H10, 559B—M0068—D01, or
559B—M0004—E08. In some embodiments, the dy competes against 559B—M0067—E02,
559B—M0039—GO7, 559B—M0044—E09, 559B—M0003—C08, 559B—M0039—H06, 0039—
D08, 559B—M0068—CO7, 559B—M0021—Gll, 006l—G06, 559B—M0036—G12, 559B—
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—GO7, 559B—M0044—E09, 559B—M0003—C08, 559B—
M0039—H06, 559B—M0039—D08, 559B—M0068—CO7, 559B—M0021—Gl l, 559B—M006l—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—GO7, 559B—M0044—E09, 0003—C08, 559B—M0039—H06, 559B—
M0039—D08, 559B—M0068—CO7, 559B—M0021—Gll, 006l—G06, 559B—M0036—G12,
559B—M0042—E06, 0070—H10, 0068—D01, and 559B—M0004—E08.
In other embodiments, the antibody that binds both intact and cleaved HMWK also 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—
M0035—F05, 559B—M0035—H09, 559B—M0043—C06, 559B—M0003—A08, 559B—M0054—Bll,
559B—M0067—Gl l, 559B—M0064—H02, or 559B—M0065—B 10. In some embodiments, the
dy competes t 559B—M0069—C09, 559B—M0038—F04, 559B—M0044—C05, 559B—
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—Bll, 559B—M0067—Gl l, 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 ses the same heavy chain and light chain
CDRs 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—M0035—F05, 559B—M0035—H09, 559B—M0043—C06, 559B—M0003—
A08, 559B—M0054—B l l, 0067—Gl l, 559B-M0064-H02, or 559B-M0065-B 10. 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, 0054—Bll, 559B—
M0067—Gl l, 559B—M0064—H02, and 559B—M0065-B10.
The details of one or more embodiments of the disclosure are set forth in the description
below. Other features or ages of the present disclosure will be apparent from the
following drawings and detailed description of several embodiments, and also from the
ed claims.
BRIEF DESCRIPTION OF DRAWINGS
The following drawings form part of the present ication and are included to further
demonstrate certain aspects of the present disclosure, which can be better understood by
reference to one or more of these gs in combination with the ed description of
specific embodiments presented herein.
is a graph showing g of 559B—M0004—B04 to intact HMWK (dark gray
bars) or cleaved HMWK (light gray bars) under the indicated ELISA conditions.
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
display screening methods described herein. Intact HWMK is shown in dark gray bars, cleaved
HMWK in light gray bars, and LMWK in medium gray bars. B: binding for several example
Fab clones. LWMK is shown in dark gray bars, intact HMWK in light gray bars, and cleaved
HWMK in medium gray bars.
is a graph showing specificity of 559B—M0004—B04 s 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 presents the ELISA signal in absorbance units, and the X—axis ts the concentration of
kininogen in ug/mL.
is a graph showing detection of 2—Chain HMWK (cleaved HMWK) in plasma or
assay buffer. Purified cleaved HMWK was spiked into SBT assay buffer (open circles), SBT
assay buffer and analyzed in the presence of 10% plasma (squares), or HMWK—deficient plasma
and analyzed in the presence of 10% plasma (triangles). Purified cleaved HMWK was also
spiked into assay buffer and analyzed in the ce of 2.5% plasma (diamonds) or HMWK
deficient plasma and ed in the presence of 2.5% plasma (closed circles). The y—aXis
ts the ELISA signal in absorbance units, and the X—axis presents the concentration of
kininogen in ug/mL.
is a graph showing levels of cleaved HMWK in the ted human plasma
samples prior to and after contact system activation. A: prior to and after contact system
activation with FXIIa or ellagic acid. B: prior to and after t system tion with FXIIa,
pKal, or ellagic acid.
is a graph showing levels of cleaved HMWK in plasma samples from 12 normal
human donors prior to and after activation of the contact system with ellagic acid.
presents graphs showing levels of cleaved HMWK following inhibition with a
pKal inhibitor. A: inhibition with landadelumab/DX—2930 or C l—INH prior to contact system
activation with ellagic acid. B: inhibition of pooled sodium e plasma samples with
landadelumab/DX—2930 prior to contact system activation with 10 nM FXIIa,
is a graph showing cleaved HMWK generation at the indicated time points
following contact system activation with FXIIa or ellagic acid.
is a graph showing levels of 2—chain HMWK in plasma samples from normal
subjects and subjects having HAE.
is photo showing results obtained from a HMWK Western blot analysis, which
are consistent with the results obtained from the 2—Chain HMWK ELISA assay described herein.
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 ouse detection antibody. The analyzed plasma samples were either
untreated or activated with 100 nM pKal, 10 nM FXIIa, or 10% c acid.
is a graph showing that the on 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 on.
presents schematics of the discovery and development of assays using the
antibodies described herein. A: schematic of the phage display methods used to er 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
capture 2—chain HMWK in citrated plasma, followed by washing and detection with an anti—
HMWK dy conjugated to a label (anti—HMWK—HRP).
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).
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.
inant Fab fragments were passively immobilized onto 384—well plates prior to on
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.
is a graph showing ition of 2—chain HMWK and kininogen es
(HKH20 and GCP28) for binding to 559B-M0004-B04.
is a graph showing a standard curve for an zed sandwich ELISA for the
detection of 2—chain HMWK in human plasma s.
presents graphs of Western blotting analyses comparing the level of 2—chain
HMWK in ed plasma samples from healthy subjects and HAE ts. A: scatter plot
comparing the percent 2—chaim HMWK in s from healthy subjects (“HV”) and HAE
patients between HAE s (“Basal”) and during an HAE attack (“Attack”). B: ROC
(receiver ing 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 ts (AUC = l). D: ROC analysis comparing the sensitivity and
specificity for the ion of HAE attack samples versus HAE basal s (AUC = 0.625).
presents graphs of Western ng analyses comparing the level of 2—chain
HMWK in SCATl69 plasma samples from healthy subjects and HAE patients. A: scatter plot
comparing the percent 2—chaim HMWK in samples from y ts (“HV”) and HAE
patients between HAE attacks (“Basal”) and during an HAE attack ck”). 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
attack samples versus HAE basal samples (AUC = 0.597).
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 ing the sensitivity and specificity for the
detection of HAE attack samples versus s 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 = .
presents graphs of ELISA analyses comparing the level of 2—chain HMWK in
SCATl69 samples from healthy subjects and HAE patients. A: scatter plot comparing the
percent m 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 y
subjects (AUC = 0.999). C: ROC analysis ing the sensitivity and specificity for the
detection of HAE attack samples versus s from healthy subjects (AUC = l). D: ROC
analysis comparing the sensitivity and icity for the detection of HAE attack samples versus
HAE basal samples (AUC = 0.8176).
DETAILED DESCRIPTION OF PRESENT DISCLOSURE
Plasma kallikrein (PKal) is a serine protease ent of the contact system and is the
primary bradykinin—generating enzyme in the circulation. The contact system is ted by
either factor XIIa (the active form of Factor XII or FXII) upon exposure to foreign or negatively
charged surfaces or on elial cell surfaces by prolylcarboxypeptidases (Sainz I.M. et al.,
Thromb t 98, 77—83, 2007). Activation of the plasma kallikrein amplifies intrinsic
coagulation via its feedback activation of factor XII and proteolytically cleaves the gen
precursor, high molecular weight kininogen (HMWK), releasing the proin?ammatory
ptide bradykinin and a cleaved HMWK, which contains two polypeptide chains linked by
a disulfide bond (also known as 2—chain HMWK).
As the primary kininogenase in the circulation, plasma kallikrein is y responsible
for the generation of bradykinin in the vasculature. A genetic deficiency in the Cl—inhibitor
protein (Cl—INH) leads to hereditary angioedema (HAE). Patients with HAE suffer from acute
attacks of painful edema often precipitated by unknown triggers (Zuraw B.L. et al., N Engl J
Med 359, 1027—1036, 2008). Through the use of pharmacological agents or genetic studies in
animal , the plasma kallikrein—kinin system (plasma KKS) has been implicated in various
diseases.
The level of d HMWK was found to be elevated in HAE attack, as well as in other
ssociated disorders. Thus, cleaved HMWK can serve 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 HMWK from its cleaved version.
The present disclosure is based, at least in part, on the development of specific
immunoassays that allows for detection of cleaved HMWK with high icity and sensitivity.
It was observed that a Sandwich ELISA in which an agent that specifically binds cleaved
HMWK is immobilized on a support member (e.g., a multi—well plate) unexpectedly ed
detection efficiency as compared to the setting of ELISA in which the antigen (in this case, the
cleaved HMWK) is lized on the support member. r, 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
sensitivity during the initial screening to discover antibodies specific for cleaved HMWK.
Moreover, the detection specificity and sensitivity was r enhanced when a l plate
was used, as compared with a 384—well plate. The present disclosure is also based on, at least in
part, the ion of antibodies that specifically bind a cleaved HMWK.
Accordingly, provided herein are immunoassays for detecting the ce or measuring
the level of a cleaved HMWK in a biological sample suspected of ning HMWK species,
using an agent (e.g., an dy) that specifically binds a cleaved HMWK (e.g., the cleaved
HMWK having a molecular weight of 46 kDa). Given the correlation between the level of
cleaved HMWK and disorders associated with or mediated by pKal (e.g., HAE), the
imunoassays bed herein can be applied to identify patients who are at risk of such
diseases, to monitor disease progression, and/or to monitor efficacy of a ent against such a
disorder.
I. Immunoassays for Specific Detection of d HMWK
One aspect of the present disclosure relates to immunoassays for detecting d
HMWK with high sensitivity and specificity. Such immunoassays may involve a Sandwich
ELISA in which an agent that specifically binds a cleaved HMWK is immobilized on a support
member, which can be a 96—well plate. The immunoassays described herein allows for selective
detection of cleaved HMWK in biological samples, e.g., serum samples or plasma samples,
which may n both intact and cleaved HMWK, as well as LMWK.
(i) High Molecular-Weight Kininogen
High molecular—weight kininogen (HMWK) exists in the plasma as a single polypeptide
(l—chain) domain (domains 1—6) protein with a molecular weight of approximately 110
kDa, referred to herein as intact HWMK. The human gene encoding HMWK is kininogen l
(KNGl). KNGl is transcribed and alternatively spliced to form mRNAs that encode either
HMWK or low molecular weight kininogen (LMWK). An exemplary protein sequence of
HMWK is provided below:
>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)
Intact HMWK, also referred to herein as “intact kininogen,” can be assayed, for example,
using ant 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 HMWK is known. See, e.g., ari, S.R. & Kaplan, A.P., Blood, 1999, 74:695—
702. An assay for HMWK that relies 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.
HMWK is cleaved by pKal within domain 4 to release the 9 amino acid, pro—
in?ammatory 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 s 1—3, and the
light chain, which contains domains 5 and 6, joined by a disulfide bond. Upon initial 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 kDa light
chain.
Exemplary sequences of the heavy and light chains of cleaved kininogen are provided
below.
> d kininogen-l heavy chain
QESQSEEIDCNDKDLFKAVDAALKKYNSQNQSNNQFVLYRITEATKTVGSDTFYSFKYEI
VQSGKTWQDCEYKDAAKAATGECTATVGKRSSTKFSVATQTCQITPAEGPVVTA
QYDCLGCVHPISTQSPDLEPILRHGIQYFNNNTQHSSLFMLNEVKRAQRQVVAGLNFRIT
YSIVQTNCSKENFLFLTPDCKSLWNGDTGECTDNAYIDIQLRIASFSQNCDIYPGKDFVQ
PPTKICVGCPRDIPTNSPELEETLTHTITKLNAENNATFYFKIDNVKKARVQVVAGKKYF
IDFVARETTCSKESNEELTESCETKKLGQSLDCNAEVYVVPWEKKIYPTVNCQPLGMISL
MK (SEQ ID NO: 2)
> cleaved gen-l light chain
IKEETTVSPPHTSMAPAQDEERDSGKEQGHTRRHDWGHEKQRKHNLGHGHKHER
DQGHGHQRGHGLGHGHEQQHGLGHGHKFKLDDDLEHQGGHVLDHGHKHKHGHGHGKHKNK
GKKNGKHNGWKTEHLASSSEDSTTPSAQTQEKTEGPTPIPSLAKPGVTVTFSDFQDSDLI
ATMMPPISPAPIQSDDDWIPDIQIDPNGLSFNPISDFPDTTSPKCPGRPWKSVSEINPTT
QMKESYYFDLTDGLS (SEQ ID NO: 3)
(ii) Antibodies Specific to Cleaved HMWK
The immunoassays described herein may use any agent that can specifically bind a
cleaved HMWK, for example, an agent that recognizes a tope on cleaved HMWK that is
not present on intact HMWK. In some embodiments, the cleaved HMWK—binding agent is an
40 antibody.
An antibody (interchangeably used in plural form) is an immunoglobulin molecule
capable of specific binding to a target, such as a carbohydrate, polynucleotide, lipid,
polypeptide, etc., through at least one antigen recognition site, located in the le region of
the immunoglobulin molecule. As used herein, the term “antibody” encompasses not only intact
(i. e., full—length) polyclonal or monoclonal antibodies, but also n—binding fragments
thereof (such as Fab, Fab', F(ab')2, Fv), single chain (scFv), mutants f, fusion proteins
comprising an antibody portion, humanized dies, chimeric antibodies, diabodies, linear
antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies) and any
other ed configuration of the immunoglobulin molecule that comprises an antigen
recognition site of the required specificity, including glycosylation variants of antibodies, amino
acid sequence ts 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 f), 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 ent s.
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., IgGl, IgG2, IgG3, IgG4, IgAl and
IgA2. The heavy—chain constant domains that correspond to the different classes of
immunoglobulins are called alpha, delta, epsilon, gamma, and mu, tively. The subunit
structures and three—dimensional configurations of different classes of immunoglobulins are well
known.
Any of the antibodies described herein can be either monoclonal or polyclonal. A
“monoclonal antibody” refers to a nous antibody population and a lonal 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.
An antibody that “specifically binds” a cleaved HMWK or an epitope thereof is a term
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 on and/or with greater ty 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
affinity, avidity, more readily, and/or with greater duration than it binds to other substances. For
example, an antibody that specifically (or entially) binds to cleaved HMWK or an epitope
therein is an antibody that binds this target n with greater affinity, avidity, more readily,
and/or with greater duration than it binds to other antigens (e.g. intact HMWK or LMWK) or
other es in the same antigen. It is also understood by reading this definition that, for
example, an antibody that specifically binds to a first target antigen may or may not ically
or preferentially bind to a second target antigen. As such, "specific binding" or "preferential
g" does not necessarily require (although it can e) exclusive binding. Generally, but
not necessarily, reference to g means preferential binding.
In some embodiments, the antibodies that specifically binds cleaved HMWK (as well the
other dies that bind both cleaved and intact, and ally LMWK) described herein have
a suitable binding affinity to a cleaved HMWK (or another target antigen as described herein).
As used herein, "binding affinity" refers to the apparent association constant or KA. The KA is
the reciprocal of the dissociation constant (KD). The antibody described herein may have a
binding affinity (KD) of at least 105, 10"), 10”, 10*, 109, 10'10 M, or lower. An increased
binding affinity corresponds to a decreased KD. Higher ty binding of an antibody to a first
target relative to a second target can be indicated by a higher KA (or a smaller cal value
KD) for binding the first target than the KA (or numerical value KD) for binding the second
target. In such cases, the antibody has specificity for the first target (e.g., a protein in a first
conformation or mimic thereof) relative to the second target (e.g., the same protein in a second
conformation or mimic thereof; or a second protein). Differences 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 105 fold. For example, the binding ty of an antibody that
specifically binds a cleaved HMWK as described herein may be 10—fold, 100—fold, —fold,
or 105—fold higher than the binding affinity of that antibody to intact HMWK and/or LMWK.
Binding affinity can be determined by a variety of methods including equilibrium
is, equilibrium g, gel filtration, ELISA, e plasmon resonance, or spectroscopy
(e. g., using a ?uorescence assay). Exemplary conditions for evaluating binding affinity are in
HBS—P buffer (10 mM HEPES pH7.4, 150 mM NaCl, 0.005% (v/v) Surfactant P20). These
techniques can be used to measure the concentration of bound binding protein as a on 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 the
binding protein on the target where (N) is the number of binding sites per target molecule by the
following equation:
[Bound] = [N] [Free]/(Kd+[Free])
It is not always necessary to make an exact determination of KA, though, since
sometimes it is sufficient to obtain a quantitative measurement of affinity, e.g., ined using
a method such as ELISA or FACS analysis, is proportional to KA, and thus can be used for
isons, such as determining Whether a higher affinity is, e.g., 2—fold higher, to obtain a
qualitative measurement of affinity, or to obtain an inference of affinity, e.g., by activity in a
onal assay, e.g., an in vitro or in vivo assay.
In some embodiments, the antibody that specifically binds to cleaved HMWK (also
referred to as an anti—cleaved HMWK antibody) binds to the same epitope of a cleaved HMWK
as 559B—M004—B04. An “epitope” refers to the site on a target antigen that is bound by a
binding protein (e.g., an dy such as a Fab or full length antibody). The site can be entirely
ed of amino acid components, entirely composed of chemical modifications of amino
acids of the n (e.g., glycosyl es), or composed of combinations thereof. Overlapping
epitopes include at least one common amino acid residue, glycosyl group, phosphate group,
sulfate group, or other molecular feature. In some cases, the epitope is ; in other instances,
the epitope is conformational.
A first antibody “binds to the same e” as a second antibody if the first antibody
binds to the same site on a target antigen that the second antibody binds, or binds to a site that
overlaps (e.g., 50%, 60%, 70%, 80%, 90%, or 100% overlap, e.g., in terms of amino acid
sequence or other lar feature (e.g., glycosyl group, phosphate group, or sulfate group)
with the site that the second antigen binds.
In some embodiments, the antibody that specifically binds to cleaved HMWK competes
against 559B—M004—B04 for binding to HMWK. A first antibody “competes for binding” with a
second antibody if the binding of the first antibody to its epitope decreases (e.g., by 10%, 20%,
%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or more) the amount of the second antibody that
binds to its epitope. The competition can be direct (e.g., the first antibody binds to an epitope
that is the same as, or overlaps with, the epitope bound by the second antibody), or indirect (e.g.,
the binding of the first antibody to its e causes a steric change in the target n that
decreases the ability of the second antibody to bind to its epitope).
In some examples, the antibody that specifically binds to cleaved HMWK comprises a
VH chain that includes a VH CDRl, a VH CDR2, and/or a VH CDR3 at least 75% (e.g., 80%,
85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the corresponding VH CDRs of 559B—
04. Alternatively or in addition, the antibody that specifically binds to cleaved HMWK
comprises a VL CDRl, a VL CDR2, and/or a VL CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%,
96%, 97%, 98%, or 99%) identical to the corresponding VL CDRs of 559B—M004—B04. In some
embodiments, the dy that ically binds to cleaved HMWK has the same heavy chain
and/or light chain mentarity determining regions (CDRs) as 004—B04.
“Complementarity determining regions” or “CDRs” are known in the art as referring to
non—contiguous sequences of amino acids within antibody variable regions, which confer
antigen specificity and binding affinity. In general, 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
ce ries 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 Ed. Public Health Service,
National Institutes of , Bethesda, Md. (the Kabat" numbering scheme), Al—Lazikani et al.,
(1997) JMB 273,927—948 (the Chothia" ing scheme), MacCallum et al., J. Mol. Biol.
262:732—745 (1996) (the Contact numbering scheme), Lefranc M P et al., Dev Comp Immunol,
2003 January; 27(1):55—77 (the IMGT numbering scheme), and Honegger A and Pluckthun A, J
Mol Biol, 2001 Jun. 8; 309(3):657—70, (the AHo numbering scheme).
The ries of a given CDR may vary depending on the scheme used for
identification. For example, the Kabat scheme is based structural ents, 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 . Thus,
unless otherwise specified, the term “complementary determining region” or “CDR” of a given
antibody should be understood to encompass the complementary determining region as defined
by any of the known schemes described herein above.
If, determined by the same numbering scheme, an antibody has the same VH and/or VL
CDRs as 559B—M004—B04 (as well as other exemplary antibodies disclosed herein), such an
antibody is deemed as having the same CDRs as 559B—M004—B04 (or the other exemplary
antibodies disclosed herein) and is within the scope of the present disclosure. For example, such
an antibody may have the same VH and/or VL CDRs as clone 559B—M004—B04 as determined by
the Chothia numbering scheme. In another example, an anti—cleaved HMWK dy within
the scope of the t disclosure may have the same VH and/or VL CDRs as clone 559B—
M004—B04, as determined by the Kabat numbering scheme.
Alternatively or in addition, the anti—cleaved HMWK antibody comprises a VH chain at
least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the VH chain of
559B—M004—B04 and/or a VL chain at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%,
or 99%) identical to the VL chain of 559B—M004—B04. In some embodiments, the antibody is
559B—M004-B04.
The “percent identity” of two amino acid ces is determined using the algorithm of
Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264—68, l990, modified as in Karlin and
Altschul Proc. Natl. Acad. Sci. USA 90:5873—77, 1993. Such an algorithm is orated into
the NBLAST and XBLAST ms (version 2.0) of Altschul, er al. J. M01. Biol. 215 :403—10,
1990. BLAST protein searches can be performed with the XBLAST program, 50,
wordlength=3 to obtain amino acid sequences homologous to the protein molecules of interest.
Where gaps exist between two sequences, Gapped BLAST can be utilized as bed in
Altschul et al., Nucleic Acids Res. 25(17):3389—3402, 1997. When utilizing BLAST and
Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST
and NBLAST) can be used.
The sequences of the heavy chain variable region and the light chain variable region of
559B—M004—B04 are shown below. Heavy chain CDRl, CDR2, and CDR3 sequences and light
chain CDRl, CDR2, and CDR3 ces are underlined and in boldface ified by one
scheme as an example).
>559B—R0048-A01 (559B—M0004—B04) Heavy Chain Amino Acid Sequence (SEQ ID NO: 4)
ZVQT. T.LuSGGGTVQPGGSLRLSCAASGFTF—SFYVMVWVRQAPGKGL_LWVSGISPSGGNTAYADSV
KGRFT: SRDNSKNTLYLQMNSLRALDTAVYYCARKLFYYDDTKGYFDFWGQGTLVTVSS
>559B—R0048—A01 (559B—M0004—B04) Light Chain Amino Acid Sequence (SEQ ID NO: 5)
QYL‘LIQBPSASGIPGQRVILSCSGSSSNIGSNYVYWYQQLPGTAPKLL_Y_RNNQRPSGVPDRFS
GSKSGTSAST.A SGT.QS Li)LADYYCAAWDDSLNGRVFGGGTKLTVL
In some instances, the antibody that specifically binds a cleaved HMWK may contain
one or more (e.g., up to 5, up to 3, or up to l) conservative mutations in one or more of the
heavy chain CDRs, or one or more of the light chain CDRs in 559B—M0004—B04, e.g., at
positions where the residues are not likely to be involved in interacting with the cleaved
HMWK. As used herein, a “conservative amino acid substitution” refers to an amino acid
substitution that does not alter the relative charge or size characteristics of the protein in which
the amino acid substitution is made. Variants can be prepared according to methods for altering
polypeptide sequence known to one of ordinary skill in the art such as are found in references
which compile such s, e.g. Molecular Cloning: A Laboratory Manual, J. Sambrook, et
al., eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York,
1989, or Current ols in Molecular Biology, F.M. Ausubel, et al., eds., John Wiley &
Sons, Inc., New York. vative substitutions of amino acids include substitutions made
amongst amino acids within the following : (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.
Antibodies capable of binding to cleaved HMWK (as well as antibodies capable of
binding to intact HMWK and/or LMWK) as described herein can be made by any method
known in the art. See, for example, Harlow and Lane, (1988) Antibodies: A Laboratory Manual,
Cold Spring Harbor Laboratory, New York.
In some embodiments, antibodies specific to a target antigen (a cleaved HMWK, the
intact HMWK, and/or LMWK) can be made by the conventional hybridoma technology. The
full—length target antigen or a fragment thereof, optionally coupled to a carrier protein such as
KLH, can be used to immunize a host animal for generating dies binding to that n.
The route and schedule of immunization of the host animal are generally in keeping with
ished and conventional techniques for antibody stimulation and production, as further
described . General techniques for production of mouse, humanized, and human
antibodies are known in the art and are described herein. It is contemplated that any mammalian
subject including humans or antibody producing cells therefrom can be manipulated to serve as
the basis for production of mammalian, including human hybridoma cell lines. Typically, the
host animal is inoculated intraperitoneally, uscularly, orally, subcutaneously, intraplantar,
and/or intradermally with an amount of immunogen, including as described herein.
Hybridomas can be prepared from the lymphocytes and immortalized myeloma cells
using the general somatic cell hybridization technique of Kohler, B. and Milstein, C. (1975)
Nature 5—497 or as modified by Buck, D. W., et al., In Vitro, 18:377—381 (1982).
Available myeloma lines, including but not limited to X63—Ag8.653 and those from the Salk
Institute, Cell Distribution Center, San Diego, Calif., USA, may be used in the hybridization.
Generally, the technique involves fusing myeloma cells and lymphoid cells using a fusogen such
as polyethylene glycol, or by electrical means well known to those skilled in the art. After the
fusion, the cells are separated from the fusion medium and grown in a selective growth medium,
such as hypoxanthine—aminopterin—thymidine (HAT) medium, to eliminate unhybridized parent
cells. Any of the media described , supplemented with or without serum, can be used for
culturing omas that secrete monoclonal antibodies. As another alternative to the cell
fusion technique, EBV alized B cells may be used to produce the anti—PKal monoclonal
antibodies described herein. The hybridomas are ed and subcloned, if d, and
supernatants are d for anti—immunogen activity by conventional immunoassay ures
(e. g., radioimmunoassay, enzyme immunoassay, or fluorescence immunoassay).
Hybridomas that may be used as source of antibodies encompass all derivatives, progeny
cells of the parent hybridomas that produce monoclonal antibodies capable of interfering with
the PKal activity. Hybridomas that produce such antibodies may be grown in vitro or in vivo
using known procedures. The monoclonal antibodies may be isolated from the culture media or
body ?uids, by conventional globulin purification ures such as ammonium sulfate
precipitation, gel electrophoresis, dialysis, chromatography, and ultrafiltration, if desired.
Undesired activity if present, can be removed, for example, by running the ation over
adsorbents made of the immunogen attached to a solid phase and eluting or releasing the desired
antibodies off the immunogen. Immunization of a host animal with a target antigen or a
fragment containing the target amino acid sequence conjugated to a protein that is immunogenic
in the species to be zed, e.g., e limpet hemocyanin, serum albumin, bovine
thyroglobulin, or soybean trypsin inhibitor using a tional or derivatizing agent, for
example maleimidobenzoyl uccinimide ester (conjugation through cysteine residues), N—
hydroxysuccinimide (through lysine residues), glutaraldehyde, succinic anhydride, SOCl, or
RlN=C=NR, Where R and R1 are different alkyl groups, can yield a population of antibodies
(e. g., monoclonal antibodies).
If desired, an antibody (monoclonal or polyclonal) of interest (e.g., produced by a
oma) may be sequenced and the polynucleotide sequence may then be cloned into a vector
for expression or propagation. The sequence ng the antibody of interest may be
maintained in vector in a host cell and the host cell can then be expanded and frozen for future
use. In an alternative, the cleotide sequence may be used for genetic manipulation to
improve the affinity (affinity maturation), or other characteristics of the antibody. It may be
desirable to genetically manipulate the antibody sequence to obtain r ty and/or
specificity to the target antigen. It Will be apparent to one of skill 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.
In other embodiments, fully human antibodies can be obtained by using commercially
available mice that have been engineered to express ic human globulin proteins.
Transgenic animals that are designed to produce a more desirable (e.g., fully human antibodies)
or more robust immune response may also be used for generation of humanized or human
antibodies. Examples of such technology are XenomouseRTM from Amgen, Inc. (Fremont,
Calif.) and HuMAb—MouseRTM and TC MouseTM from Medarex, Inc. (Princeton, NJ.) In
r alternative, antibodies may be made recombinantly by phage display or yeast
technology. See, for e, US. Pat. Nos. 5,565,332; 5,580,717; 743; and 6,265,150;
and Winter et al., (1994) Annu. Rev. Irnmunol. 12:433—455, and . Alternatively, the 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.
Antigen—binding fragments of an intact antibody (full—length antibody) can be prepared
via routine methods. For example, F(ab')2 fragments can be produced by pepsin ion of an
antibody molecule, and Fab fragments that can be generated by reducing the disulfide bridges of
F(ab')2 fragments.
A single—chain antibody can be ed via recombinant technology by linking a
nucleotide sequence coding for a heavy chain variable region and a nucleotide sequence coding
for a light chain le region. Preferably, a ?exible linker is incorporated between the two
le regions. Alternatively, techniques described for the tion of single chain
dies (U.S. Patent Nos. 778 and 692) can be adapted to produce a phage or
yeast scFv library and scFv clones specific to a PKal can be identified from the library following
routine procedures. Positive clones can be subjected to further screening to identify those that
specifically bind a target antigen, such as a cleaved HMWK.
In some embodiments, the antibodies specific to a cleaved HMWK (or to intact HMWK
or LMWK) may be isolated from an antibody library, which may 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) following routine practice. A synthetic antibody library refers to a library the
members of which are designed following predetermined rules (e.g., having a complete
randomized CDR region such as CDRs or a semi randomized CDR region such as CDRl or
CDR2 of the heavy chain, the light chain, or both).
In some instances, the antibody library is a display library (e.g., a phage y y
or a yeast display library). A display y is a collection of es; each entity includes an
accessible polypeptide component and a recoverable component that encodes or identifies the
polypeptide component. The polypeptide component is varied so that different amino acid
sequences are represented. The polypeptide component can be of any length, e.g., from three
amino acids to over 300 amino acids. A display library entity can include more than one
polypeptide component, for example, the two ptide chains of a sFab. In one ary
implementation, a display library can be used to identify proteins that bind to a cleaved HMWK
(as well as other target antigens described herein). In a ion, the polypeptide component of
each member of the library is probed with a cleaved HMWK (or a fragment thereof) and if the
polypeptide component binds to the cleaved HMWK, the y library member is identified,
typically by retention on a support. An exemplary illustration for identifying antibodies specific
to cleaved HMWK using a phage display dy library is provided in Figure 12.
Retained display library members are recovered from the support and analyzed. The
analysis can e amplification and a subsequent selection under similar or dissimilar
conditions. For example, positive and negative selections can be alternated. The analysis can
also include determining the amino acid sequence of the polypeptide component and purification
of the polypeptide component for ed terization.
Antibodies obtained following a method known in the art and described herein can be
terized 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 es on proteins, including solving the
crystal structure of an antibody—antigen complex, competition assays, gene fragment expression
assays, and tic peptide—based assays, as described, for example, in Chapter ll of Harlow
and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold
Spring Harbor, N.Y., 1999. In an additional example, e g can be used to determine
the sequence to which an antibody binds. The epitope can be a linear epitope, i.e., contained in a
single stretch of amino acids, or a conformational epitope formed by a three—dimensional
interaction of amino acids that may not necessarily be contained in a single stretch (primary
ure linear sequence). Peptides of varying lengths (e.g., at least 4—6 amino acids long) can
be isolated or sized (e.g., inantly) and used for binding assays with an antibody. In
another example, the epitope to which the antibody binds can be determined in a systematic
screening by using overlapping peptides derived from the target antigen sequence and
determining binding by the dy. According to the gene fragment expression assays, the
open reading frame encoding the target antigen is fragmented either randomly or by specific
genetic constructions and the reactivity of the sed fragments of the antigen with the
antibody to be tested is determined. The gene nts may, for example, be produced by PCR
and then transcribed and translated into protein in vitro, in the presence of radioactive amino
acids. The binding of the antibody to the radioactively d antigen fragments is then
determined by precipitation and gel electrophoresis.
Certain epitopes can also be identified by using large libraries of random peptide
sequences displayed on the surface of phage particles (phage libraries). Alternatively, a defined
library of overlapping peptide fragments can be tested for binding to the test antibody in simple
binding assays. In an additional example, mutagenesis of an n binding domain, domain
swapping experiments and alanine scanning nesis can be performed to fy residues
required, sufficient, and/or necessary for epitope g. For example, domain swapping
experiments can be performed using a mutant of a target antigen in which various fragments of
the HMWK polypeptide have been replaced (swapped) with sequences from a closely related,
but antigenically distinct protein. By assessing binding of the antibody to the mutant HMWK,
the ance of the particular antigen fragment to antibody binding can be assessed.
Alternatively, competition assays can be performed using other antibodies known to bind
to the same antigen to determine whether an antibody binds to the same epitope as the other
antibodies. Competition assays are well known to those of skill in the art.
Any of the anti—cleaved HMWK antibodies is also within the scope of the present
disclosure.
(iii)Immzmoassays
Provided herein are immunoassays for detecting a cleaved HMWK. As used herein,
the term “immunoassay” may be referred to hangeably as an —based assay or
immuno—based assay. In general, an immunoassay detects the presence and/or concentration
(level) of a molecule (e.g., HMWK), in a sample using an agent that binds to the molecule, such
as an antibody. Examples of immunoassays include Western blots, enzyme linked
immunosorbent assays s), lateral flow assay, radioimmunoassays,
ochemiluminescence—based detection assays, magnetic immunoassays, and related
ques. In some embodiments, the immunoassay is an ELISA assay. In some embodiments,
the immunoassay is a sandwich ELISA assay. In some embodiments, the assay is a
lateral ?ow assay.
ELISAs are 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 ELISAs are also known in the art
and cially available (see, e.g., ELISA kits from Life Technologies and BD Biosciences).
To perform the immunoassay bed herein, a sample may be obtained from a
subject. As used herein, a “sample” refers to a composition that comprises tissue, e.g., blood,
plasma or protein, from a subject. A sample includes both an initial unprocessed sample taken
from a t as well as subsequently processed, e.g., partially purified or preserved forms.
Exemplary samples include blood, plasma, tears, or mucus. In some embodiments, the sample is
a body ?uid sample such as a serum or plasma sample. A sample to be analyzed by the
immunoassay described herein can be either an initial unprocessed sample taken from a subject
or subsequently processed, e.g., partially purified or preserved forms. In some ments,
multiple (e.g., at least 2, 3, 4, 5, or more) samples may be collected from the subject, over time
or at particular time intervals, for example to assess the progression of a disease or disorder or
evaluate the efficacy of a treatment. The multiple samples may be obtained before and after a
treatment, or during the course of a treatment.
A sample can be ed from a subject using any means known in the art. In some
embodiments, the sample is obtained from the subject by collecting the sample (e.g., a blood
sample) into an ted collection tube (e.g., an evacuated blood collection tube). In some
ments, the evacuated collection tube contains one or more protease inhibitors, for
e, to reduce or prevent ex vivo activation of the contact system during sample collection.
Such protease tors may be contained in a liquid formulation. In some embodiments, the
protease inhibitors comprise at least one serine protease inhibitor and at least one cysteine
protease inhibitor. Such evacuated tion tubes are known in the art. See, for example, PCT
Application No. US2016/046681. Optionally, an evacuated blood collection tube may further
comprise one or more anti—coagulants.
A “patient,:9 6‘subject” or “host” (these terms are used interchangeably) to be d by
the subject method may mean either a human or non—human animal. In some embodiments, a
subject is suspected of or is at risk for or s from a kallikrein—mediated disorder, e.g., a
bradykinin—mediated disorder, such as hereditary angioedema (HAE), non—histamine—dependent
idiopathic angioedema, rheumatoid arthritis, Crohn’s e, lupus, Alzheimer’s e, septic
shock, burn injury, brain ischemia/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 ry heart disease with unstable angina pectoris,
edema, eye disease, gout, intestinal bowel disease, oral mucositis, neuropathic pain,
in?ammatory pain, spinal stenosis—degenerative spine disease, post operative ileus, aortic
aneurysm, osteoarthritis, hereditary angioedema, pulmonary sm, stroke, head trauma or
peri—tumor brain edema, sepsis, acute middle cerebral artery (MCA) ischemic event (stroke),
osis (e.g., after angioplasty), systemic lupus erythematosis nephritis, an autoimmune
disease, an in?ammatory disease, a cardiovascular disease, a neurological disease, a disease
associated with protein misfolding, a disease associated with angiogenesis, hypertensive
nephropathy and diabetic nephropathy, allergic and respiratory diseases (e.g., anaphylaxis,
asthma, chronic obstructive pulmonary disease, acute atory distress me, cystic
fibrosis, persistent, rhinitis) and tissue injuries (e.g., burn or chemical injury).
Alternatively or in addition, the subject who needs the analysis described herein may
be a patient of the disease or disorder. Such a subject may be under the attack of the disease
(e.g., HAE) currently, or may suffer from the e in the past (e.g., during disease quiescence
currently). In some examples, the subject is a human t who may be on a treatment of the
disease, for example, a ent ing a Cl esterase tor (Cl—INH), a plasma kallikrein
inhibitor, or a inin inhibitor. In other instances, such a human patient may be free of such
a treatment.
The sample described herein can be subject to analysis using an agent that specifically
binds a cleaved HMWK to determine the level of the cleaved HMWK in the sample. In some
embodiments, the immunoassays described herein may in the format of a sandwich ELISA, in
which a first agent (e.g., the antibody bed herein) that specifically binds the cleaved
HMWK is immobilized on a support member. The support member can then be incubated with
a sample as described herein for a suitable period of time under conditions that allow for the
formation of cleaved HMWK/first agent (e.g., antibody) complex. Such a complex can then be
detected using a second agent that binds HMWK. The second agent can be conjugated to a
label, which can release a signal directly or ctly. The intensity of the signal represents the
level of the cleaved HMWK in the sample.
Any support member known in the art may be used in the method, including but not
limited to a membrane, a bead, a slide, or a multi—well plate. Selection of an appropriate support
member for the assay will depend on various factor such as the number of samples and
method of detecting the signal released from label conjugated to the second agent.
In some embodiments, the support member is a membrane, such as a ellulose
membrane, a polyvinylidene ?uoride (PVDF) membrane, or a cellulose acetate ne. In
some examples, the immunoassay may be in a Western blot assay format or a lateral ?ow assay
format.
In some embodiments, the support member is a multi—well plate, such as an ELISA plate.
In some embodiments, the immunoassays bed herein can be carried out on high
throughput platforms. In some embodiments, multi—well plates, e.g., 24—, 48—, 96—, 384— or
r well plates, may be used for high hput assays. Individual immunoassays
can be carried out in each well in parallel. Therefore, it is generally desirable to use a plate
reader to measure multiple wells in parallel to increase assay throughput. In some embodiments,
plate readers that are capable of imaging multi—wells (e.g., 4, 16, 24, 48, 96, 384, or greater
wells) in parallel can be used for this platform. For example, a commercially available plate
reader (e.g., the plate::vision system available from Perkin Elmer, Waltham, MA) may be used.
This plate reader is capable of kinetic—based ?uorescence analysis. The plate::vision system has
high tion efficiency optics and has special optics designed for the analysis of 96 wells in
parallel. Additional suitable parallel plate readers include but are not limited to the SAFIRE
(Tecan, San Jose, CA), the FLIPRTETRA® ular Devices, Union City, CA), the
FDSS7000 (Hamamatsu, Bridgewater, NJ), and the CellLux (Perkin Elmer, m, MA).
As bed in Example 1, it was unexpectedly discovered that the surface area and/or
volume of the wells of the multi—well plate may affect the results of the immunoassay. In some
embodiments, the bed immunoassays are med in 96—well plates, such as a 96—well
ELISA plate.
In other embodiments, high—throughput screening immunoassays of the present
disclosure can be automated (e.g., adapted to robotic assays).
In some embodiments, the immunoassays may be performed on low—throughput
platforms, including single immunoassay format. For example, a low—throughput platform may
be used to measure the ce and amount of cleaved HMWK in biological s (e.g.,
biological tissues, tissue extracts) for diagnostic methods, monitoring of disease and/or treatment
progression, and/or predicting whether a disease or disorder may t from a particular
treatment.
Any method known in the art can be used to immobilize an agent that specifically binds
a d HMWK such as the antibodies described herein onto a support member as also
described herein. In some embodiments, the immobilization involves binding the agent (e.g.,
the antibody) to the support member. In other embodiments, the immobilization involves
ing the antibody to the support member. Such adsorption methods may be performed, for
example, by incubating the antibody in a buffer in the wells of a multi—well plate. In some
embodiments, the agent such as the antibody is provided in a coating buffer and ted in the
wells of a multi—well plate. Coating buffers will be evident to one of skill in the art and may be
prepared or ed from a commercial source. Non—limiting examples of coating buffers
include 50 mM sodium bicarbonate, pH 9.6; 0.2 M sodium bicarbonate, pH 9.4; phosphate
buffered solution (50 mM phosphate, pH 8.0, 0.15 M NaCl); carbonate—bicarbonate solution;
and TBS (50 mM TRIS, pH 8.0, 0.15 M NaCl).
In some embodiments, the first agent is immobilized on the support member by
hydrophobic interactions between the first agent and the support member. In some
embodiments, the first agent is immobilized on the support member using electrophoretic
Either before or after immobilization, or both, the support member may be ted
with a blocking buffer. In general, blocking buffers are used to block any of the exposed surface
of the support ne (e.g., sites on the support ne unoccupied by the first agent).
Use of a blocking buffer may reduce the baseline signal detected (i. 6., “background
interference”) and/or improve the sensitivity of the immunoassay and/or reduce non—specific
binding of ents of the sample to the support membrane. As described in Example 1,
selection of the blocking buffer affected the results of the assay. 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 (e.g., 2% BSA in PBS
buffer). In some embodiments, the blocking buffer is free from serum albumin, such as bovine
serum albumin or human serum albumin. In some ments, the blocking buffer comprises
casein fragments, and ally NaCl and Tween and may have a pH 7.0—7.4. In some
embodiments, the casein fragments are high purity casein fragments. Such a blocking buffer
may be prepared or obtained from a commercial source (e.g., The Blocking Solution LowCross
from CANDOR Bioscience).
The support member, on which the agent specific to a cleaved HMWK is attached, can
be brought in contact (incubated) with a sample as described herein, which is ted of
containing the cleaved HMWK. In general, the term “contact” refers to an re of the
support member with the biological sample or agent for a suitable period sufficient for the
formation of complexes n the agent, such as an antibody, and the cleaved HMWK in the
sample, if any. Afterwards, the sample may be removed from the support , which can
then be washed for multiple times to remove any unbound cleaved HMWK. In some
embodiments, the contacting is performed by capillary action in which a biological sample or
agent is moved across a surface of the support membrane.
The support member can then be incubated with a second agent that binds HMWK for a
suitable period allowing for the binding of the second agent to HMWK attached to the support
member.
The second agent can be any agent capable of binding to HMWK, such as an antibody
capable of binding to HMWK (either specific to the cleaved form of HMWK or can cross react
to both the cleaved HMWK and intact HMWK). In some ments, the second agent
comprises one or more antibodies that bind HWMK ed and/or intact). In some
embodiments, the antibody is a mouse monoclonal antibody or a monoclonal sheep antibody. It
is conjugated with a label, which is a compound capable of releasing a signal either directly or
indirectly (e.g., via interaction with one or more onal compounds).
In some embodiments, the label is a signal releasing agent, which is an agent that either
directly releases a signal (e.g., a dye or ?uorophore) or releases a signal upon interacting with a
substrate (e.g., an enzyme such as HRP or B—galactosidase, which can convert a colorless
substrate to a colored product). As used herein, the term “?uorophore” (also referred to as
“?uorescent label” or “?uorescent dye”) refers to moieties that absorb light energy at a defined
excitation wavelength and emit light energy at a different wavelength.
In other embodiments, the label can be a member of a receptor—ligand pair. As used
herein, a “ligand—receptor pair” refers to a pair of molecules (e.g., biological molecules) that
have a specific affinity for each other, e.g., —streptavidin. In this case, the support member
carrying the first agent—cleaved HMWK—second agent may be further incubated with the other
member of the —receptor pair for a suitable period such that the two members of the
or—ligand pair ct. The other member of the or—ligand pair is conjugated with a
signal releasing agent as described herein. In one example, the second agent is conjugated to
biotin and HRP—conjugated streptavidin is used for detection.
After washing away any unbound conjugate, a substrate on may be added to aid in
detection. For example, after a set interval, the reaction may be d (e.g., by adding 1 N
NaOH) and the concentration of colored product formed may be measured in a
spectrophotometer. The intensity of color is proportional to the concentration of bound antigen.
Next, the signal released from the label as described herein can be detected/measured by
routine methodology, which would depend on the specific format of an immunoassay and the
signal releasing agent used therein. As used herein, the terms “measuring” or “measurement,”
or alternatively “detecting” or “detection,” means assessing the presence, absence, quantity or
amount (which can be an effective amount) of a substance within a sample, including the
tion of ative or quantitative concentration levels of such nces, or otherwise
evaluating the values or categorization of a subject.
, e.g., Western blot assays, may further involve use of a quantitative imaging
system, e.g., LICOR imaging technology, which is commercially ble (see, e.g., the
Odyssey® CLX infrared imaging system from LI—COR Biosciences). In some embodiments, an
electrochemiluminescence detection assay or an assay relying on a combination of
electrochemiluminescence and patterned array logy is used (e.g., an ECL or MULTI—
ARRAY technology assay from Meso Scale Discovery (MSD)).
Any of the immunoassays described herein, e.g., one or more steps of the immunoassays,
may be carried out in a suitable assay buffer, which will be evident to one of skill in the art. In
some embodiments, the assay buffer contains or has been supplemented with ZnClz. In some
embodiments, the assay buffer contains at least about 10 uM, 20 uM, 30 uM, 40 uM, 50 uM, 60
uM, 70 uM, 80 uM, 90 uM, 100 uM, 150 uM, 200 uM, 250 uM, 300 uM, 350 uM, 400 uM,
450 uM, 500 uM or more ZnClz. In some embodiments, such a ZnClz—containing assay buffer is
used in the step in which the agent specific to cleaved HMWK (e.g., an antibody specific to
cleaved HMWK) binds a cleaved HMWK. ZnClz es the binding activity of the agent
(e.g., antibody) to the cleaved HMWK.
In some embodiments, the assay buffer contains serum albumin, such as bovine serum
albumin or human serum albumin. In some embodiments, the assay buffer ns 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%,
, 0.16%, 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 of a
surfactant. In one example, the assay buffer contains 0. 1% BSA and 0.05% Tween—20 in PBS.
(iv) Diagnostic and Prognostic Applications
The assay methods and kits described herein can be applied for evaluation of a e or
er associated with plasma kallikrein, such as those described herein (e.g., HAE), given the
correlation between the level of cleaved HMWK and such diseases or disorders (6.g. as a
biomarker). Alternatively or in addition, the assay methods and kits described herein may be
used to monitor the progress of such a disease, assess the efficacy of a treatment for the disease,
identify patients suitable for a ular treatment, and/or predict disease status (6.g. , attack
versus quiescence) in a subject.
In some embodiments, the level of cleaved HMWK determined by the immunoassay
described herein can be relied on to evaluate whether a subject (e.g., a human t) from
whom the biological sample is ed, has or is at risk for a disease or disorder associated with
plasma kallikrein, such as HAE or autoimmune disease such as RA, UC, and Crohn’s disease).
The level of cleaved kininogen can then be compared with either the intact kininogen or the total
amount of kininogen in the sample to determine a value (e.g., percentage) of cleaved kininogen,
a value of intact kininogen, or both, in the sample. The value of cleaved kininogen and/or intact
kininogen can be compared to a reference value to determine whether the subject has or is at risk
for the PKal—mediated disorder, e.g., HAE or an autoimmune disease, such as RA, UC, and
Crohn’s disease. For example, if the percentage of cleaved gen is at or higher than a
reference , the subject can be identified as having or at risk for a pKal—mediated disorder
such as HAE, RA, UC, and Crohn’s disease. Alternatively, if the percentage of intact kininogen
is at or lower than a reference number, the subject can be identified as having or at risk for a
pKal—mediated disorder such as HAE, RA, UC, and Crohn’s disease.
In some embodiments, the sample for analysis of the methods described herein is
derived from a human subject who has or is at risk of having hereditary angioedema (HAE).
HAE is also known as "Quincke edema," Cl esterase inhibitor deficiency, Cl tor
deficiency, and tary angioneurotic edema (HANE). HAE is characterized by recurrent
episodes of severe swelling (angioedema), which can affect, e.g., the limbs, face, genitals,
intestinal tract, and airway. Symptoms of HAE include, e.g., swelling in the arms,
legs, lips, eyes, tongue, and/or ; airway ge that can involve throat swelling and
sudden hoarseness; repeat es of abdominal cramping without obvious cause; and/or
swelling of the ines, which can be severe and can lead to abdominal cramping,
vomiting, dehydration, diarrhea, pain, and/or shock. About one—third of individuals with this
HAE develop a non—itchy rash called erythema marginatum during an attack.
Swelling of the airway can be life threatening and causes death in some ts.
Mortality rates are ted at 15—33%. HAE leads to about 15,000—30,000 emergency
department visits per year.
Trauma or stress, e.g., dental procedures, sickness (e.g., viral illnesses such as colds
and the ?u), menstruation, and surgery can trigger an attack of angioedema. To prevent
acute attacks of HAE, patients can attempt to avoid specific stimuli that have previously
caused attacks. However, in many cases, an attack occurs without a known trigger.
Typically, HAE ms first appear in childhood and worsen during puberty. On
average, untreated individuals have an attack every 1 to 2 weeks, and most episodes last for
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.
There are three types of HAE, known as types I, II, and III. It is estimated that HAE
affects l in 50,000 people, that type I accounts for about 85 percent of cases, type II
accounts for about 15 percent of cases, and type III is very rare. Type III is the most newly
described form and was originally t to occur only in women, but families with
affected males have been identified.
HAE is inherited in an autosomal dominant pattern, such that an ed person can
inherit the mutation from one affected parent. New mutations in the gene can also occur,
and thus HAE can also occur in people with no history of the disorder in their . It is
estimated that 20—25% of cases result from a new spontaneous mutation.
Mutations in the SERPINGl gene cause hereditary dema type I and type II.
The Gl gene provides instructions for making the Cl inhibitor protein, which is
important for controlling in?ammation. Cl inhibitor blocks the activity of certain proteins
that e in?ammation. Mutations that cause hereditary angioedema type I lead to
reduced levels of Cl inhibitor in the blood. In st, mutations that cause type II result in
the production of a Cl inhibitor that functions abnormally. Without the proper levels of
functional Cl inhibitor, excessive amounts of bradykinin are generated. Bradykinin
promotes in?ammation by sing the leakage of ?uid through the walls of blood vessels
into body tissues. Excessive accumulation of ?uids in body tissues causes the episodes of
swelling seen in individuals with tary angioedema type I and type II.
Mutations in the F12 gene are ated with some cases of hereditary angioedema
type III. The F12 gene provides instructions for making coagulation factor XII. In addition
to playing a critical role in blood clotting (coagulation), factor XII is also an ant
stimulator of in?ammation 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 generated and blood vessel walls become more leaky, which leads
to episodes of swelling. The cause of other cases of hereditary dema type III remains
unknown. Mutations in one or more as—yet unidentified genes may be responsible for the
disorder in these cases.
HAE can present similarly to other forms of angioedema resulting from allergies or
other medical conditions, but it differs significantly in cause and treatment. When HAE is
misdiagnosed as an allergy, it is most commonly treated with antihistamines, steroids, and/or
epinephrine, which are typically ineffective in HAE, although epinephrine can be used for
life—threatening reactions. Misdiagnoses have also ed in unnecessary exploratory
y for ts with abdominal swelling, and in some HAE patients abdominal pain has
been incorrectly diagnosed as psychosomatic.
Cl inhibitor therapies, as well as other therapies for HAE, are described in Kaplan,
A.P., JAllergy Clin Immunol, 2010, l26(5):918—925.
Acute treatment of HAE attacks is provided to halt ssion of the edema as
quickly as possible. Cl inhibitor concentrate from donor blood, which is stered
intravenously, is one acute ent; r, this treatment is not available in many
countries. In emergency situations where Cl tor concentrate is not available, fresh
frozen plasma (FFP) can be used as an alternative, as it also contains Cl inhibitor.
Purified Cl inhibitor, derived from human blood, has been used in Europe since
1979. Several Cl inhibitor treatments are now available in the U.S. and two Cl inhibitor
products are now available in Canada. Berinert P (CSL Behring), which is pasteurized, was
approved by the FDA. in 2009 for acute attacks. CINRYZE®, which is nanofiltered, was
ed by the FDA. in 2008 for prophylaxis. Rhucin/Ruconest (Pharming) is a
recombinant Cl inhibitor under pment that does not carry the risk of infectious
disease transmission due to human blood—borne pathogens.
Treatment of an acute HAE attack also can include medications for pain relief and/or
IV ?uids.
Other treatment modalities can stimulate the synthesis of Cl inhibitor, or reduce Cl
inhibitor consumption. Androgen medications, such as danazol, can reduce the frequency
and severity of attacks by stimulating production of Cl inhibitor.
Helicobacter pylori can trigger abdominal attacks. Antibiotics to treat H. pylori will
decrease abdominal attacks.
Newer ents attack the contact cascade. Ecallantide (KALBITOR®) inhibits
plasma kallikrein and has been approved in the U.S.. ant (FIRAZYR®, Shire) inhibits
the bradykinin B2 receptor, and has been approved in Europe and the U.S.
Diagnosis of HAE can rely on, e.g., family history and/or blood tests. Laboratory
findings associated with HAE types I, II, and III are described, e.g., in Kaplan, A.P., J
Allergy Clin Immunol, 2010, l26(5):918—925. In type I HAE, the level of Cl inhibitor is
decreased, as is the level of C4, whereas Clq level is normal. In type II HAE, the level of
Cl tor is normal or increased; however, Cl inhibitor function is abnormal. C4 level is
decreased and Clq level is . In type III, the levels of Cl inhibitor, C4, and Clq can
all be normal. The present disclosure is based, at least in part, on the identification of
additional proteins that have differential levels in samples from HAE patients as compared
to healthy individuals (Table 1). Measuring the level or presence of 2—HMWK can be used
to identify whether a subject has a e, such as HAE. In some embodiments, the
methods may be used to ine r a patient has had or is having an HAE attack.
Symptoms of HAE can be assessed, for example, using questionnaires, e.g.,
questionnaires that are completed by patients, ians, or family members. Such
questionnaires are known in the art and include, for example, visual analog . See, e.g.,
McMillan, C.V. et al. Patient. 2012;5(2):ll3—26.
The value of cleaved kininogen and/or intact kininogen detected in a sample from a
subject can be compared to a reference value to determine whether the subject has or is at risk
for the PKal—mediated disorder (6.g.
, HAE). Alternatively or in addition, the level of the cleaved
kininogen and/or intact kininogen ed in a sample from the subject can be compared to a
nce value to assess the efficacy of a treatment for the disorder, the prognosis or severity of
the er, and/or identifying a subject as a candidate for treatment.
The reference value can be a control level of cleaved kininogen percentage. In some
embodiments, the control level is the percentage of cleaved kininogen in a control sample, such
as a sample (e.g., blood or plasma sample) obtained from a healthy subject or population of
healthy subjects, which preferably are of the same species as the candidate subject. As used
herein, a healthy subject is a subject that is apparently free of the target e (e.g., a PKal—
mediated disorder such as HAE or autoimmune diseases such as RA, US, and Crohn’s disease)
at the time the level of cleaved and/or intact kininogen is measured or has no history of the
disease.
The control level can also be a predetermined level or threshold. Such a predetermined
level can represent the tage of cleaved gen in a population of subjects that do not
have or are not at risk for the target disease. It can also represent the percentage of cleaved
kininogen in a population of subjects that have the target disease.
The predetermined level can take a variety of forms. For example, it can be single cut—
off value, such as a median or mean. In some embodiments, such a predetermined level can be
established based upon comparative groups, such as where one d group 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, for example, a range enting the percentages of
cleaved kininogen in a control population Within a predetermined tile.
The control level as described herein can be determined by routine technology. In some
examples, the control level can be obtained by performing a conventional method (e.g., the same
assay for obtaining the level of d and/or intact kininogen in a test sample as described
herein) on a control sample as also described herein. In other examples, levels of cleaved and/or
intact gen can be obtained from s of a control population and the results can be
analyzed by, e.g., a ational program, to obtain the l level (a predetermined level)
that represents the level of cleaved and/or intact kininogen in the control population.
By comparing the percentage of d kininogen in a sample obtained from a
candidate subject to the reference value as described herein, it can be determined as to Whether
the candidate subject has or is at risk for the PKal—mediated disease (e.g., HAE or an
mune disease such as RA, UC, and Crohn’s disease). For e, if the percentage of
cleaved kininogen in a sample of the candidate subject deviates from the reference value (e.g.,
increased as compared to the reference value or decreased as compared to the reference value),
the candidate subject might be identified as having or at risk for the disease. When the reference
value represents represent the percentage range of cleaved kininogen in a population of subjects
that have the target disease, the percentage of d kininogen in a sample of a candidate
falling in the range indicates that the candidate t has or is at risk for the target disease. In
some instances, a reference value may represent a background level indicating e of
cleaved kininogen. Presence of cleaved kininogen is deemed as a deviation from such a
background reference value. As used herein, a “deviation from” a control sample or reference
value asses levels of cleaved HMWK as well as the presence or absence of cleaved
HMWK in the sample.
As used herein, “an ed level or a level above a reference value” means that the
level/percentage of cleaved kininogen is higher than a reference value, such as a pre—determined
threshold of a level/percentage of cleaved kininogen in a control sample. Control levels are
described in detail herein.
An elevated percentage of cleaved kininogen includes a cleaved kininogen percentag
that is, for example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%,
200%, 300%, 400%, 500% or more above a reference value. An elevated tage of cleaved
kininogen also includes increasing a phenomenon from a zero state (e.g., no or undetectable
cleaved kininogen and/or intact kininogen that binds to a capture reagent in a sample) to a non—
zero state (e.g., some or detectable cleaved kininogen and/or intact kininogen).
As used , “a decreased percentage/level or a percentage/level below a reference
value” means that the percentage/level of cleaved is lower than a reference value, such as a pre—
determined threshold of cleaved kininogen in a control sample. l levels are described in
detail herein.
An decreased level of d kininogen includes a cleaved kininogen that is, for
example, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,
300%, 400%, 500% or more lower than a reference value. A decreased level of cleaved
kininogen that binds to a e reagent also includes decreasing a phenomenon from a non—
zero state (e.g., some or detectable cleaved kininogen in a ) to a zero state (e.g., no or
undetectable cleaved kininogen in a sample).
In some embodiments, the candidate subject is a human patient having a symptom of a
pKal—mediated disorder, e.g., such as HAE or an autoimmune disease such as RA, UC, and
Crohn’s disease. For example, the subject has edema, swelling wherein said swelling is
completely or predominantly peripheral; hives; redness, pain, and swelling in the absence of
evidence of infection; non—histamine—mediated edema, recurrent attacks of ng, or a
combination thereof. In other embodiments, the subject has no symptom of a pKal—mediated
er at the time the sample is collected, has no history of a symptom of a pKal—mediated
disorder, or no history of a ediated disorder such as HAE. In yet other embodiments, the
subject is resistant to an anti—histamine therapy, a corticosteroid therapy, or both.
A subject identified in the s described herein may be subject to a suitable
treatment.
The assay methods and kits described herein can be applied for evaluation of the efficacy
of a treatment for a disease ated with plasma rein, such as those described herein,
given the correlation between the level of cleaved HMWK and such diseases. For examples,
multiple biological samples (e.g., blood or plasma samples) can be collected from a subject to
whom a treatment is performed either before and after the treatment or during the course of the
treatment. The levels of d and/or intact kininogen can be measured by any of the assay
methods as described herein and values (e.g., percentages) of cleaved and/or intact gen
can be determined accordingly. If the percentage of the cleaved gen decreases after the
treatment or over the course of the treatment (the cleaved gen percentage in a later
collected sample as compared to that in an earlier collected sample) or the percentage of intact
kininogen increases after the treatment or over the course of the treatment, it indicates that the
treatment is effective. In some examples, the ent involves a therapeutic agent, such as a
kallikrein inhibitor, a bradykinin B2 receptor antagonist, or a Cl—INH replacement agent.
Examples of the therapeutic agents include, but not limited to, landadelumab (DX—2930),
ecallantide (DX—88), icantibant, and human plasma—derived Cl—INH.
If the subject is identified as not sive to the treatment, a higher dose and/or
frequency of dosage of the therapeutic agent are administered to the subject identified. In some
embodiments, the dosage or frequency of dosage of the therapeutic agent is maintained, lowered,
or ceased in a subject identified as responsive to the treatment or not in need of further
treatment. Alternatively, a different ent can be applied to the subject who is found as not
responsive to the first treatment.
In other embodiments, the values of cleaved kininogen, either alone or in combination
with that of intact kininogen, can also be relied on to identify a disorder that may be treatable by
a pKal inhibitor. To practice this method, the level of cleaved kiniogen and/or the level of intact
kininogen in a sample collected from a t (e.g., a blood sample or a plasma sample) having
a target disease can be measured by a suitable assay, e.g., those described herein such as a
Western blot or ELISA assay. Values such as percentages of the cleaved and/or intact kininogen
can be determined as described herein. The values 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 (6.g. , elevated or decreased), it
indicates that a pKal tor may be effective in treating the e. For example, if the
percentages of cleaved kininogen are decreasing after the treatment or over the course of the
treatment, the treatment can be identified as being effective. Alternatively, if the percentages of
intact kininogen are increasing after the treatment or over the course of the treatment, the
treatment is identified as being effective.
If the disease is fied as being susceptible (can be treated by) to a pKal inhibitor, the
method can further comprise administering to the subject having the disease an ive amount
of a pKal tor, e.g., ecallantide (DX—88), EPIKAL—Z, or landadelumab 30).
Also within the scope of the present disclosure are methods of ting the severity of
a disease or disorder associated with plasma kallikrein or the disease state. For example, as
described herein, HAE may be in the ent state (basal , during which the t does
not experience symptoms of the disease. HAE attacks are typically recurrent episodes in which
the subject may experience pain and swelling, for example in the hands, feet, face,
gastrointestinal tract, genitals, and larynx t) that can last from two to five days. In some
embodiments, the level of 2—HMWK is indicative of whether the subject will experience, is
encing, or will soon experience an HAE . In some embodiments, the methods
involve comparing the level of 2—HMWK in a sample obtained from a subjecting having HAE to
the level of 2—HMWK in a sample from the same t, for example a sample obtained from
the same subject at basal state or a sample obtained from the same subject during a HAE attack.
(v) Non-Clinical Applications
Further, assays for detecting the levels of cleaved 2—HMWK described herein may be
used for research purposes. Although many diseases and disorders associated with or mediated
by plasma kallikrein have been identified, it is possible that other es are mediated by
similar mechanisms or involve similar components. In some embodiments, the methods
described herein may be used to identify a e as being associated with or mediated by
plasma kallikrein or with components of the contact activation . In some embodiments,
the s described herein may be used to study mechanisms (e.g., the discovery of novel
biological pathways or processes involved in disease development) or progression of a disease.
In some embodiments, the levels of cleaved 2—HMWK as measured using the assays
described herein, may be relied on in the development of new therapeutics for a disease
associated with the contact activation system. For example, the levels of cleaved 2—HMWK may
be measured in samples obtained from a subject having been administered a new therapy (e.g., a
clinical trial). In some ments, the levels of cleaved 2—HMWK may te the efficacy
of the new therapeutic or the progression of the disease in the subject prior to, during, or after
the new therapy.
11. Treatment of Diseases Associated with Plasma Kallikrein
A subject at risk for or suffering from a disease associated with plasma kallikrein, as
identified using the methods and assays described herein, may be treated with any
appropriate therapeutic agent. In some embodiments, provided methods include selecting a
treatment for a subject based on the output of the described method, e.g., measuring the level
of cleaved .
In some embodiments, the method comprises one or both of selecting or
administering a therapeutic agent, e.g., a kallikrein inhibitor, a inin B2 or
inhibitor, and/or a Cl esterase inhibitor, for administration to the subject based on the output
of the assay, e.g., 2—HMWK detection.
In some ments, the therapeutic agent is administered one or more times to the
subject. In some embodiments, a plasma kallikrein inhibitor is administered to a subject. In
some embodiments, kallikrein inhibitor is a peptide, a small molecule inhibitor, a kallikrein
antibody, or a fragment thereof. In some embodiments, an antagonist of bradykinin B2
receptor is administered to a subject. In some embodiments, a Cl—INH is administered to a
subject.
The therapeutic agent, 6. g. kallikrein inhibitor, bradykinin B2 or inhibitor,
and/or Cl—INH, may be administered along with another therapy as part of a combination
therapy for ent of the disease or condition that es the contact activation system.
Combination therapy, e.g., with one or more of a kallikrein inhibitor, bradykinin B2 receptor
antagonist, or C l—INH replacement agent, 6.g. , with one or more of a kallikrein inhibitor,
bradykinin B2 receptor antagonist or Cl—INH replacement agent and another therapy, may
be provided in multiple different configurations. The first agent may be administered before
or after the stration of the other therapy. In some situations, the first agent and
another therapy (e.g., a therapeutic agent) are administered rently, or in close
temporal proximity (e.g., a short time interval n the injections, such as during the
same treatment session). The first agent and the other therapy may also be administered at
greater temporal intervals.
Plasma kallikrein g 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 eutic agents for a variety of diseases and conditions,
e.g., diseases and conditions that involve plasma rein activity. For example, in some
embodiments, the disease or condition that involves plasma kallikrein activity is hereditary
angioedema (HAE). In some ments a plasma kallikrein binding agent such as a
plasma rein inhibitor is administered to a subject at risk or ing from a e
associated with the contact activation system.
A number of useful protein inhibitors of kallikrein, either tissue and/or plasma
kallikrein, include a Kunitz domain. As used herein, a “Kunitz domain” is a polypeptide
domain having at least 51 amino acids and containing at least two, and preferably three,
disulfides. The domain is folded such that the first and sixth cysteines, the second and
fourth, and the third and fifth cysteines form disulfide bonds (e.g., in a Kunitz domain
having 58 amino acids, cysteines can be present at positions ponding to amino acids 5,
14, 30, 38, 51, and 55, according to the number of the BPTI homologous sequences
provided below, and disulfides can form between the cysteines at position 5 and 55, 14 and
38, and 30 and 51), or, if two disulfides are present, they can form between a corresponding
subset of cysteines thereof. The spacing n respective cysteines can be within 7, 5, 4,
3, 2, l or 0 amino acids of the following g between positions corresponding to: 5 to
55, 14 to 38, and 30 to 51, according to the numbering of the BPTI sequence provided
below. The BPTI sequence can be used as a reference to refer to specific positions in any
generic Kunitz domain. Comparison of a Kunitz domain of interest to BPTI can be
med by identifying the best fit ent in which the number of aligned nes in
maximized.
The 3D structure (at high resolution) of the Kunitz domain of BPTI is known. One
of the X—ray ures is ted in the Brookhaven Protein Data Bank as "6PTI". The
3D structure of some BPTI homologues (Eigenbrot et al., Protein Engineering (1990)
3(7):59l—598; Hynes et al., Biochemistry (1990) 29:10018—10022) are known. At least
eighty one Kunitz domain sequences are known. Known human homologues include 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):l8832—l8837) two Kunitz domains of Inter—oc—
Trypsin Inhibitor, APP—l (Kido et al. J. Biol. Chem. (1988) 263(34):l8lO4—l8lO7), a Kunitz
domain from en, three Kunitz domains of TFPI—2 (Sprecher et al., PNAS USA (1994)
91:3353—3357), the Kunitz domains of cyte growth factor activator inhibitor type 1,
the Kunitz domains of Hepatocyte growth factor activator inhibitor type 2, the Kunitz
domains 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)
containing three Kunitz domains.
Table l: Exemplary Natural Kunitz Domains
LACI 1 MIYTMKKVHA LWASVCLLLN LAPAPLNAds eedeehtiit
(SEQ ID dtelpplklM
NO: 78) 51 HSFCAFKADD GPCKAIMKRF FFNIFTRQCE CEGN
QNRFESLEEC
101 KKMCTRDnan riiktthqe kpdfoleed pgiCrgxitr
xfxnngtkgc
151 erkaggC1g nmnnfetlee CkniCedgpn gfqunygtq
1navnnsltp
201 qstkvpslfe fhgpswCltp adrglCrane nrfyynsvig
kCrpfkysgC
251 ggnennftsk qulraCkkg fiqriskggl iktkrkrkkq
rvkiayeeif
301 vknm
The signal sequence (1-28) is uppercase and
underscored
LACI-K1 (50-107) is uppercase
LACI-K2 (121-178) is underscored
LACI-K3 (211-270) is bold
(SEQ ID
NO:79) 1234567890123456789012345678901234567890123456789012345678
RPDFCLEPPYTGPCKARIIRYFYNAKAGLCQTFVYGGCRAKRNNFKSAEDCMRTCGGA
TheKmnudommns?mvemerdknmhoasLACLKlOe?mms?)ml07LLACL
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) reports mutational s in which the Pl residues of each of the three
Kunitz domains were altered. l inhibits Factor Vlla a) when F.Vlla is
complexed to tissue factor and LACI—K2 inhibits Factor Xa.
A variety of methods can be used to identify a Kunitz domain from a sequence
database. For example, a known amino acid sequence of a Kunitz domain, a consensus
sequence, or a motif (e.g., the ProSite Motif) can be searched against the GenBank sequence
databases nal Center for Biotechnology Information, National Institutes of Health,
Bethesda MD), e. g., using BLAST; against Pfam database of HMMs (Hidden Markov
Models) (e.g., using t ters for Pfam searching; t the SMART database; or
against the ProDom database. For example, the Pfam Accession Number PF00014 of Pfam
Release 9 provides numerous Kunitz domains and an HMM for identify Kunitz domains. A
description of the Pfam database can be found in mer et al. Proteins (1997)
28(3):405—420 and a detailed ption of HMMs can be found, for example, in ov
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—3 14. The SMART database (Simple Modular Architecture ch Tool,
EMBL, Heidelberg, DE) of HMMs as described in Schultz et al. Proc. Natl. Acad. Sci. USA
(1998) 95:5857 and z et al. Nucl. Acids Res (2000) 28:231. The SMART database
contains domains identified by ing with the hidden Markov models of the HMMer2
search program (R. Durbin et al. (1998) Biological sequence analysis: probabilistic models
ofproteins and nucleic acids. Cambridge University Press). The database also is annotated
and monitored. The ProDom protein domain database consists of an automatic compilation
of homologous domains (Corpet et al. Nucl. Acids Res. (1999) —267). Current
versions of ProDom are built using recursive AST searches (Altschul et al. Nucleic
Acids Res. (1997) 25:3389—3402; Gouzy et al. Computers and Chemistry (1999) 23:333—
340.) of the SWISS—PROT 38 and TREMBL protein databases. The database automatically
generates a consensus sequence for each domain. Prosite lists the Kunitz domain as a motif
and identifies proteins that e a Kunitz domain. See, e. g., Falquet et al. c Acids
Res. (2002) 30:235—238.
Kunitz domains ct with target protease using, primarily, amino acids in two
loop regions (“binding loops”). The first loop region is between about residues
corresponding to amino acids 13—20 of BPTl. The second loop region is between about
residues corresponding to amino acids 3 l—39 of BPTl. An exemplary library of Kunitz
domains varies one or more amino acid positions in the first and/or second loop regions.
Particularly useful positions to vary, when screening for Kunitz s that interact with
kallikrein or when selecting for improved affinity variants, include: ons 13, 15, l6, l7,
l8, l9, 3 l, 32, 34, and 39 with respect to the sequence of BPTl. At least some of these
positions are expected to be in close contact with the target protease. It is also useful to vary
other positions, e. g., positions that are adjacent to the aforementioned positions in the three—
dimensional structure.
The “framework ” of a Kunitz domain is defined as those residues that are a
part of the Kunitz domain, but ically excluding residues in the first and second binding
loops regions, i.e., about residues corresponding to amino acids 13—20 of BPTl and 3 l—39 of
BPTl. Conversely, residues that are not in the binding loop may te a wider range of
amino acid substitution (e.g., conservative and/or non—conservative substitutions).
In one embodiment, these Kunitz domains are variant forms of the looped structure
including Kunitz domain 1 of human lipoprotein—associated coagulation inhibitor (LACl)
protein. LACI ns three internal, well—defined, peptide loop structures that are
paradigm Kunitz domains (Girard, T. et al., Nature (1989) 338:518—520). ts of
Kunitz domain 1 of LACI described herein have been screened, ed and bind kallikrein
with enhanced ty and specificity (see, for example, U.S. Pat. Nos. 5,795,865 and
287). These methods can also be applied to other Kunitz domain frameworks to
obtain other Kunitz domains that interact with rein, e.g., plasma kallikrein. Useful
modulators of kallikrein function typically bind and/or inhibit kallikrein, as ined
using kallikrein binding and inhibition .
In some aspects, the plasma kallikrein inhibitor binds to the active form of plasma
kallikrein. In some embodiments, the plasma kallikrein inhibitor, binds to and inhibits
plasma kallikrein, e.g., human plasma kallikrein and/or murine kallikrein. Exemplary
ptide plasma kallikrein agents are disclosed in U.S. Patent No. 5,795,865, U.S. Patent
No. 5,994,125, U.S. Patent No. 6,057,287, U.S. Patent No. 6,333,402, U.S. Patent No.
7,628,983, and 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.
7,81 1,991, and U.S. Publication No. 20110086801, the entire contents of each of which is
incorporated herein by reference. In some embodiments, the plasma kallikrein inhibitor is
an inhibitory polypeptide or peptide. In some embodiments, the inhibitory peptide is
ecallantide (also referred to as DX—88 or KALBITOR®; SEQ ID NO:80). In some
embodiments, the kallikrein inhibitor comprises or consists of an about 58—amino acid
sequence of amino acids 3—60 of SEQ ID NO: 80 or the DX—88 polypeptide haVing the 60—
amino acid sequence of SEQ ID NO: 80.
Glu Ala Met His Ser Phe Cys Ala Phe Lys Ala Asp Asp Gly Pro Cys Arg Ala Ala
His Pro Arg Trp Phe Phe Asn Ile Phe Thr Arg Gln Cys Glu Glu Phe Ile Tyr Gly Gly Cys
Glu Gly Asn Gln Asn Arg Phe Glu Ser Leu Glu Glu Cys Lys Lys Met Cys Thr Arg Asp
(SEQ ID NO: 80).
The plasma kallikrein inhibitor can be full—length antibodies (e.g., an IgG (e.g., an
IgGl, IgG2, IgG3, IgG4), IgM, IgA (e.g., IgAl, 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 globulins, or can be a
single chain antibody. The plasma rein inhibitor can be recombinant proteins such as
humanized, CDR grafted, chimeric, deimmunized, or in vitro generated antibodies, and may
optionally include constant regions derived from human germline immunoglobulin
sequences. In one embodiment, the plasma kallikrein inhibitor is a monoclonal antibody.
Exemplary plasma kallikrein g proteins are disclosed in US. ation 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 dy) having
the light and/or heavy chains of antibodies selected from the group consisting of Ml62—A04,
Ml60-Gl2, Ml42-H08, X63-G06, XlOl-AOl (also referred to as DX-2922), X8l-B01,
X67-D03, X67-G04, 1, X67-D03, X67-G04, Xl , Xl 15-D05, Xl 15-E09,
Xl 15—H06, Xl , Xl 15—D01, Xl 15—F02, Xl24—G01 (also referred to herein as DX—
2930 or lanadelumab), X1 , M29-D09, Ml45-Dl l, M06-D09 and M35-G04. In
some embodiments, the plasma kallikrein binding protein competes with or binds the same
epitope as Ml62—A04, Ml60—Gl2, Ml42—H08, X63—G06, XlOl—AOl (also referred to herein
as DX-2922), X8l-B01, X67-D03, X67-G04, X8l-B01, X67-D03, X67-G04, Xl 15-B07,
XllS-D05,X115-E09, XllS-H06,X115-A03, XllS-D01,X115-F02,Xl24-G01,,X115-
G04, M29—D09, Ml45—Dl l, M06—D09 and M35—G04. In some embodiments, the plasma
kallikrein binding protein is lanadelumab. See US 20110200611 and US 20120201756,
which are incorporated by reference herein.
An example of a plasma kallikrein inhibitory antibody is lanadelumab. The amino
acid ces of the heavy chain and light chain variable regions of lanadelumab are
provided below with the CDR regions identified in boldface and underlined.
Lanadelumab heavy chain variable region sequence (SEQ ID NO: 81)
mVQLLmSGGG LVQPGGSLRL FTFS HYIMMWVRQA PGKGLEWVSG IYSSGGITVY
RFTI SRDNSKNTLY LQMNSLRAED TAVYYCAYRR IGVPRRDEFD MVTV SS
Lanadelumab light chain variable region sequence (SEQ ID NO: 82)
DIQMTQSPS TASASVGDRV TITCRASQSI SSWLAWYQQK PGKAP
SRFSGSGSGT EFTLTISSLQ PDDFATYYCQ QYNTYWTFGQ GTKVEI
In some embodiments, a plasma kallikrein inhibitor can have about 85%, 90%, 91%,
92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher sequence identity to a plasma
kallikrein inhibitor described herein. In some ments, a plasma kallikrein inhibitor
can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher
ce identity in the HC and/or LC framework regions (e.g., HC and/or LC FR 1, 2, 3,
and/or 4) to a plasma kallikrein inhibitor described herein. In some embodiments, a plasma
kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher sequence identity in the HC and/or LC CDRs (e.g., HC and/or LC CDRl, 2,
and/or 3) to a plasma rein inhibitor described herein. In some embodiments, a plasma
kallikrein inhibitor can have about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher sequence identity in the constant region (6.g. and/or CLl)
, CH1, CH2, CH3,
to a plasma kallikrein inhibitor bed herein.
In some aspects, a small molecule binds and inhibits the active form of plasma
kallikrein.
inin BZ Receptor Inhibitors
In some embodiments, a bradykinin B2 receptor inhibitor (e.g., antagonist) is
administered to a subject. Exemplary bradykinin B2 receptor antagonists include icatibant
(Firazyr®), which is a peptidomimetic drug ning 10 amino acids which block binding
of native bradykinin to the bradykinin B2 receptor.
C1-INH Replacement Agents
In some embodiment, a Cl esterase inhibitor (Cl—INH), such as a replacement Cl—
INH agent is administered to a subject. Exemplary Cl—INH replacement agents are publicly
ble and include, for example, human plasma—derived , e. g. Berinert® and
CINRYZE®.
III. Kits for Detection of Cleaved HMWK
The present disclosure also provides kits for use in evaluating cleaved HMWK in
samples suspected of containing a cleaved HWMK, e.g., biological s from human
patients. Such kits can se a first agent that specifically binds to cleaved HMWK as
compared to intact HMWK or LMWK. In some embodiments, the first agent is an antibody,
such as any of the antibodies described herein that specifically bind cleaved HMWK (e.g.,
559B—M004 or onal variants thereof as described herein). In some embodiments, the kits
further comprise a second agent (e.g., an antibody binding to HMWK) for detecting binding of
the first agent to the cleaved HMWK. The second agent can be ated to a label. In some
embodiments, the second agent is an antibody that specifically binds cleaved HMWK. In other
embodiments, the second agent is an antibody that cross reacts with both cleaved and intact
HMWK.
The kit may further comprise a support member for ming the immunoassay and
immobilizing the first agent. In some ments, the support member is a 96—well plate, such
as a 96—well ELISA plate. The kit can also comprise one or more buffers as described herein but
not limited to a coating buffer; an assay buffer, such as an assay buffer containing ZnClz; a
ng ; a wash buffer; and/or a stopping buffer.
In some ments, the kit can comprise instructions for use in accordance with any
of the methods described herein. The included instructions can comprise a description of how to
use the components contained in the kit for measuring the level of cleaved and/or intact HMWK
in a sample, which can be a biological sample collected from a human patient. Alternatively or
in addition, the kit may comprise may comprise a description of how to use components
contained in the kit for measuring the level of LMWK.
The instructions relating to the use of the kit generally include information as to the
amount of each component and suitable conditions for performing the assay methods described
herein. The components in the kits may be in unit doses, bulk packages (e.g., multi—dose
packages), or sub—unit doses. Instructions supplied in the kits of the present disclosure are
lly written instructions on a label or package insert (e.g., a paper sheet included in the kit),
but machine—readable instructions (e.g., instructions carried on a magnetic or optical storage
disk) are also acceptable.
The label or package insert indicates that the kit is used for evaluating the level of
cleaved and/or intact HMWK. In some embodiments, the kit is used for evaluating the level of
LWMK. Instructions may be provided for practicing any of the methods described herein.
The kits of this present disclosure are in le packaging. Suitable packaging
includes, but is not limited to, vials, bottles, jars, ?exible packaging (e.g., sealed Mylar or plastic
bags), and the like. Also contemplated are packages for use in ation with a specific
device, such as an inhaler, nasal administration device (e.g., an atomizer) or an infusion device
such as a minipump. A kit may have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a stopper able by a hypodermic injection needle).
The container may also have a sterile access port (for example the container may be an
intravenous solution bag or a vial having a r pierceable by a hypodermic ion needle).
Kits may ally provide additional components such as interpretive information,
such as a control and/or standard or reference sample. Normally, the kit ses a container
and a label or package insert(s) on or associated with the container. In some embodiments, the
present disclosure es articles of manufacture comprising contents of the kits described
above.
IV. Other Antibodies Binding to Cleaved HMWK
Also provided herein are isolated antibodies that bind both cleaved HMWK and intact
HMWK. In some ments, such antibodies do not bind LMWK or bind to LMWK with a
low ty. In other embodiments, such antibodies also bind to LMWK.
In some embodiments, the antibodies that specifically binds a cleaved HMWK and intact
HMWK (or onally LMWK) described herein have a suitable binding affinity to one or
more of the target antigens. The antibody bed herein may have a binding affinity (KD) of
at least 105, 106, 10”, 10*, 109, 10'10 M, or lower.
Examples of the antibodies noted above and their binding icities are provided in
Table 2 in Example 2 below. The amino acid sequences of the heavy chain and light chain
U variable regions are provided below with the CDR s identified in boldface and underlined
(determined by one scheme as an example):
>559B—R0049—A01 (559B—M0067—EO2) Heavy Chain Amino Acid Sequence (SEQ ID NO: 6)
'VQTLSGGGTVQPGGSLRLSCAASGFTFSLYPMVWVRQAPGKGLLWVSSIYPSGGFTTYADSV
KGRFT: SRDNSKNTLYLQMNSLRAEDTAVYYCARSSRYYYYGMDVWGQGTTVTVSS
>559B—R0049—A01 (559B—M0067—EO2) Light Chain Amino Acid Sequence (SEQ ID NO: 7)
QY_LLlQPPSMSGlPGQRVl__SCSGSSSNIGSEYVYWFQQLPGTAPKLL:YRNDQRPSGVPDRFS
GSKSGTSAST.A SGLRSLD'lDYYCSTWDDTLRTGVFGGGTKVTVL
>559B—R0049—G05 (559B—M0039—GO7) Heavy Chain Amino Acid Sequence (SEQ ID NO: 8)
'VQT.LSGGGTVQPGGSLRLSCAASGFTFSRYRMRWVRQAPGKGLLWVSGISPSGGWTYYADSV
KGRFT: SRDNSKNTLYLQMNSLRALDTAVYYCTTDNGDYALAHWGQGTLVTVSS
>559B—R0049—G05 (559B—M0039—GO7) Light Chain Amino Acid Sequence (SEQ ID NO: 9)
QDIQMTQSPSSLSASVGDKVL lCRASQRIINYLNWYQQKPGKAPKLL__YAASSLQSGVPSRFS
GSGSGlDblTl SSLQPLDbAiYYCQQSYSAPLTbGGGlRVi K
>559B—R0048—A09 (559B—M0044—E09) Heavy Chain Amino Acid Sequence (SEQ ID NO: 10)
LVQLLLSGGGTVQPGGSLRLSCAASGFTFSQYSMGWVRQAPGKGLLWVSSIYSSGGSTQYADSV
EERFT:SRDNSKNTLYLQWNSLRAL3TATYYCARTRRGWFGEDYYYYMDVWGKGTTVTVSS
>559B—R0048—A09 (559B—M0044—E09) Light Chain Amino Acid Sequence (SEQ ID NO: 11)
QDIQMTQSPSSLSASVGDL I ICRASQGIRNDVGWYQQKPGKAPQRL__YAASSLQSGVPSRFS
GSGSGIbTTI SSTQPLDbAIYYCLQHNSYPLTbGGGIKVL K
>559B-R0048—E01 (559B—M0003—C08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 12)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSPYMMYWVRQAPGKGLLWVSSISPSGGKTWYADSV
KGRFTT SRDNSKNTLYLQMNSLRALDTAVYYCARLGGSSSYYYYYYYGMDVWGQGTTVTVSS
>559B-R0048—E01 M0003—C08) Light Chain Amino Acid Sequence (SEQ ID NO: 13)
QSALTQSPSASGTPGQRVTT SCSGSSSNIGGNTVNWYQQFPGTAPK.LTYSNNQRPSGVPDRFS
GSKSGTSASTA SGLQSLDLA YYCASWDDRLNGHWVFGGGTRLTV_L
>559B—R0049-G01 (559B—M0039—H06) Heavy Chain Amino Acid Sequence (SEQ ID NO:14)
LVQTJ'SGGGLVQPGGS R SCAASGFTFSAYDMHWVRQAPGKGL_LWVSSIWPSGGGTYYADSV
KGRFTTSRDNSKNTLYLQWWSLRALDTAVYYCARGDYDYGDFTDAFDIWGQGTMVTVSS
>559B—R0049-G01 (559B—M0039—H06) Light Chain Amino Acid Sequence (SEQ ID NO: 15)
QSALTQPASVSGSPGQS I SCTGTSSDVGSYNLVSWYQQHPGKAPKLMTYEGSKRPSGVPDRF
SGSKSGNIASL SGLQALDLADYYCCSYAGSYSYVFGTGTRVTVL
>559B—R0049—E05 (559B—M0039—D08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 16)
LVQT.LSGGGTVQPGGSLRLSCAASGFTFSNYAMQWVRQAPGKGLLWVSWIYSSGGPTYYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARGLPGQPFDYWGQGTLVTVSS
>559B—R0049—E05 (559B—M0039—D08) Light Chain Amino Acid Sequence (SEQ ID NO: 17)
QS.LLiQPPSASGIPGQRVI__SCSGSSSNIGNNYVYWYQQFPGTAPKLLTYRNNQRPSGVPDRFS
GSKSGTSASLA SGLRSL3LADYYCATWDDRLSGWVFGGGTKLTVL
R0048-A11 (559B—M0068—CO7) Heavy Chain Amino Acid Sequence (SEQ ID NO: 18)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSSYQMHWVRQAPGKGLLWVSGIYSSGGSTPYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARGHHGMDVWGQGTTVTVSS
>559B—R0048—A11 (559B—M0068—CO7) Light Chain Amino Acid Sequence (SEQ ID NO: 19)
QDIQMTQSPSSVSASVGDLVI ICRASQGISSWLAWYQQKPGKAPKLL__YAASNLQSGVPSRFS
GSGSGIDbTTI DbAIYYCQKYNIAPYTbGQGIKTL K
R0048-A03 (559B—M0021—G1 1) Heavy Chain Amino Acid Sequence (SEQ ID NO: 20)
SGGGTVQPGGSLRLSCAASGFTFSPYPMTWVRQAPGKGLLWVSGISSSGGFTPYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARMVRGVIKAFDIWGQGTMVTVSS
>559B—R0048-A03 (559B—M0021—G1 1) Light Chain Amino Acid Sequence (SEQ ID NO: 21)
QY_LLIQPPSASGIPGQRVI__SCSGSSSNIGSHYVFWYQQLPGAAPKLLLYRNNQRPSGVPDRFS
SAST.A SGT.RSL3LADYYCATWDNSLSAWVFGGGTKLTVL
>559B—R0048—C05 M0061—GO6) Heavy Chain Amino Acid Sequence (SEQ ID NO: 22)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSKYTMWWVRQAPGKGLLWVSVISSSGGKTYYADSV
KGRFTL SRDNSKNTLYLQMNSLRALDTAVYYCARTANRAFDIWGQGTMVTVSS
>559B—R0048—C05 (559B—M0061—GO6) Light Chain Amino Acid Sequence (SEQ ID NO: 23)
Q3:QMTQSPAALSVSPGL{AILSCRASQSVSSDLAWYQQKPGQAPRLL_HGASTRATGLPARFS
GSGSGRbiTI SSTQSLDFAVYYCQQYNDWPPLFGPGTKVN_K
>559B—R0049-A03 (559B—M0036—G12) Heavy Chain Amino Acid Sequence (SEQ ID NO: 24)
LVQTTSGGGTVQPGGSLRLSCAASGFTFSRYYMAWVRQAPGKGLLWVSGIVPSGGQTGYADSV
U KGRFTL SRDNSKNTLYLQMNSLRAL3TAVYYCARTRRGWFGEDYYYYMDVWGKGTLVTVSS
>559B—R0049-A03 (559B—M0036—G12) Light Chain Amino Acid Sequence (SEQ ID NO: 25)
Q3-QMIQSPGILSLSPGL{AIVSCRASQSVGSTYLAWYQHKPGQAPRLL_YGASSRATGLPDRF
SGSGSGIDbTTI SSTLPLDE'A YYCQHFHTSPPGITb'GQGIRT.L K
>559B—R0048-C09 (559B—M0042—E06) Heavy Chain Amino Acid Sequence (SEQ ID NO: 26)
LVQTTSGGGTVQPGGSLRLSCAASGFTFSMYKMSWVRQAPGKGLLWVSVISPSGGRTYYADSV
KGRFT NTLYLQMNSLRALDTAVYYCARGTRTSGLDYWGQGTLVTVSS
>559B—R0048-C09 (559B—M0042—E06) Light Chain Amino Acid ce (SEQ ID NO: 27)
QSALTQPASVSGSPGQS I SCTGTSSDVGGYKYVSWYQQ
SGSKSGNTASLT"SGLQALDLADYYCSSYTSSTTVVFGGGTKLTVL
>559B—R0048—E09 (559B—M0070—H10) Heavy Chain Amino Acid Sequence (SEQ ID NO: 28)
LVQTTSGGGTVQPGGSLRLSCAASGFTFSTYGMRWVRQAPGKGLLWVSVISPSGGKTNYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARGRPDYYAMDVWGQGTTVTVSS
>559B—R0048—E09 (559B—M0070—H10) Light Chain Amino Acid Sequence (SEQ ID NO: 29)
QSALTQPPSASGAPGQRVT SCSGSSSNIGSNTVNWYQKLPGTAPKLLLYYNDRRPSGVPDRFS
GSKSGNTAST.T SGLQALDLADYYCAAWDDSLSGPVFGGGTKLTVL
>559B—R0048—E05 (559B—M0068—D01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 30)
LVQTTSGGGTVQPGGSLRLSCAASGFTFSIYPMSWVRQAPGKGLLWVSGISPSGGKTAYADSV
40 KGRFT SRDNSKNTLYLQMNSLRAL3TAVYYCARGQGRAVRGKLYYYGMDVWGQGTTVTVSS
>559B—R0048—E05 (559B—M0068—D01) Light Chain Amino Acid Sequence (SEQ ID NO: 31)
QSALTQPPSASQTPGQTVT: SCSGSSSNIGTNNVNWYQQLPGTAPKLLISSHHRRPSGVP3RFS
ASKSGTSASTA L3LA3YYCAAWDDSLNGPVFGGGTKLTVL
>559B—R0048—C01 (559B—M0004—E08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 32)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSMYHMNWVRQAPGKGLLWVSSIYSSGGSTRYADSV
KGRFT: SR3NSKNTLYLQMNSLRAL3TAVYYCARGVRYGMDVWGQGTTVTVSS
>559B—R0048—C01 (559B—M0004—E08) Light Chain Amino Acid Sequence (SEQ ID NO: 33)
QSPSSVSASVG3
GSGSGJ3bJTJ SSTQBL3bAJYYCQQANSFPITbGQGJRTL K
>559B—R0049—C01 (559B—M0069—C09) Heavy Chain Amino Acid Sequence (SEQ ID NO: 34)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSMYDMHWVRQAPGKGLLWVSSISSSGGYTQYADSV
U KGRFT SR3NSKNTLYLQMNSLRAL3TAMYYCARDRGLIAAAGGFDPWGQGTLVTVSS
>559B—R0049—C01 (559B—M0069—C09) Light Chain Amino Acid Sequence (SEQ ID NO: 35)
Q3IQMTQSPSSLSASVG3
GSGSGJ3bJLJLSSLQP33bAJYYCQRTYGRPLTbGGGJKVL K
R0049—A05 (559B—M0038—F04) Heavy Chain Amino Acid Sequence (SEQ ID NO: 36)
LVQT.LSGGGTVQPGGSLRLSCAASGFTFSKYEMMWVRQAPGKGLLWVSSISPSGGYTMYADSV
KGRFT SR3NSKNTLYLQMNSLRAL3TAVYYCARHRSKWNDAPFDSWGQGTLVTVSS
>559B—R0049—A05 (559B—M0038—F04) Light Chain Amino Acid Sequence (SEQ ID NO: 37)
Q3IQMTQSPSSLSASVG3
GSGlGi 3J:' TL 1 RST QPL.3J:'ASYJLCQQSYSSPGITJLGPGTKVL. K
>559B—R0048—G05 (559B—M0044—C05) Heavy Chain Amino Acid Sequence (SEQ ID NO: 38)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSIYQMYWVRQAPGKGLLWVSSIYSSGGRTFYADSV
KGRFT SR3NSKNTLYLQMNSLRA_L3TAVYYCATRGSWYVGGNEYFQHWGQGTLVTVSS
>559B—R0048—G05 (559B—M0044—C05) Light Chain Amino Acid Sequence (SEQ ID NO: 39)
QSVLTQSPSLSLSPGQJASLPCSG3JLGNKEVSWYQQKBGQSPVLVLYQ3JKRPSG PLRESGS
NSGNJAJLJLJGJQAM3LA3YYCQVWDSNSYAFGPGTKVTVL
>559B—R0048—C11 (559B—M0047—H01) Heavy Chain Amino Acid ce (SEQ ID NO: 40)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSFYMMYWVRQAPGKGLLWVSSISSSGGFTRYADSV
KGRFT SR3NSKNTLYLQMNSLRA_L3TAVYYCARVRGLAVAAPDYWGQGTLVTVSS
>559B—R0048—C11 (559B—M0047—H01) Light Chain Amino Acid Sequence (SEQ ID NO: 41)
QSELTQPASVSGSPGQS I SCIGTSSDIGTYNYVSWYQQHPGKAPKLMLYDVNTRPSGVS3RF
SGSKSGNTASLT"SGLQAL3LA3YYCSSYTTSVTWVFGGGTTLTVL
>559B—R0048—C03 (559B—M0019—E12) Heavy Chain Amino Acid Sequence (SEQ ID NO: 42)
LVQTJ'SGGGLVQPGGSLRLSCAASGFTFSGYNMYWVRQAPGKGLEWVSRISPSGGWTSYADSV
KGRFTL SR3NSKNTLYLQMNSLRAL3TAVYYCTRGQWMDWWGQGTWVTVSS
>559B—R0048—C03 (559B—M0019—E12) Light Chain Amino Acid Sequence (SEQ ID NO: 43)
QSPSSLSASVG3
3Y T.I 3 GIYECQHTDDFSVTFGGGTKV3LK
>559B—R0048—A05 (559B—X0004—B05) Heavy Chain Amino Acid Sequence (SEQ ID NO: 44)
LVQTJSGGGTVQPGGSLRLSCAASGFTFHYRMMWVRQAPGKGLLWVSYISSSGGYTAYADSVK
U ERFTL SR3NSKNTLYLQMNSLRAL3TAVYYCAAKRNRAFDIWGQGTMVTVSS
>559B—R0048—A05 (559B—X0004—B05) Light Chain Amino Acid Sequence (SEQ ID NO: 45)
QSP3SLAVS.GLRAI NCKSSQSVLYSSNNKNYLAWYQQKPGQPPKLL__YWASTRESG
VP3RFSGSGSGT3FT.T'SSTQAL3VAVYYCQQYYSTPLGE'GQGIKLL K
>559B—R0048—E11 (559B—M0048—D12) Heavy Chain Amino Acid Sequence (SEQ ID NO: 46)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSRYQMTWVRQAPGKGLLWVSSIGSSGGFTNYADSV
KGRFT SR3NSKNTLYLQMNSLRAL3TAVYYCARLPANFYYYMDVWGKGTTVTVSS
>559B—R0048—E11 (559B—M0048—D12) Light Chain Amino Acid ce (SEQ ID NO: 47)
Q3IQMTQSPSSLSASVG3KVI ICRASQNIYSFLNWYQQKPGKAPKLL__YATSSLQSGVPSRFS
GSGSGI3bITI SSTQPL3bASYYCQQNYNIPWTbGQGIKVL K
>559B—R0048-G11 (559B—M0053—G01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 48)
LVQTHTSGGGTVQPGGSLRLSCAASGFTFSWYMMKWVRQAPGKGLLWVSSIVPSGGWTTYADSV
KGRFT SR3NSKNTLYLQMNSLRAL3TAVYYCATEGNLWFGEGRAFDIWGQGTMVTVSS
R0048-G11 (559B—M0053—G01) Light Chain Amino Acid Sequence (SEQ ID NO: 49)
Q3-QMIQSPGILSLSPGL{AILSCRASQSVSSSYLAWYQQKPGQAPRLL_YGASSRATGLP3RF
SGSGSGI 3J:' IT. I SRL LI’L.3J:'AVYYCQQRSNWPPSFGQGTRL3LK
>559B—R0049—C05 (559B—M0038—H03) Heavy Chain Amino Acid Sequence (SEQ ID NO: 50)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSKYDMHWVRQAPGKGLLWVSRISSSGGKTEYADSV
KGRFT SR3NSKNTLYLQMNSLRA_L3TAVYYCAREYRYCTANTCSLYGMDVWGRGTTVTVSS
>559B—R0049—C05 (559B—M0038—H03) Light Chain Amino Acid Sequence (SEQ ID NO: 51)
Q3:QMTQSPSSLSASVGDKVALICRTSQGVRSDFAWYQQTPGKAPRRL:YAAFILDNGVPSRFS
GSGSGIbITI LDEAIYYCQQSYSTPLTFGGGTKVEMK
>559B-R0048—E03 (559B—M0017—H08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 52)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSPYWMHWVRQAPGKGLLWVSVISPSGGGTGYADSV
KGRFT: SRDNSKNTLYLQMNSLRALDTAVYYCARESRGSGSHEDYWGQGTLVTVSS
>559B-R0048—E03 (559B—M0017—H08) Light Chain Amino Acid Sequence (SEQ ID NO: 53)
Q3-QMIQSPAILSLSPGL{AILSCRASQSVSSYLAWYQQKPGQAPRLL_YGASNRGTG:PARFS
GSGSGIbITI SSTQSLDFAVYFCQQYKNWPNLTFGGGTKVD:K
>559B-R0049—E03 (559B—M0035—F05) Heavy Chain Amino Acid Sequence (SEQ ID NO: 54)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSHYPMAWVRQAPGKGLLWVSGIVSSGGRTVYADSV
U KGRFT: SRDNSKNTLYLQMNSLRAL3TAVYYCARDPYDFWSEGAFDIWGQGTMVTVSS
>559B-R0049—E03 (559B—M0035—F05) Light Chain Amino Acid Sequence (SEQ ID NO: 55)
QSVLTQPPSASGTPGQRVT SCSGSSSNIGNNFVYWYHQVPGTAPK.L"YKNNQRPSGVPDRFS
GSKSAASASLA SGLRSL3LADYYCAAWDNSLSGFYVFGAGTKVTVL
>559B—R0049-GO3 (559B—M0035—H09) Heavy Chain Amino Acid Sequence (SEQ ID NO: 56)
LVQT.LSGGGTVQPGGSLRLSCAASGFTFSWYGMHWVRQAPGKGLLWVSRIGPSGGPTSYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARGYYGTGRYFQHWGQGTLVTVSS
R0049-GO3 (559B—M0035—H09) Light Chain Amino Acid Sequence (SEQ ID NO: 57)
QSPDSLSLSPGDRATLSCRASQSVGSDYLAWYQQKPGQAPRLL_YDASNRATG:PARF
SGSGSGIDb'ITI SSLLPLDb'AVYYCQQRSNWPPTb'GGGIKVL K
>559B—R0048—AO7 (559B—M0043—C06) Heavy Chain Amino Acid Sequence (SEQ ID NO: 58)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSAYAMRWVRQAPGKGLLWVSYISSSGGETMYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCANGYGRIDYWGQGTLVTVSS
>559B—R0048—AO7 (559B—M0043—C06) Light Chain Amino Acid Sequence (SEQ ID NO: 59)
QSVLTQPASVSGSPGQS I SCTGTSSDIGGYNYVSWYQQ
SGSKSGNTASLT"SGLQALDLADYYCSSYTSGSTRVFGTGTRVTVL
>559B—R0048-G01 M0003—A08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 60)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSAYVMRWVRQAPGKGLLWVSSIGSSGGPTYYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARRGGSGSSHAFDIWGQGTMVTVSS
>559B—R0048-G01 (559B—M0003—A08) Light Chain Amino Acid Sequence (SEQ ID NO:61)
QSPSSLSASVGDKVI ICRASQSISSYLNWYQQKPGKAPKLL__YAASSLQSGVPSRFS
GSGSGIDbTTI SSLQPLDSGiYYCQQYNSFPLTb'GGGIKVL K
>559B—R0048—GO9 (559B—M0054—B 1 1) Heavy Chain Amino Acid Sequence (SEQ ID NO: 62)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSYYGMNWVRQAPGKGLLWVSVISPSGGLTVYADSV
KGRFT: SRDNSKNTLYLQMNSLRALDTAMYYCATGFAVQHGGGAFDIWGQGTMVTVSS
>559B—R0048—GO9 (559B—M0054—B 1 1) Light Chain Amino Acid Sequence (SEQ ID NO: 63)
QSPAILSMSPGL{AILSCRASQSVTTYLAWYQQKPGQAPRLL_YDASIRATGVPARFS
GSGSGIDbiTI SRT.LPLDb'AVYYCQQRTIWPLTb'GGGIKVL K
>559B—R0048—EO7 (559B—M0067—G1 1) Heavy Chain Amino Acid Sequence (SEQ ID NO: 64)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSPYEMVWVRQAPGKGLLWVSSIVPSGGWTVYADSV
U KGRFT: NTLYLQMNSLRALDTAVYYCASPSGRGLAFDIWGQGTMVTVSS
>559B—R0048—EO7 (559B—M0067—G1 1) Light Chain Amino Acid Sequence (SEQ ID NO: 65)
Q3-QMIQSPGILSLSPGL{AILSCRASQSISSSYLAWYQQKPGQAPRLL_YGASSRATGVPDRF
SGSGSGIbTTI SSTQPLDFATYYCLQQKSYPYTE'GQGIKVL K
R0048—CO7 M0065—B10) Heavy Chain Amino Acid Sequence (SEQ ID NO: 66)
LVQT.LSGGGTVQPGGSLRLSCAASGFTFSKYFMTWVRQAPGKGLLWVSWISSSGGYTNYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCARGAYYYDAFDIWGQGTMVTVSS
R0048—CO7 (559B—M0065—B10) Light Chain Amino Acid Sequence (SEQ ID NO: 67)
QDIQMTQSPSSLSASVGDKVI ICRASQSIAIFLNWYQQTPGKPPKLL_XGASTLQSGVPSRFS
GSGSGADFTTT SNTQTLDFTTYYCQQSYSTLYTEGQGIKTL K
>559B—R0049—C03 (559B—M0037—E08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 68)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSRYSMSWVRQAPGKGLLWVSVISSSGGMTYYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAMYYCARDYYGNMDVWGKGTTVTVSS
>559B—R0049—C03 (559B—M0037—E08) Light Chain Amino Acid Sequence (SEQ ID NO: 69)
QD_QMIQSPSSLSISVGDKVI ICRTSQDISGALAWYQQKPGKAPRLL__FGASSLESGVPSRFS
GSGSGIDbTTI SSTQPLDbAIYYCQQFNKYPLTbGGGIKVL K
>559B-R0049—E01 (559B—M0035—A01) Heavy Chain Amino Acid Sequence (SEQ ID NO: 70)
LVQTJ.SGGGTVQPGGSLRLSCAASGFTFSWYTMGWVRQAPGKGLLWVSYIYPSGGYTMYADSV
KGRFT SRDNSKNTLYLQMNSLRALDTAVYYCANPYSSGGYWGQGTLVTVSS
>559B-R0049—E01 (559B—M0035—A01) Light Chain Amino Acid Sequence (SEQ ID NO: 71)
QDLQMIQSPLSLPVIPGLPAS SCRSSQSLLDSNGYNYLDWFLQKPGQSPQLL__YLGFNRASGV
PDRFSGSGSGIDETLK SRVLALDVGVYYCMQALQTPYTb'GQGIKT.L I
>559B—R0048-G03 (559B—M0003—E08) Heavy Chain Amino Acid Sequence (SEQ ID NO: 72)
'VQTJSGGGTVQPGGSLRLSCAASGFTFSAYLMTWVRQAPGKGLLWVSGISPSGGITKYADSV
KGRFTL SRDNSKNTLYLQMNSLRALDTAVYYCARDIPNWIYGMDVWGQGTTVTVSS
>559B—R0048-G03 (559B—M0003—E08) Light Chain Amino Acid Sequence (SEQ ID NO: 73)
QSALTQPPSVSVSPGQTASL TCSGDKLGNKYASWYQQKPGQSPVLVLYQDRRRPSG S
NSGNIAILI-SGIQAMDLADYYCQAWDSGVVFGGGTKLTVL
R0048—G07 (559B—M0052—E02) Heavy Chain Amino Acid Sequence (SEQ ID NO: 74)
'VQTJSGGGTVQPGGSLRLSCAASGFTFSNYLMLWVRQAPGKGLLWVSGISPSGGGTAYADSV
U KGRFTL SRDNSKNTLYLQMNSLRALDMAVYYCAKVAYSGSYYYYYYMDVWGKGTTVTVSS
>559B—R0048—G07 (559B—M0052—E02) Light Chain Amino Acid Sequence (SEQ ID NO: 75)
QDLQMTQSPSSLSASVGDKVI ICRASQSISSYLNWYQQKPGKAPKLL__YAASSLQSGVPSRFS
GSGSGIDbTTI SSTQPLDEAIYYCQQSYSTHSITEGQGIRTL K
>559B—M0064—H02 Heavy Chain Amino Acid ce (SEQ ID NO: 76)
LVQTJSGGGTVQPGGSLRLSCAASGFTFSQYIMGWVRQAPGKGLLWVSSIGSSGVTVYADSVK
ERFT SRDNSKNTLYLQMNSLRALDTAVYYCARGGGVTVLHAFDIWGQGTMVTVSSASTKGPSV
FPLAPSSKS
>559B—M0064—H02 Light Chain Amino Acid Sequence (SEQ ID NO: 77)
QSALTQPASVSGSPGQS I SDVGGYNYVSWYQQHPGKVPKT YEGNKRPSGVPDRF
SGSKAGNTASLTVSGLQALDLADYYCTAYGGHSRFYVE'GIGIKVIVLGQPKANP
Also Within the scope of this disclosure are functional lents of any of the
exemplary antibodies listed above. Such a functional equivalent may bind to the same epitope
of a cleaved HMWK and/or intact HMWK, or the sample epitope of LMWK as one of the above
listed exemplary antibodies. In some embodiments, the functional equivalent competes against
one of the above—listed exemplary dies for g to a target antigen.
In some embodiments, the functional equivalent ses a VH chain that includes a VH
CDRl, a VH CDR2, and/or a VH CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%,
98%, or 99%) identical to the corresponding VH CDRs of one of the above—listed exemplary
antibodies. Alternatively or in addition, the functional equivalent comprises a VL CDRl, a VL
CDR2, and/or a VL CDR3 at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%)
identical to the exemplary antibody as listed above. In some embodiments, the functional
equivalent has the same heavy chain and/or light chain complementarity determining regions
(CDRs) as one of the above—listed exemplary antibodies.
Alternatively or in addition, the functional equivalent comprises a VH chain at least 75%
(e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical to the VH chain of an exemplary
antibody and/or a VL chain at least 75% (e.g., 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%)
identical to the VL chain of the exemplary antibody.
In some instances, the functional equivalent may contain one or more (e.g., up to 5, up to
3, or up to l) conservative mutations in one or more of the heavy chain CDRs, or one or more of
the light chain CDRs in an exemplary antibody, e.g., at positions where the residues are not
likely to be involved in interacting with a target antigen.
Without further elaboration, it is ed that one skilled in the art can, based on the
above description, utilize the present present disclosure to its fullest extent. The following
specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of
the remainder of the disclosure in any way whatsoever. All publications cited herein are
incorporated by nce for the purposes or subject matter referenced herein.
Examyle 1: pment oflmmunoassays for Specific Detection of Cleaved HMWK
An ELISA—based immunoassay screen was initially developed to fy Fab fragments
in a phage display y that bound to cleaved or intact HMWK. In general, the assay
conditions relied on biotinylated intact or cleaved HMWK immobilized on avidin coated
384—well assay plates, ng using a bovine serum albumin (BSA) blocking buffer, and
contacting the immobilized HMWK with Fab displayed on phage from an overnight culture in
E. coli ted with anti—Ml3—HRP dy).
As shown in , panel A, the selection was directed s obtaining n
HMWK specific antibodies by first preforming a negative selection of the library with an input
of approximately 1 x 1012 phage against biotinylated l—chain HMWK immobilized streptavidin
coated magnetic beads (Dynabeads M280, Thermo Fisher). The depleted library was then
contacted with biotinylated 2—chain HMWK immobilized on streptavidin coated magnetic beads.
The beads were extensively washed with PBS buffer and used to infect E. coli for phage output
ication to complete a round of selection. Three rounds of selection were performed prior
to screening individual phage colonies by ELISA with biotinylated l—chain and 2—chain HMWK
immobilized on avidin coated plates followed by ion with horse radish peroxidase
(HRP) conjugated anti—M13 antibody and absorbance detection due to substrate hydrolysis for
3,3’,5,5’—Tetramethylbenzidine (TMB). Recombinant Fab fragments were expressed in E. coli
and ed by protein A sepharose chromatography (Wassaf et al. Anal. Biochem. (2006) 351:
241—253). The specificity of each purified Fab was ined by coating 384 well plates and
measuring binding to biotinylated l—chain HMWK, to biotinylated 2—chain HMWK, or to
ylated LMWK, followed by detection with streptavidin conjugated to HRP and TMB
detection. These assay ions led to the identification of the 559B—M004—B04 isolate, which
specifically binds cleaved HMWK over intact HMWK (.
The immobilized HMWK was also ted with a crude (unpurified) 559B—M004—B04
Fab preparation from an overnight culture in E. coli. Fab bound to the HMWK was detected
using an anti—human Fab—HRP antibody, but did not result in specific binding to cleaved HMWK
(.
The configuration of the immunoassay was reversed by passively immobilizing the
purified Fab fragment of 559B—M004—B04 on polystyrene ll assay plate. The Fab was
contacted with biotinylated HMWK, and the bound HMWK were detected with streptavidin—
HRP. (.
Unexpectedly, the specificity of the 559B—M004—B04 Fab to cleaved HMWK was
enhanced when the BSA blocking buffer was replaced with a commercially available blocking
buffer, the LowCross Blocking Solution from Candor Biosciences during the initial screening
analyses (. Further, performing the immunoassay using 96—well assay plates rather than
384—well plates r increased the observed specificity of 559B —M004—B04 to cleaved
HMWK (.
The s obtained using the 559B—M004—B04 isolate led to the development of an
immunoassay (ELISA) for the detection of 2—HMWK in samples (, panel B). This assay
can also be used to further evaluate binding characteristics of other Fab fragments and
antibodies. Brie?y, a Fab is coated on to a multiwell plate ght. The following day the
plate is washed then blocked with BSA Buffer. Following a wash samples, rds, and QCs
diluted in LowCross Buffer are added to the plate and after a subsequent incubation and then
wash, any bound 2—Chain HMWK is detected by adding HRP—labeled sheep anti—HMWK
polyclonal detection dy. Following incubation with the ion antibody, the plate is
washed and TMB substrate is added to the plate. After a short incubation the on is stopped
with phosphoric acid. The optical density is then measured at 630nm.
e 2: Evaluation ofBinding Speci?city ofFab Clones Using Immunoassays Described
Herein
Thirty—six purified Fab clones (see Table 2 below) were assessed for binding to cleaved
HWMK, intact HMWK, and LWMK using the immunoassay bed in Example 1.
Specifically, each of the ed Fab clones was immobilized on 96—well assay plates at a
concentration of l ug/L) in a total volume of 100 uL in PBS and incubated overnight at 2—8°C.
The assay plates were blocked using LowCross blocking buffer. Biotinylated intact HMWK,
biotinylated cleaved HMWK or biotinylated LMWK (lug/L each) was added to each well in a
total volume of 100 uL and incubated for 2 hours prior to washing with a wash buffer. HRP—
d streptavidin was added to each well at a concentration of lOOng/mL, and the signal was
developed using Ultra TMB Substrate. The signal to noise ratio was calculated using the signal
observed upon the on of the biotinylated protein to an uncoated well. ( panels A and
B). Based on the ELISA results, the antibodies can be divided among 5 categories (Table 2).
Table 2: Binding characteristics of Fab nts
ELISA Binding Fab Fragment
Low ty binder 559B-M0035-A01, 559B—M0052—E02, 559B—M0003—E08
Bind to cleaved and intact 559B—M0067—EO2, 559B-M0039-GO7, 559B—M0044—E09, 559B—
HMWK, not LMWK M0003—C08, 559B—M0039—H06, 559B—M0039—D08, 559B—
M0068—C07, 559B—M002l—Gll, 006l—G06, 559B—
M0036—Gl2, 559B—M0042—E06, 559B—M0070—H10, 559B—
M0068—D01, 559B—M0004—E08
Bind to cleaved and intact 559B—M0069—C09, 559B—M0038—F04, 559B—M0044—C05, 559B—
HMWK and LMWK M0047—H01, 559B—M0019—El2, 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—B l l, 559B—M0067—Gl 1, 559B—
M0065-B10, 559B-M0064—HO2
Mainly bind to LMWK 559B—M0037—E08
Specifically Bind to 559B—M0004-B04
cleaved HMWK
l antibodies were ed that bound to both l—chain, 2—chain HMWK and
LMWK, such as 0064—H02. These antibodies are likely to bind an epitope in domains 1
through 4, which are shared n HMWK and LMWK. M070—H10 is an example of an
antibody presumed to bind an epitope shared between l—chain and 2—chain HMWK but not on
LMWK. LMWK is a kininogen splice variant leads to a truncated protein composed of domains
1 through 4 and part of domain 5 (Colman et al. Blood (1997) 90: 3819—3843). Consequently,
antibodies such as 10 are likely to bind domain 5 or domain 6.
As shown in , panel A, 559B—M0004—B04 exhibited selectivity for 2—chain over
both l—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 uL of 2 ug/mL) was passively immobilized on a 96 well plate (Nunc
Maxisorp plate) (, panel B). The following day, the plate was washed and then blocked
with 2% BSA (Protease/IgG free) in PBS buffer. Following a wash, samples containing cleaved
HMWK in 0.1% BSA buffer in PBS with 0.05% Tween—20 (2—Chain HMWK assay buffer).
Purified protein standards (e.g., 2—chain HMWK, intact HMWK or LMWK) were spiked into
HNKW HMWK—deficient plasma and d 1:320 in 2—chain HMWK assay buffer. Following
plate washing with PBST, a mixture of 2 mouse monoclonal antibodies (1 lH05 and 13B 12) at l
ug/mL in 2—chain HMWK assay buffer were added for 1 hour at room temperature. d
detection antibodies were washed and a 1:2000 dilution of goat anti—mouse secondary antibody
conjugated to adish peroxidase (HRP) was added. The assay containing the ary
antibody was incubated for 1 hour at room temperature, and unbound secondary antibody was
removed by washing with 2—chain HMWK assay buffer. Signal was detected by the addition of
3, 3’,5,5’—tetramethylbenzidine (TMB), an HRP substrate. The reaction was stopped with
phosphoric acid. Hydrolysis of a TMB substrate was ed using a microplate reader at 450
nm (. Additionally, ming the ELISA assay using samples containing
cleaved HMWK in 2—chain HMWK assay buffer buffer or HMWK—deficient plasma and
analyzed in the presence of either 2.5% or 10% plasma resulted in similar binding (.
Using these immunoassay conditions, specifically binding to cleaved HMWK was detected.
The assay resulted in comparable performance when HMWK was provided in either 2—chaim
HMWK assay buffer or HMWK—deficient plasma (FIGs. 3 and 4). Furthermore, there was no
g of 559B-M0004-B04 to LMWK.
The ELISA assay was evaluated for detection of cleaved HMWK generated upon t
activation in human plasma (FIGs. 5A and 5B). The amount of cleaved HMWK in normal
human plasma was measured in the absence or presence of a catalytic amount of FXIIa, pKal, or
ellagic acid, which causes FXII ctivation to FXIIa and uently generation of cleaved
HMWK (FIGs. 5, panels A and B). Consistent with the role of plasma kallikrein as the primary
plasma enzyme required for the tion of 2—chain HMWK, neither ellagic acid nor FXIIa
addition lead to the generation of cleaved HMWK in prekallikrein—deficient plasma. The contact
system in FXI deficient plasma was y activated using either FXIIa, pKal, or ellagic; a
result consistent with the understanding that FXIa is generated by FXIIa and does not produce 2—
chain HMWK.
The results from the 2—Chain HMWK ELISA were corroborated by detecting cleaved
HMWK generated upon contact activation in human plasma by n blot analysis using the
mouse monoclonal antibody, 11H05 (). The 11H05 antibody specifically binds the light
chain of HMWK and nates both the 56 kDa light chain and the further proteolyzed 46 kDa
light chain, which is subsequently generated through the lytic activity of plasma kallikrein
at a site near the inus of the HMWK light chain (Colman et al. Blood (1997) 90: 3819—
3843).
The ELISA assay was also evaluated for the ability to detect cleaved HMWK generated
in plasma from 12 normal donors (. Following ellagic acid tion of the t
activation system, cleaved HMWK was detected in each of the 12 samples. The amount of
cleaved HMWK was also measured after the contact activation system was inhibited in normal
plasma using s concentrations of landadelumab (DX—2930; a specific inhibitor of plasma
kallikrein) or an inhibitor of the serpin Cl—INH, then activated with ellagic acid ( panels
A and B). Landadelumab (DX—2930) is a fully human antibody potent (Ki 2 0.12 nM) and
specific inhibitor of plasma kallikrein that was discovered using phage and is in clinical
development for the prophylactic treatment of HAE—ClINH attacks (Chyung et al. Ann. Allergy
Asthma Immunol. (2014) 113: 460—466; Kenniston et al. J. Biol. Chem. (1994) 289: 23596—
23608). When lanadelumab was spiked into citrated plasma at different concentrations it
effectively inhibited the generation of 2—chain HMWK induced by FXIIa as shown by Western
blot and sandwich ELISA (). The IC50 for lanadelumab inhibition of n HMWK
generation was 212 i 28 nM, which is consistent with the value expected for the activation of all
prekallikrein in neat plasma (approximately 500 nM). The complete inhibition of signal by
elumab in plasma treated with a contact system activator confirms that M004—B04 is
specific for 2—chain HMWK generated by plasma kallikrein.
Activation of the contact system in kininogen—deficient plasma did not yield an increase
in ELISA signal in this preliminary assay using M004—B04 as the capture dy and a HRP—
conjugated sheep polyclonal anti—kininogen as the detection antibody (data not shown).
It is also evident from that plasma collected from a healthy subject using EDTA
as an anti—coagulant was activated similarly as citrated plasma; supporting the observation that
metal ions are not required for contact system activation (Colman et al. Blood (1997) 90: 3819—
3843). However, 2—chain HMWK was not detected by ELISA in EDTA plasma ()
ting that M004—B04 antibody binding to 2—chain HMWK is dependent upon a metal ion.
A zinc binding site on HMWK in domain 5 (amino acids 479—498) of the light chain was
previously identified and shown to mediate gen interactions with the endothelial cell
surface receptors gClqR, cytokeratin l, and the urokinase plasminogen activator receptor and
thereby enhance contact system activation n et al. Adv. Immunol. (2014) 121: 41—89;
vist et al. Biol. Chem. (2013) 394: 1195—1204). The addition of ZnClz to the assay buffer
was tested at various concentrations and was found to enhance binding of the dy to
cleaved HMWK (). Increasing concentrations of ZnCl2 on the ELISA signal observed
with ellagic acid activated citrated and EDTA plasma was investigated. The ELISA signal in
EDTA plasma increased to an apparent maximum at ZnClz concentrations above 400 uM (in
well concentration).
Binding of l—chain HMWK to zinc was usly shown using electron microscopy to
promote a more compact and spherical quaternary structure (Herwald et al. Eur J. m.
(2001) 268: 4). It was also shown by on microscopy that 2—chain HMWK adopts a
more elongated, less spherical, quaternary ure than l—chain HMWK in a buffer containing
EDTA (Herwald et al. Eur J. Biochem. (2001) 268: 396—404). Though the effect of zinc on the
ure of n HMWK was not previously reported, the apparent zinc dependent binding
of M004—B04 described herein suggests the 2—chain HMWK exists in a unique conformation in
the presence of zinc.
The EDTA concentration in plasma collected in commercially available spray coated
KZEDTA tubes is approximately 4 mM, which following a 1:20 dilution converts to an in—well
concentration of approximately 200 uM and is consistent with the restoration of Zinc—dependent
binding upon addition of sufficient ZnClz to overwhelm the chelating capacity of EDTA. In
contrast, the ELISA signal ed plasma activated using ellagic acid was not increased in the
presence of 25 or 50 uM ZnClz (in well concentrations) but at trations above 100 uM
ZnClz the ELISA signal increased to a maximum above 200 uM ZnClz. () The normal
tration for zinc in plasma from healthy volunteers is 10—17 uM (Wessells et al. J. Nutr.
(2014) 144: 1204—1210). Since the ELISA signal observed in activated citrated plasma only
increased when with in—well ZnClz concentrations > 50 uM, which would equates to in—plasma
concentrations >l mM, it appears that the ELISA is not susceptible to physiologic fluctuations in
the concentration of zinc in the plasma. Consequently, the subsequent ments did not add
ZnClz to the assay buffer.
As described above, the binding of 004—B04 to 2—chain HMWK was enhanced
by supra—physiologic concentrations of ZnClz and inhibited by metal chelation with high
concentrations of EDTA. A zinc binding site has been described in domain of 2—chain HWMK
and a synthetic peptide encompassing this site (HKH20, HKHGHGHGKHKNKGKKNGKH
(SEQ ID NO: 83) was shown to inhibit contact system activation via an attenuation of cell
surface association (Nakazawa et al. Int. Immunopharmacol. (2002) 2: 1875-1885).
Consequently, the HKH20 peptide, as well as the GCP28 peptide corresponding to sequences in
domain 3 were tested for their ability to inhibit 2—chain HMWK binding to 559B—M004—B04 by
ELISA. As shown in , the HKH20 peptide but not the GCP28 peptide inhibits n
HMWK binding to M004—B04, which suggests that the M004—B04 e could reside within
domain 5 in the vicinity of the zinc g site. To perform the assay, the kininogen peptides
were diluted to 250 ug/mL and allowed to preincubate on assay plate. Then, purified 2—chain
HMWK in deficient human plasma was d 160 and then added to plate.
Time dependence of generation of cleaved HMWK in normal citrated human plasma was
assessed at various time points following tion of the contact tion system with ellagic
acid or FXIIa (. Finally, the ELISA assay was used to assess the presence and quantity
of cleaved HMWK in plasma samples from patients with hereditary angioedema (HAE)
compared to citrated plasma samples from normal patients (without HAE). The samples from
patients with HAE were found to contain elevated levels of 2—chain HMWK (1423 i 603
ng/mL) relative to s from normal donors (432.4 i 186 ng/mL) (, which are
statistically different (P: 0.017) by one way ANOVA analysis.
Having determined that M004—B04 specifically binds a neo—epitope on 2—chain HMWK
that is not present on l—chain HMWK or LMWK and demonstrating that the antibody binding is
dependent on plasma kallikrein activity, the assay was also tested using a pair of mouse
monoclonal antibodies (1 lH05 and 13B 12) for the detection (). Antibody 13B 12
appears to bind the heavy chain of HMWK and llH05 appears to bind the light chain of
HMWK, the combination of both antibodies for detection ed in a signal boost, ly due
to their non—overlapping binding epitopes in the antigen.
The importance of plasma collection on the assessment of contact system has been
previously described (Suffritti et al. Clin. Exp. Allergy (2014) 44: 1503—1514). It is well known
that contact of plasma with glass or other polar es results in extensive ex vivo contact
system activation that can mask the accurate determination of endogenous contact system
activation (Colman et al. Blood (1997) 90: 3819—3 843). The ability of the optimized sandwich
ELISA to detect 2—chain HMWK was compared in different plasma types, including a
customized plasma containing a mixture of se inhibitors in acid citrate dextrose in an
evacuated, plastic blood collection tube referred to as 9 (HTI, Essex Vt). As shown in
the standard curve prepared in SCATl69 plasma is less sensitive than the curve prepared
in citrated plasma, likely due to the inclusion of 2 mM EDTA in the collected plasma. At the
plasma on used in this assay (1:320) this tration of EDTA (3.1 uM) does not
interfere significantly with the n HMWK and may assist in stabilizing the plasma from
proteolytic degradation due to metalloproteases.
Citrated and SCATl69 plasma from y volunteers was compared to samples from
HAE patients by n blot and the sandwich ELISA assay. In , panels A—C, the
Western blot method of detecting 2—chain HMWK (i.e. cleaved kininogen) in citrated plasma
was capable of entiating samples from HAE ts from healthy volunteers (HV), as
shown by receiver operator characteristic (ROC) analysis with an area under the curve (AUC)
value of 0.977 for the comparison of basal to HV, or 1.0 for the comparison of attack to HV.
Citrated plasma samples from HAE patients during quiescence (basal) were differentiated from
attack samples with an AUC of 0.625 (, panel D).
As shown in , panels A—C, the Western blot method of detecting 2—chain HMWK
in SCATl69 plasma was capable of differentiating samples from HAE ts from samples
from healthy volunteers (HV), as shown by ROC analysis an AUC value of 0.915 for the
comparison of basal to HV, or 0.967 for the comparison of attack to HV. SCATl69 samples
from HAE patients during quiescence (basal) were differentiated from from attack samples with
an AUC of 0.597 (, panel D).
In , panels A—C, the 2—chain ELISA method of detecting 2—chain HMWK in
citrated plasma was capable of differentiating samples from HAE patients 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 attack to HV. Citrated plasma samples from HAE
patients during quiescence (basal) were differentiated from from attack samples with an AUC of
0.709 (, panel D).
As shown in , panels A—C . the 2—chain ELISA method of detecting 2—chain
HMWK in SCATl69 samples was capable of differentiating samples from HAE patients from
y 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 attack to HV. Citrated plasma samples from HAE
patients during quiescence (basal) were differentiated from from attack s with an AUC of
0.8176 (, panel D).
For the above ROC analysis, both the 2—chain HMWK Western blot and the 2—chain
HMWK ELISA trated herein may be useful in differentiating patients having or at risk
of having HAE based on the levels of d kininogen in plasma, as compared to healthy
volunteers. The presence of protease inhibitors in SCATl69 plasma reduced the ex vivo plasma
activation during blood collection.
OTHER MENTS
All of the features disclosed in this specification may be combined in any combination.
Each feature disclosed in this specification may be replaced by an alternative feature serving the
same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature
disclosed is only an example of a generic series of equivalent or similar features.
From the above ption, one skilled in the art can easily ascertain the essential
characteristics of the present disclosure, and without departing from the spirit and scope thereof,
can make various changes and modifications of the present disclosure to adapt it to various
usages and conditions. Thus, other embodiments are also within the claims.
EQUIVALENTS AND SCOPE
Those skilled in the art will ize, or be able to ascertain using no more than routine
mentation, many lents to the specific embodiments of the t disclosure
described herein. The scope of the present disclosure is not intended to be limited to the above
description, but rather is as set forth in the appended claims.
In the claims articles such as 6‘ :9 en
a an,” and “the” may mean one or more than one unless
indicated to the ry or otherwise evident from the context. Claims or descriptions that
include “or” n one or more members of a group are considered satisfied if one, more than
one, or all of the group members are present in, employed in, or otherwise relevant to a given
product or process unless indicated to the ry or otherwise evident from the context. The
present disclosure includes embodiments in which exactly one member of the group is present
in, employed in, or otherwise relevant to a given product or process. The present disclosure
includes embodiments in which more than one, or all of the group members are present in,
employed in, or otherwise relevant to a given product or process.
Furthermore, the present sure encompasses all variations, combinations, and
permutations in which one or more limitations, elements, clauses, and descriptive terms from
one or more of the listed claims is introduced into another claim. For e, any claim that is
ent on another claim can be modified to include one or more limitations found in any
other claim that is dependent on the same base claim. Where elements are presented as lists, e.g.,
in Markush group format, each subgroup of the elements is also disclosed, and any element(s)
can be removed from the group. It should it be understood that, in general, where the present
disclosure, or aspects of the present disclosure, is/are ed to as comprising particular
elements and/or features, certain embodiments of the present disclosure or aspects of the present
disclosure consist, or consist essentially of, such elements and/or features. For es of
simplicity, those ments have not been specifically set forth in haec verba herein. It is
also noted that the terms “comprising” and “containing” are ed to be open and permits the
inclusion of additional elements or steps. Where ranges are given, endpoints are included.
Furthermore, unless otherwise indicated or otherwise evident from the context and
understanding of one of ordinary skill in the art, values that are expressed as ranges can assume
any specific value or sub—range within the stated ranges in different ments of the present
disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly
dictates otherwise.
This ation refers to various issued patents, published patent ations, journal
articles, and other publications, all of which are incorporated herein by reference. If there is a
con?ict between any of the incorporated references and the instant specification, the
specification shall control. In on, any particular embodiment of the present present
sure that falls within the prior art may be explicitly excluded from any one or more of the
claims. Because such ments are deemed to be known to one of ordinary skill in the art,
they may be excluded even if the exclusion is not set forth explicitly herein. Any particular
embodiment of the present disclosure can be excluded from any claim, for any reason, whether
or not related to the existence of prior art.
Those skilled in the art will recognize or be able to ain using no more than routine
experimentation many equivalents to the specific embodiments bed herein. The scope of
the present embodiments described herein is not intended to be limited to the above Description,
but rather is as set forth in the appended claims. Those of ry skill in the art Will appreciate
that various changes and modifications to this description may be made Without departing from
the spirit or scope of the present disclosure, as defined in the following claims.
Claims (28)
1. An immunoassay method for detecting a cleaved high molecular weight kininogen (HMWK), the method comprising: (i) providing a support member, on which a first agent that specifically binds a cleaved HMWK is immobilized; (ii) contacting the support member of (i) with a biological sample suspected of containing a cleaved HMWK; (iii) contacting the support member obtained in (ii) with a second agent that binds HMWK, wherein the second agent is conjugated to a label; and (iv) ing a signal released from the label of the second agent that is bound to the support member, directly or indirectly, to determine the level of the cleaved HMWK in the biological sample; wherein the first agent is an antibody comprising a heavy chain complementarity determining region (CDR) 1 sequence FSFYVMV, a heavy chain CDR2 sequence GISPSGGNTAYADSVK, and a heavy chain CDR3 sequence KLFYYDDTKGYFDF and a light chain CDR1 ce SGSSSNIGSNYVY, a light chain CDR2 sequence RNNQRPS, and a light chain CDR3 sequence AWDDSLNGRV.
2. The immunoassay of claim 1, wherein the dy comprises a heavy chain le region (VH) sing the amino acid sequence of SEQ ID NO: 4 and a light chain variable region (VL) comprising the amino acid of SEQ ID NO: 5.
3. The immunoassay method of claim 1 or 2, wherein the support member is a 96- well plate.
4. The immunoassay method of any one of claims 1-3, n, prior to step (ii), the support member of (i) is incubated with a blocking buffer.
5. The immunoassay method of any one of claims 1-4, n the second agent is a polyclonal antibody, a monoclonal antibody, or a mixture of two or more monoclonal antibodies that bind to HMWK.
6. The immunoassay method of any one of claims 1-5, n the label is a signal releasing agent.
7. The immunoassay method of any one of claims 1-6, wherein the label is a member of a receptor-ligand pair and the immunoassay further comprises, prior to step (iv), contacting the second agent in (iii) that is bound to the support member, with the other member of the receptor-ligand pair, wherein the other member is conjugated to a signal releasing agent.
8. The immunoassay method of claim 7, wherein the or-ligand pair is biotin and streptavidin.
9. The immunoassay method of any one of claims 1-8, wherein the immunoassay is a Western blot assay, an ELISA assay, or a lateral flow assay.
0. The immunoassay method of any one of claims 1-9, wherein step (ii) is performed in the presence of ZnCl2.
11. The immunoassay method of any one of claims 1-10, wherein the biological sample is ed from a human subject.
12. The immunoassay method of claim 11, wherein the biological sample is a serum sample or plasma sample, which is processed from a blood sample collected in an evacuated blood collection tube comprising one or more se inhibitors.
13. The immunoassay method of claim 11 or claim 12, wherein human subject has a disease and wherein the immunoassay further comprises determining whether the e is mediated by plasma kallikrein (pKal) based on the level of the cleaved HMWK determined in step (iv), a deviation of the level of the cleaved HMWK in the biological sample from that of a control sample being indicative that the disease is mediated by pKal.
14. The immunoassay method of claim 11 or claim 12, further comprising determining whether the human subject has or is at risk for a disease ed by plasma kallikrein based on the level of the cleaved HMWK determined in step (iv), wherein if the level of the cleaved HMWK of the biological sample from the subject deviates from the level of the d HMWK of a control sample, the subject is identified as having or at risk of having the disease.
15. The immunoassay method of claim 14, wherein the subject is to be administered an effective amount of a therapeutic agent for treating the e, if the subject is identified as having the disease.
16. The immunoassay method of claim 15, wherein the therapeutic agent is a plasma kallikrein (pKal) inhibitor, a bradykinin 2 receptor (B2R) inhibitor, and/or a C1 esterase inhibitor.
17. The immunoassay method of claim 16, wherein the pKal inhibitor is an anti-pKal antibody or an inhibitory peptide.
18. The immunoassay method of claim 17, wherein the therapeutic agent is lanadelumab, ecallantide, icatibant, or human plasma-derived C1-INH.
19. The immunoassay method claim 11 or claim 12, wherein the human subject is on a treatment for the disease, and wherein the method further comprises assessing the efficacy of the ent based on the level of the d HMWK determining in step (iv), a deviation of the level of the cleaved HMWK in the biological sample from the subject from that of a control sample being indicative of the treatment efficacy.
20. The immunoassay method of claim 11 or claim 12, further comprising identifying a suitable treatment for the subject based on the level of the cleaved HMWK.
21. The immunoassay method of claim 11 or claim 12, further sing identifying the subject as a ate for a ent of the disease based on the level of the cleaved HMWK.
22. The immunoassay method of any one of claims 11, 12, or 14-21, wherein the human subject has a history of the e.
23. The immunoassay method of claim 22, wherein the e is hereditary angioedema (HAE).
24. The immunoassay method of claim 11 or claim 12, n the human subject has a y of HAE, and wherein the immunoassay 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 biological sample from the subject from that of a control sample being indicative of the risk of disease attack.
25. The immunoassay method of claim 24, wherein the subject is to be administered a therapeutic agent, if the t is at risk of disease attack.
26. An ed antibody, which specifically binds a cleaved high molecular weight kininogen (HMWK); wherein the antibody comprises a heavy chain complementarity determining region (CDR) 1 sequence FSFYVMV, a heavy chain CDR2 sequence GISPSGGNTAYADSVK, and a heavy chain CDR3 ce KLFYYDDTKGYFDF, and a light chain CDR1 sequence SGSSSNIGSNYVY, a light chain CDR2 sequence RNNQRPS, and a light chain CDR3 sequence AWDDSLNGRV.
27. The isolated antibody of claim 26, n the antibody comprises a heavy chain variable region (VH) comprising the amino acid sequence of SEQ ID NO: 4 and light chain variable region (VL) comprising the amino acid sequence of SEQ ID NO: 5.
28. A kit for detecting a cleaved high molecular weight kininogen (HMWK), the kit comprising a first agent that specifically binds a cleaved HMWK; wherein the first agent is an antibody of claim 26 or claim 27.
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PCT/US2016/057640 WO2017070170A1 (en) | 2015-10-19 | 2016-10-19 | Immunoassay to detect cleaved high molecular weight kininogen |
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