DETECTION OF CLEAVAGE ACTIVITY OF AN ENZYME
FIELD OF THE INVENTION
The present invention relates to detecting cleavage activity of an enzyme. The various
aspects of the invention include an enzyme detection device, kit, method and use for
detecting or measuring the presence in a test sample of the activity of an enzyme capable of
cleaving a substrate. The invention also relates to indicator and binding molecules useful for
1 0 carrying out the invention.
BACKGROUND TO THE INVENTION
Enzymes constitute a family of proteins involved in catalysing chemical reactions within living
15 organisms. As a result of their importance, there are numerous situations in which it is
necessary and/or beneficial to measure enzyme levels, and importantly, enzyme activity.
In particular, increases in enzyme activity have been found to correlate with specific
conditions and/or diseases. For example up-regulated protease activity has been associated
20 with many aspects of cancer progression. The measurement of enzyme activity in samples
taken from individuals with a particular condition or suspected of having a specific condition
or disease may therefore be useful for prognostic or diagnostic purposes.
Within the enzyme family, there are many classes of enzyme that act by facilitating substrate
25 cleavage. For example, peptidases and proteases catalyse the hydrolysis of peptide bonds
within their respective substrates. In the past, researchers have, in some cases, sought to
measure this type of activity using kits or devices that measure release of a fragment or
'leaving group' from the initial enzyme substrate.
30 Assays based on this fundamental principle have been refined such that in some cases,
inventors have described engineered substrate molecules linked to reporter moieties.
Cleavage of the substrate by the enzyme to be detected, if present, leads to release of said
reporter, which can be detected by a range of techniques available to those skilled in the art.
An assay of this type is described for example in US2006/0003394.
35
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Others have sought to develop assays for the measurement of enzyme activity based around
the principle of discriminating between modified and unmodified forms of an enzyme
substrate. In this regard, W02009/024805 describes an enzyme detection device utilising a
"substrate recognition molecule" (SRM) carrying a detectable label, wherein the SRM
5 specifically binds to the enzyme substrate in the unmodified state and in doing so presents
binding of the enzyme to the substrate.
US 5,171 ,662 describes methods for identifying compounds that inhibit HIV protease. The
methods are based on a competitive binding radioimmunoassay. US 2005/0164311
1 0 describes methods for detecting reaction products to indicate the presence of a reaction
product inducer such as an enzyme.
DESCRIPTION OF THE INVENTION
15 The present invention results from attempts to improve sensitivity and/or specificity of
protease activity detection. In particular, in certain test samples such as urine and
environmental samples, proteases may be present in extremely low concentrations. The
devices and methods described herein aim to permit detection of protease activity at low
levels or concentrations. It has been found that use of binding molecules, such as antibodies,
20 that bind only to specific products of cleavage but not to the uncleaved indicator molecule,
enable detection of protease activity at low concentrations in test samples (in particular urine
samples). In the context of the claimed flow devices, specific assays can be performed
where the reagents can be employed in excess without impacting on specificity of detection.
It is also shown herein that the claimed flow devices provide diagnostically useful results,
25 where comparable enzyme immunocapture and activity assays do not. Without being bound
by theory this may be due to differing binding efficiencies throughout the different processes.
Accordingly, in one aspect, the invention provides an enzyme detection device for detecting
the presence in a test sample of cleavage activity of an enzyme capable of cleaving a
30 substrate, the device comprising:
(i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by said
enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
35 wherein cleavage of the at least one cleavage site produces a novel binding site;
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(ii) a capture zone to receive the test sample, wherein the capture zone comprises capture
molecules capable of binding to the capture site of the indicator molecule in order to
immobilise the indicator molecule including the novel binding site; and
(iii) binding molecules capable of binding to the novel binding site, wherein the binding
5 molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred.
Similarly, the invention provides an enzyme detection device for detecting the presence in a
test sample of cleavage activity of an enzyme capable of cleaving a substrate, the device
1 0 comprising:
(i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
15 wherein cleavage of the at least one cleavage site produces at least two parts of the
cleavage region, at least one part of which remains connected to the capture site;
(ii) a capture zone to receive the test sample, wherein the capture zone comprises capture
molecules capable of binding to the capture site of the indicator molecule; and
(iii) binding molecules capable of binding to the part of the indicator molecule containing the
20 at least one part of the cleavage region connected to the capture site, wherein the binding
molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred.
The two parts of the cleavage region are thus separated from one another at the site of
25 cleavage. The cleavage event at the site of the cleavage produces the novel binding site
The invention further provides a method for detecting the presence or absence in a test
sample of cleavage activity of an enzyme capable of cleaving a substrate, the method
comprising:
30 (i) bringing an indicator molecule into contact with the test sample, said indicator molecule
comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by said
enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
35 wherein cleavage of the at least one cleavage site produces a novel binding site;
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(ii) adding to the test sample binding molecules capable of binding to the novel binding site,
wherein the binding molecules are incapable of binding to the indicator molecule unless and
until cleavage has occurred;
(iii) capturing the part of the indicator molecule containing the novel binding site at a capture
5 zone through binding of capture molecules in the capture zone to the capture site; and
(iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the novel binding site of the indicator molecule captured in the capture zone.
Similarly, the invention also provides a method for detecting the presence or absence in a
1 0 test sample of cleavage activity of an enzyme capable of cleaving a substrate, the method
comprising:
15
(i) bringing an indicator molecule into contact with the test sample, said indicator molecule
comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site
wherein cleavage of the at least one cleavage site produces at least two parts of the
cleavage region, at least one part of which remains connected to the capture site;
(ii) adding to the test sample binding molecules capable of binding to the part of the indicator
20 molecule containing the at least one part of the cleavage region connected to the capture
site, wherein the binding molecules are incapable of binding to the indicator molecule unless
and until cleavage has occurred;
(iii) capturing the part of the indicator molecule containing the at least one part of the
cleavage region connected to the capture site at a capture zone through binding of capture
25 molecules in the capture zone to the capture site; and
(iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the part of the indicator molecule captured in the capture zone.
The devices and methods of the invention have been shown by the inventors to have specific
30 application in the field of diagnosis of respiratory conditions. Thus, the invention further
provides a method for diagnosing (the presence or absence of) a respiratory condition in a
test sample by detecting cleavage activity of an enzyme capable of cleaving a substrate, the
method comprising:
(i) bringing an indicator molecule into contact with the test sample, said indicator molecule
35 comprising
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(a) a cleavage region comprising at least one cleavage site, which can be cleaved by said
enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
wherein cleavage of the at least one cleavage site produces a novel binding site;
5 (ii) adding to the test sample binding molecules capable of binding to the novel binding site,
wherein the binding molecules are incapable of binding to the indicator molecule unless and
until cleavage has occurred;
(iii) capturing the part of the indicator molecule containing the novel binding site at a capture
zone through binding of capture molecules in the capture zone to the capture site; and
10 (iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the novel binding site of the indicator molecule captured in the capture zone
wherein an increased level of cleavage compared to a control diagnoses the respiratory
condition.
15 The invention also provides a method for diagnosing (the presence or absence of) a
respiratory condition in a test sample by detecting cleavage activity of an enzyme capable of
cleaving a substrate, the method comprising:
(i) bringing an indicator molecule into contact with the test sample, said indicator molecule
comprising
20 (a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site
wherein cleavage of the at least one cleavage site produces at least two parts of the
cleavage region, at least one part of which remains connected to the capture site;
25 (ii) adding to the test sample binding molecules capable of binding to the part of the indicator
molecule containing the at least one part of the cleavage region connected to the capture
site, wherein the binding molecules are incapable of binding to the indicator molecule unless
and until cleavage has occurred;
(iii) capturing the part of the indicator molecule containing the at least one part of the
30 cleavage region connected to the capture site at a capture zone through binding of capture
molecules in the capture zone to the capture site; and
35
(iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the part of the indicator molecule captured in the capture zone, wherein an
increased level of cleavage compared to a control diagnoses the respiratory condition.
In specific embodiments, the respiratory condition is an inflammatory respiratory condition.
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In specific embodiments, the respiratory condition is chronic obstructive pulmonary disease
or inflammation of the respiratory tract (in particular the lungs) as a result of cystic fibrosis.
The inventors have shown herein that the devices and methods of the invention can usefully
5 be applied to measure elevated cleavage activity as an indicator of a respiratory condition. In
order to take into account background levels of cleavage activity, the methods involve
comparing measured levels of cleavage in the test sample to a control. Typically, the control
represents corresponding levels of cleavage activity in a healthy subject. By "healthy
subject" is meant a subject not suffering from the respiratory condition. The control may be
10 in a corresponding test sample taken from a matched healthy control. Alternatively, the
control may be a threshold level of cleavage set by determining cleavage activity in a range
of healthy and diseased patients. Suitable methods for setting a threshold are well known to
those skilled in the art. The threshold may be mathematically derived from a training set of
patient data. The score threshold thus separates the test samples according to presence or
15 absence of the respiratory condition. The interpretation of this quantity, i.e. the cut-off
threshold may be derived in a development or training phase from a set of patients with
known outcome. The threshold may therefore be fixed prior to performance of the claimed
methods from training data by methods known to those skilled in the art.
20 The enzyme detection devices of the invention may be supplied in a format ready for
immediate use. Alternatively, the essential components may be provided as a kit of parts,
optionally together with suitable reagents and/or instructions for assembly of the enzyme
detection device. Accordingly, in another aspect, the invention provides an enzyme detection
kit for detecting the presence in a test sample of cleavage activity of an enzyme capable of
25 cleaving a substrate, the kit comprising:
(i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by said
enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
30 wherein cleavage of the at least one cleavage site produces a novel binding site;
(ii) capture molecules capable of binding to the capture site of the indicator molecule
(iii) a solid support to which the capture molecules can be attached (i.e. are attachable or
attached) to form a capture zone to receive the test sample; and
(iv) binding molecules capable of binding to the novel binding site, wherein the binding
35 molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred.
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In another aspect, the invention provides an enzyme detection kit for detecting the presence
in a test sample of cleavage activity of an enzyme capable of cleaving a substrate, the kit
comprising:
5 (i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
wherein cleavage of the at least one cleavage site produces at least two parts of the
1 0 cleavage region, at least one part of which remains connected to the capture site;
(ii) capture molecules capable of binding to the capture site of the indicator molecule,
(iii) a solid support to which the capture molecules can be attached (i.e. are attachable or
attached) to form a capture zone to receive the test sample; and
(iii) binding molecules capable of binding to the part of the indicator molecule containing the
15 at least one part of the cleavage region connected to the capture site, wherein the binding
molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred.
In related aspects, the invention also provides for use of an enzyme detection device as
20 described and defined herein for diagnosing a respiratory condition in a test sample.
Similarly, the invention also provides for use of a method as described and defined herein for
diagnosing a respiratory condition in a test sample. The invention further provides for use of
an enzyme detection kit as described and defined herein for diagnosing a respiratory
condition in a test sample. In each of these uses, the respiratory condition may be chronic
25 obstructive pulmonary disease or inflammation of the respiratory tract as a result of cystic
fibrosis.
Thus, central to many of the aspects of the invention is the indicator molecule. The indicator
molecule comprises a cleavage region comprising at least one cleavage site. The cleavage
30 site is cleaved by any enzyme or enzymes in a test sample with the relevant enzyme
cleavage activity. The cleavage region provides a suitable context for the cleavage site to
ensure cleavage is efficient, if the enzyme is present in the sample. In specific embodiments
the cleavage region is a peptide. In addition to the peptide bond representing a protease
cleavage site, the additional amino acids in the peptide may ensure specificity and sensitivity
35 of cleavage. The cleavage region may contain multiple cleavage sites in certain
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embodiments, particularly where the indicator molecule is structurally constrained, for
example where it also comprises a scaffold molecule.
The indicator molecule also comprises a capture site (intended to encompass at least one
5 capture site). The capture site is a discrete region of the indicator molecule which permits
immobilization of the indicator molecule, whether cleaved or uncleaved, at a capture zone.
The capture site is discussed herein below in greater detail.
The indicator molecule also optionally comprises a scaffold molecule, as discussed in greater
1 0 detail below.
Cleavage of the indicator molecule splits the indicator molecule to reveal or form at least one
novel binding site. The two parts of the cleavage region are thus separated from one another
at the site of cleavage. Typically the novel binding site comprises a conformational epitope
15 produced as a consequence of cleavage. Use of binding molecules that bind specifically to
the newly revealed binding site or sites but not to the indicator molecule prior to cleavage
enables specific and sensitive detection of cleavage activity of an enzyme. Accordingly, in
some embodiments, cleavage of the at least one cleavage site produces at least two parts of
the indicator molecule (or cleavage region of the indicator molecule), at least one part of
20 which contains (or remains connected to) the capture site and as a consequence of cleavage
contains a binding site for binding molecules and wherein the binding molecules are
incapable of binding to the binding site unless and until cleavage has occurred. In other
words, the binding site is hidden or is not formed until cleavage at the cleavage site occurs.
25 In some embodiments, cleavage of the at least one cleavage site produces at least two
separate parts of the (cleavage region of the) indicator molecule. Thus, cleavage may
produce at least two parts or fragments; one part or fragment that contains or is connected to
the capture site and a separate part or fragment that does not contain, or is not connected to,
the capture site. The binding molecules bind to the new binding site on the part or parts of
30 the indicator molecule that contain or include the capture site. This permits specific detection
of cleavage at the site of capture of the indicator molecule through binding to the capture
molecules (i.e. binding of the binding molecules is detected in the capture zone).
However, it is not essential that cleavage (at the cleavage site) produces at least two
35 completely separate molecules, provided that cleavage produces a novel binding site for the
binding molecules and wherein the binding molecules are incapable of binding to the binding
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site unless and until cleavage has occurred. Thus cleavage produces two parts of the
cleavage region which are separated at the cleavage site. Accordingly, in some
embodiments, cleavage of the at least one cleavage site produces at least two parts of the
cleavage region, at least two parts of which remain connected, either directly or indirectly (for
5 each part), to the capture site. This is shown schematically in Figure 16A. In specific
embodiments the indicator molecule contains a further linkage or connection away from the
cleavage site or outside of the cleavage region such that cleavage of the at least one
cleavage site produces at least two parts of the cleavage region of the indicator molecule
which remain connected to one another. This does not exclude the possibility that cleavage
1 0 produces at least three fragments, at least one of which does not remain connected via the
further linkage or connection. This is particularly the case where the cleavage region may
comprise more than one cleavage site. This is shown schematically in Figure 168. The
further linkage or connection may comprise a disulphide bond in some embodiments. It has
been found that use of scaffold molecules, linked to the indicator molecule, provides a further
15 linkage or connection within the indicator molecules. Such scaffold molecules may act as a
structural constraint that is useful for developing binding molecules that bind to the indicator
molecule only after cleavage has occurred. Without being bound by theory, the structural
constraint is believed to assist in producing a specific and reproducible binding site that is not
present unless and until cleavage at the cleavage site has occurred. The scaffold molecule
20 may enhance the differences in spatial conformation between the indicator molecule pre- and
post-cleavage, as discussed in greater detail herein. The scaffold may also constrain the
cleaved indicator molecule in a particular spatial conformation following cleavage. This may
assist in improving specificity of detection in terms of the binding molecules discriminating
between cleaved and uncleaved indicator molecules, by providing a clearly defined and
25 different molecule after cleavage against which binding molecules can be designed or raised.
30
35
Thus, in some embodiments, the binding molecules bind to the region of cleavage. In
specific embodiments, the binding site may thus encompass both sides of the cleavage site
following cleavage (i.e. at least two parts of the cleavage region). The binding molecules
may bind to both parts of the indicator molecule following cleavage.
The invention therefore also provides an indicator molecule for use in detecting the presence
in a test sample of cleavage activity of an enzyme capable of cleaving a substrate, the
indicator molecule comprising:
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present,
(b) a capture site; and
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(c) a scaffold molecule which acts to connect at least two parts of the indicator
molecule outside of the cleavage site, such as outside of the cleavage region
wherein the scaffold further acts to structurally constrain the indicator molecule in a manner
such that cleavage of the at least one cleavage site produces a novel binding site to which
5 binding molecules bind, but wherein the binding molecules are incapable of binding to the
indicator molecule unless and until cleavage has occurred.
The invention also provides an indicator molecule for use in detecting the presence in a test
sample of cleavage activity of an enzyme capable of cleaving a substrate, the indicator
10 molecule comprising:
15
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present to produce at least two parts
of the cleavage region,
(b) a capture site; and
(c) a scaffold molecule which acts to connect at least two parts of the indicator
molecule such that cleavage of the at least one cleavage site produces at least two
parts of the cleavage region of the indicator molecule which remain connected to one
another
wherein the scaffold further acts to structurally constrain the indicator molecule in a manner
20 such that cleavage of the at least one cleavage site produces a (novel) binding site to which
binding molecules bind, but wherein the binding molecules are incapable of binding to the
indicator molecule unless and until cleavage has occurred.
The scaffold molecule is typically attached to the indicator molecule away from the cleavage
25 site so that cleavage activity of the enzyme is not inhibited by the scaffold. Thus the
cleavage region may be separated from the scaffold molecule by one or more linker or
spacer regions. Those linker or spacer regions may incorporate the capture site in some
embodiments. The scaffold molecule is typically linked to the indicator molecule by two
linkages, although it is possible that additional linkages can be employed- for example 3, 4,
30 5 or 6 etc. -linkages depending upon the scaffold molecule that is used and the nature of the
indicator molecule. It is also possible that a single scaffold molecule can be linked to multiple
indicator molecules. In embodiments where the scaffold molecules contain more than two
halogen substituents, in particular bromomethyl substituents, such as 4 or 6 bromomethyl
substituents the scaffold molecule may provide a structural constraint for multiple indicator
35 molecules. Each pair of substituents may be attached to connect at least two parts of a
cleavage region. Thus, the scaffold effectively links (and structurally constrains) multiple
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separate cleavage regions. In specific embodiments, the indicator molecules comprise more
than one constrained peptide (cleavage region). The cleavage regions can also be different
resulting in a single molecule containing different cleavable sequences. Here it may be
possible to detect cleavage of each individual peptide cleavage region using two or more
5 distinct binding molecules (e.g. antibodies raised against its cleaved substrate).
Consequently where an assay signal is required only when two or more proteases are
present it is possible that binding molecule (antibody) binding only takes place when all the
distinct cleavage sites have been cleaved. In this instance the binding molecule (antibody)
would have to be raised to the form of indicator molecule after cleavage by the two or more
1 0 proteases.
The scaffold molecule assists in constraining the cleaved ends or parts of the indicator
molecule (usually a peptide) to produce a novel and specific binding site for a binding
molecule (usually an antibody binding to a newly revealed or produced epitope, in particular
15 a conformational epitope). The binding molecule may, therefore, bind specifically to either
cleaved end or part of the indicator molecule or to both sides of the cleavage site (i.e. within
the cleavage region either side of the cleavage site). In specific embodiments, the scaffold
further acts to structurally constrain the indicator molecule in a manner such that cleavage of
the at least one cleavage site produces a binding site containing both parts of the cleavage
20 region of the indicator molecule to which binding molecules bind, but wherein the binding
molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred. In specific embodiments, the binding site includes the cleavage site. In specific
embodiments, the binding site represents a novel structural conformation of the indicator
molecule. Cleavage may produce at least one new conformational epitope. The novel
25 binding site for the binding molecule may comprise any part of the indicator molecule,
provided that enzyme cleavage activity and capture are not substantially impeded. In certain
embodiments, the binding site comprises at least a portion of the cleavage region. In specific
embodiments, the binding site comprises at least a portion of the scaffold molecule.
30 In most embodiments, the cleavage site is specific for cleavage by a protease. However, as
discussed herein, the indicator molecules of the invention may be cleaved by other enzymes
such as oxidoreductases, hydrolases and lyases, and include the subcategories of protease,
peptidase, lipase, nuclease, carbohydrase, phosphatase, sulphatase, neuraminidase,
esterase, DNAse, and RNAse. In certain embodiments, the cleavage site is specific for
35 cleavage by an endopeptidase. One or more different proteases may be detected according
to the invention. In certain embodiments, the cleavage site is specific for cleavage by a
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matrix metalloproteinase (MMP). This is particularly of relevance for the diagnostic
applications of the invention including detection in urine samples. MMPs are zinc-dependent
endopeptidases. They are responsible for cleaving various proteins, including extracellular
matrix proteins. The MMPs include MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP1 0,
5 MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP19, MMP20, MMP21,
MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27 and MMP28. In other embodiments,
the cleavage site is specific for an elastase, such as (human) neutrophil elastase (HNE). In
some embodiments, there may be a cleavage site for multiple proteases, such as multiple
MMPs and/or one or more MMPs together with HNE. A suitable HNE substrate comprises,
1 0 consists essentially of or consists of the following amino acid sequence:
CQESIRLPGC (SEQ ID NO: 3)
This substrate forms a separate aspect of the invention. The substrate may contain
15 additional residues to facilitate immobilisation such as a tyrosine residue to provide phenyl
groups for attachment. In particular, the substrate may contain additional amino acid
residues excluding cysteine residues so that alternative immobilisation chemistries can be
used. Thus, the substrate may comprise the following amino acid sequence:
20 YCQESIRLPGC (SEQ ID NO: 4)
The at least one cleavage site may be biased for cleavage by specific proteases in some
embodiments. This permits the invention to be utilised in order to detect specific protease
25 activity in the test sample. Many proteases are known and their sites of preferred cleavage
well reported. In certain embodiments, the at least one cleavage site is biased for cleavage
by specific matrix metalloproteinases. More specifically, in some embodiments, the at least
one cleavage site is biased for cleavage by MMP-13 and/or MMP- 9. The at least one
cleavage site may be biased for cleavage by MM P-13, 9, 2, 12 and 8. The bias may be for
30 the group of MMPs equally or may be in that particular order of preference. As is shown
herein, it is possible to design specific indicator molecules and cleavage sites within the
indicator molecules that are biased for cleavage by these particular MMPs, in the specified
order of preference. Accordingly, in some embodiments, the cleavage site is within the
amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ). This may be considered a specific
35 example of the "cleavage region" of the indicator molecule. In those embodiments, cleavage
produces a part of the cleavage region of the indicator molecule containing the amino acid
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sequence GPQG and a part of the cleavage region of the indicator molecule containing the
amino acid sequence IFQG. Either part can be the part connected to the capture site. In
specific embodiments, the indicator molecule comprises the amino acid sequence
CGPQGIFGQC (SEQ ID NO: 2). Inclusion of the cysteine residues provides thiol groups
5 which represent a convenient linkage point for various scaffold molecules. The cleavage
region may be separated from the attachment points for the scaffold molecule by one or
more linker or spacer regions in some embodiments. Thus, the indicator molecule may
comprise the structure:
10
Spacer - Cleavage region - Spacer
~ / Scaffold molecule
The capture site may be found within one or both of the spacers in some embodiments.
15 Thus, the indicator molecules of the invention may comprise suitable amino acids at or near
the N and C terminus to facilitate linkage to the scaffold molecule. The amino acids may
comprise thiol groups. Suitable residues include cysteine and selenium. The scaffold
molecules may be attached to the indicator molecules via thioether linkages.
20 A range of suitable scaffold molecules and methods for linking the scaffold molecules to a
peptide are discussed in W02004/077062 and W02008/0 13454, the relevant disclosures of
which are hereby incorporated by reference. The present invention applies these scaffold
molecules in a new manner to present cleavage sites and produce new binding sites after
cleavage which permit detection of enzyme cleavage activity (especially protease activity) in
25 a test sample.
In certain embodiments, the scaffold molecule comprises a (hetero)aromatic molecule. In
more specific embodiments, the (hetero)aromatic molecule comprises at least two benzylic
halogen substitutents. The scaffold molecule is a halomethylarene in some embodiments,
30 such as a halomethylarene selected from the group consisting of bis(bromomethyl)benzene,
tris(bromomethyl)benzene and tetra(bromomethyl)benzene, or a derivative thereof. In
specific embodiments, the scaffold is selected from the group consisting of ortho-, meta- and
para- dihaloxylene and 1 ,2,4,5 tetra halodurene, such as meta- 1 ,3-
bis(bromomethyl)benzene (m-T2), ortho- 1,2-bis(bromomethyl)benzene (o-T2), para- 1,4-
35 bis(bromomethyl)benzene (p-T2), meta-1,3-bis(bromomethyl)pyridine (m-P2), 2,4,6-
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tris(bromomethyl)mesitylene (T3), meta- 1,3-bis(bromomethyl)-5- azidobenzene (m-T3-N3)
and/or 1 ,2,4,5 tetrabromodurene (T4).
Suitable derivatives of halomethyl arenes include ortho, meta and para bis(bromomethyl)
5 benzenes. More specifically 1 ,2-bis(bromomethyl) benzene, 1 ,3-bis(bromomethyl) benzene
and 1 ,4-bis(bromomethyl) benzene. Further substituted halomethylarenes include 1 ,3,5-
tris(bromomethyl)benzene, 1 ,2,4,5-tetrakis(bromomethyl)benzene and 1 ,2,3,4,5,6-
hexakis(bromomethyl)benzene. Polycyclic halomethylarenes include 2,7-bis(bromomethyl)naphthalene,
1 ,4-bis(bromomethyl)-naphthalene, 1 ,8-bis(bromomethyl)-naphthalene, 1 ,3-
1 0 bis(bromomethyl)-naphthalene, 1 ,2-bis(bromomethyl)-naphthalene, 2,3-bis(bromomethyl)naphthalene,
2,6-bis(bromomethyl)-naphthalene, 1 ,2,3,4-tetrakis(bromomethyl)-naphthalene,
9,1 0-bis(bromomethyl)-phenanthrene, 5,1 0-bis(bromomethyl)-anthracene, and 1-
(bromomethyl)-3-[3-(bromomethyl)benzyl]benzene. Methyl substituted halomethylarenes
include 1 ,3-bis(bromomethyl)-5-methylbenzene, 2,5-bis(bromomethyl)-1 ,3-dimethylbenzene,
15 2,5-bis(bromomethyl)-1 ,4-dimethylbenzene, 2,4-bis(bromomethyl)-1 ,3,5-trimethylbenzene
and 3,6-bis(bromomethyl)durene. Nitro substituted halomethylarenes include 3,4-
bis(bromomethyl)-nitrobenzene and 2,3-bis(bromomethyl)-nitrobenzene. Hydroxy substituted
halomethylarenes include 1 ,3-bis(bromomethyl)-5-hydroxybenzene and cyano substituted
halomethylarenes include 2,6-bis(bromomethyl)-benzonitrile. Methoxy substituted
20 halomethylarenes include 1 ,3-bis(bromomethyl)-5-methoxybenzene, 1 ,3-bis(bromomethyl)-2-
methoxy-5-methylbenzene, 1 ,3-bis(bromomethyl)-5-hydroxybenzene, 2,3-bis(bromomethyl)-
1 ,4-dimethoxybenzene, and 2,5-bis(bromomethyl)-1 ,4-dimethoxybenzene.
Some suitable scaffold molecules for use in the indicator molecules of the invention are
25 shown in Figure 14. A number of specific suitable scaffold molecules are also shown,
together with proposed nomenclature, in Figure 15.
Due to their relative rigidity and ease of synthetic use, the halomethyl arene derivatives are
preferred candidates to act as scaffold molecules in the present invention. They are
30 particularly convenient for creating constrained peptide substrates. However one can
envisage other appropriate chemistries with which to "cyclise" the indicator molecule, such as
a peptide. In the case of peptides containing thiols (eg: in the form of cysteine), a simple
disulphide bond formation or a diepoxide derivative can be used to affect covalent closure of
the structure. Another appropriate chemistry includes the "click chemistry" method, involving
35 the cycloaddition reaction between azides and alkynes forming stable triazoles. Here for
example a peptide bearing two azido lysine amino acids could be intramolecularly cross
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linked by a dialkyne reagent. Such reactions can be catalysed by copper. However in some
examples such as those where a strained alkyne is used, no catalyst is required. A further
chemical route includes that of stable hydrazone formation. Indicator molecules (in particular
peptides) containing two phenyl hydrazine moieties may be cross linked intramolecularly via
5 a dialdehyde reagent. A further chemical route is possible through peptide-based indicator
molecules containing two tyrosine amino acids. These peptides can be intramolecularly
crosslinked using a bis(diazo) scaffold to form the corresponding diazo adduct.
The scaffold molecules may also include further functionalities or reactive groups to facilitate
1 0 generation of a novel binding site following enzymatic cleavage of the cleavage site. Thus,
following cleavage at the cleavage site there are at least two parts of the cleavage region of
the indicator molecule which are no longer connected to one another through the cleavage
site. One or more of those "free" parts may become further constrained by interaction with
the scaffold molecule. This may produce a significant change in structure of the overall
15 molecule. This in turn permits specific binding molecules to be generated which will not
cross-react with the indicator molecule prior to cleavage. Thus, by way of example, in the
case of peptides constrained by a scaffold molecule one can envisage a specific
conformational change after cleavage of the cleavage site. The afforded degrees of freedom
in the peptide chain may allow it to self-assemble via non covalent interactions in a new
20 stable conformation, creating a new conformational epitope unique to the molecule and
recognised by the binding molecule (such as an antibody raised against the cleaved
substrate). These non-covalent interactions may comprise hydrophobic interactions between
the amino acid side chains and the aromatic rings in the scaffold molecule. The non-covalent
interactions can be further enhanced in scaffolds with extended substitution patterns such
25 that for example a negatively charged nitro substituent can interact with positively charged
amino acids such as lysine, arginine or histidine included within the cleavage region.
Hydrogen bond interactions are also possible between methoxy and/or hydroxyl aryl
substituents and a number of amino acids, including serine, threonine and tyrosine. In
addition the two cleaved peptide parts of the cleavage region may be free to self-assemble
30 with each other inducing a secondary structure such as a helix or beta stranded structure
after cleavage. In further embodiments, a combination of both peptide-peptide interactions
and peptide-scaffold interactions, as described above, may produce a novel binding site
recognised by a binding molecule. Such interactions serve to differentiate the structure in 3
dimensional space between its uncleaved "closed" form and its "open" form following
35 cleavage and hence significantly enhance the specificity of interaction between the cleaved
indicator molecule and the binding molecule (e.g. an antibody raised against the cleaved
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peptide product). The resulting high specificity of interaction is beneficial to the sensitivity of
detection of enzyme cleavage activity within the sample because it facilitates use of the
indicator molecule in excess without the risk of the binding molecule binding to uncleaved
indicator molecule (e.g. the antibody raised against the cleaved peptide from binding to the
5 u ncleaved peptide).
The scaffold should not prevent cleavage at the one or more cleavage sites. In some
embodiments, the scaffold may orientate the (cleavage region of the) indicator molecule to
optimise or improve efficiency of cleavage at the cleavage site. The scaffold may effectively
10 fix or constrain the cleavage region to present the cleavage site in a favourable manner for
the enzyme activity to be detected. The effect of the scaffold molecule on cleavage of any
given substrate can readily be tested by a simple time course experiment. A test may
determine whether cleavage occurs in the presence of the enzyme within a reasonable time
(e.g. 5-10 minutes). This testing can be qualified, for example through mass spec analysis,
15 optionally in combination with HPLC as it should evolve a new hydrolysed molecule (with a
different molecular mass) which should also retain differently on a reverse phase analytical
column. Those indicator molecules incorporating a scaffold molecule can, for example, then
be prepared as an immunogen in its purified cleaved form. This can be used to raise
antibodies in a suitable animal such as a sheep, either as free peptide or conjugated to a
20 carrier protein. Antisera may then be characterised by ELISA to immobilised antigen and an
antigen column may be used to affinity purify and refine the polyclonal response specifically
to the cleaved indicator molecule. The complete indicator molecule may then be tested
according to the methods of the invention.
25 A specific indicator molecule of the invention is shown in Figure and comprises the amino
acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 in which the thiol groups of the cysteine
residues are used to cyclise the peptide via linkage to a halomethylarene group. Thus, an
indicator molecule of the invention may comprise the structure set forth as formula I below
(and shown in Figure 23).
30
(I)
The cleaved version is shown in formula II below (and also shown in Figure 23).
5
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s-' A,t..
-~- 'P,
C_o G,c
~- ........ "- \_s~s_} o,
v (II)
In some embodiments, the indicator molecule lacks the tyrosine residue shown in formulas I
and II. This tyrosine residue is not required for HNE activity.
A range of suitable binding molecules for use in the invention are disclosed herein, which
discussion applies mutatis mutandis here. Typically, the binding molecule comprises an
antibody.
1 0 For the avoidance of doubt, these indicator molecules may be employed in any of the other
aspects of the invention (devices, kits, methods, uses etc.).
In the context of the invention as a whole, the one or more cleavage sites may be any site at
which an enzymatically-cleavable bond is present. For example, this bond may be present
15 between neighbouring residues of the indicator molecule. Such residues may be selected
from nucleotides, monosaccharides, and amino acids. The indicator molecule typically
comprises a peptide cleavage region. Thus, in some embodiments, the cleavage region
comprises a sequence of amino acids. In a preferred embodiment of the invention, the
cleavage site is a specific peptide bond located between two amino acid residues.
20
In further embodiments of the invention, the at least one cleavage site is located within a
peptide, a protein, a carbohydrate, a lipid or a nucleic acid cleavage region. In certain
embodiments, the indicator molecule may be engineered such that it comprises the enzyme's
natural substrate or a portion thereof, such that the enzyme is presented with its native
25 cleavage site, optionally in its native state within the cleavage region. In certain other
embodiments, the indicator molecule may be engineered such that it comprises an artificial
or non-native cleavage site and/or substrate region. For example, the cleavage site in the
indicator molecule may be engineered or mutated such that the rate of cleavage activity or
specificity of cleavage activity exhibited by the enzyme is increased (or decreased) relative to
30 the rate and/or specificity of cleavage activity of the enzyme measured under comparable
conditions against the enzyme's natural substrate.
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In certain embodiments of the invention, the cleavage region may comprise multiple cleavage
sites, wherein cleavage at any one of the sites produces at least two parts of the cleavage
region, at least one part of which remains connected to the capture site. In the context of the
present invention, the term 'multiple' means at least two, at least three, at least four, and so
5 forth. In certain embodiments, the cleavage region of the indicator molecule includes
between 2, 3, 4, 5 and 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 50, 100,500 or 1000
cleavage sites. In some embodiments, the indicator molecule includes between 2 and 5, 6,
7, 8, 9 or 10 cleavage sites.
10 In one embodiment, the multiple cleavage sites may all be identical. In this configuration, the
repeated cleavage site may be relatively non-specific or may be highly specific for one
enzyme or enzyme subtype as defined above. Moreover, use of an indicator molecule of this
type may help to increase the sensitivity of the enzyme detection device by providing a
means to increase the concentration of cleavage sites present within the test sample.
15
In other embodiments, the cleavage region of the indicator molecule may comprise multiple
cleavage sites wherein there are at least two different cleavage sites present within the same
indicator molecule. In preferred embodiments of the invention, the indicator molecule may
comprise at least three, at least four, at least five, and up to at least 8 different cleavage
20 sites.
In a further preferred embodiment, the different cleavage sites are recognised by different
enzymes or different categories, subcategories or subtypes of enzymes as defined above,
such that the device of the invention can be used to detect the activity of multiple different
25 enzymes. The activities may be grouped, such that the detection of enzyme activity gives a
useful result. For example, a group of enzymes may be involved in a disease state such that
detection of the relevant activity of one or more of the enzyme group is diagnostically useful.
Use of multiple cleavage sites (whether identical or non-identical) may be particularly useful
30 for situations in which very low levels of enzyme activity are to be detected in a test sample.
35
For example, an indicator molecule having multiple cleavage sites as defined above may be
used to detect enzyme activity in a urine sample containing low levels of protease. Use of
multiple cleavage sites may also be particularly applicable where the indicator molecule
incorporates a scaffold molecule.
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In addition to a cleavage region containing at least one cleavage site, the indicator molecule
comprises a capture site. The capture site mediates binding of the indicator molecule to a
capture molecule present within a capture zone. Thus, the capture site is the portion of the
indicator molecule responsible for retaining or localising the indicator molecule within the
5 capture zone. Following cleavage of the indicator molecule, the capture site may remain
intact or substantially intact, such that the site is still recognised and bound by a capture
molecule present within the capture zone of the device. Under these circumstances, both
intact indicator molecules and the part of the indicator molecules comprising the capture site
following cleavage will be bound to capture molecules within the capture zone. The capture
1 0 site may comprise any suitable molecule, for example a biotin molecule. It is also possible
for the scaffold molecule to form a part, or the entirety, of the capture site in order to permit
immobilization of the indicator molecule at a capture zone. For example, the capture zone
may comprise antibodies raised against the scaffold molecule, preferably in the form as
attached to the indicator molecule. In these embodiments, the scaffold molecule is not
15 substantially involved in binding to the binding molecules. Key to effectiveness of the
indicator molecules is immobilization via the interaction between capture site and capture
molecules at the capture zone and simultaneous binding by binding molecules after cleavage
has occurred. In those embodiments in which the scaffold molecule defines a part of the
binding site for the binding molecules after cleavage, the capture site must be sufficiently
20 distinct to prevent either or both binding events from being impeded.
As noted above, the cleavage site may be within a peptide, a protein, a carbohydrate, a lipid
or a nucleic acid cleavage region. In specific embodiments of the invention, the cleavage
region and capture site are defined by discrete amino acids or groups of amino acids within a
25 peptide or protein. As used herein the term "peptide" is intended to mean a length of amino
acids of no more than (about) 20, 30, 40 or 50 amino acids.
Alternatively, the capture site may be present in a region of the indicator molecule which is
separate to the region in which the cleavage site is located. Thus, in certain embodiments of
30 the invention, the capture site may be present within a capture region, and the cleavage site
may be present within a separate cleavage region of the indicator molecule. In embodiments
wherein the capture site is in a separate region of the indicator molecule to the cleavage site,
the capture site may comprise materials or residues entirely distinct from those found in the
region of the molecule containing the cleavage site. For example, the cleavage region may
35 comprise amino acid residues whilst the capture site may comprise or consist of a biotin
moiety. Moreover, in embodiments wherein the indicator molecule comprises separate
5
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regions bearing the cleavage site and capture site, said regions may be associated by any
means known to one of skill in the art. In a preferred embodiment, said regions may be
associated via a direct covalent linkage. Said regions may be immediately adjacent or may
be separated by a linker or spacer, for example, a polyethylene glycol moiety.
The enzyme or enzymes to be detected according to the invention must be capable of
cleaving the indicator molecule at the cleavage site. This activity is required in order for the
indicator molecule to be cleaved at the cleavage site, to produce at least two parts of the
cleavage region of the indicator molecule, at least one part of which remains connected to
10 the capture site. Thus, in some embodiments of the present invention, the enzyme or
enzymes to be detected are selected from the following categories:- oxidoreductases,
hydrolases and lyases, and include the subcategories of protease, peptidase, lipase,
nuclease, carbohydrase, phosphatase, sulphatase, neuraminidase, esterase, DNAse, and
RNAse. In specific embodiments, the enzyme is a protease. In certain embodiments, the
15 protease comprises an endopeptidase. One or more different proteases may be detected
according to the invention. In certain embodiments, the protease is a matrix
metalloproteinase (MMP). MMPs are zinc-dependent endopeptidases. They are responsible
for cleaving various proteins, including extracellular matrix proteins. The MMPs include
MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP1 0, MMP11, MMP12, MMP13, MMP14,
20 MMP15, MMP16, MMP17, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25,
MMP26, MMP27 and MMP28. In other embodiments, the cleavage site is specific for an
elastase, such as (human) neutrophil elastase (HNE). In some embodiments, there may be
a cleavage site for multiple proteases, such as multiple MMPs and/or one or more MMPs
together with HNE. A suitable HNE substrate comprises, consists essentially of or consists
25 of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 and may be incorporated into
an indicator molecule, such as shown in formula I or II (including forms lacking the tyrosine
residue).
As discussed herein, the at least one cleavage site may be biased for cleavage by specific
30 proteases in some embodiments. This permits the invention to be utilised in order to detect
specific protease activity in the test sample. Many proteases are known and their sites of
preferred cleavage well reported. In certain embodiments, the at least one cleavage site is
biased for cleavage by specific matrix metalloproteinases. More specifically, in some
embodiments, the at least one cleavage site is biased for cleavage by MMP-13 and/or MMP-
35 9. The at least one cleavage site may be biased for cleavage by MMP-13, 9, 2, 12 and 8.
The bias may be for the group of MMPs equally or may be in that particular order of
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preference. As is shown herein, it is possible to design specific indicator molecules and
cleavage sites within the indicator molecules that are biased for cleavage by these particular
MMPs, in the specified order of preference. Accordingly, in some embodiments, the
cleavage site is within the amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ), which thus
5 forms the cleavage region. In those embodiments, cleavage produces a part of the indicator
molecule containing the amino acid sequence GPQG and a part of the indicator molecule
containing the amino acid sequence I FOG. Either part can be the part containing the capture
site. As is shown herein, the inventors have produced binding molecules which specifically
recognise either resultant sequence, but not the original (pre-cleavage) amino acid
1 0 sequence.
Within the context of the present invention the indicator molecules (via the capture site) may
bind to the capture molecules with relatively high affinity. In some embodiments, the
dissociation constant (kd) for the indicator molecule will be relatively low and preferably
15 between OM and 1 x 1o-7M (depending on the sensitivity required of the assay). In certain
embodiments of the invention, the dissociation constant for the indicator molecule will be
between 1 x 10-15M and 1 x 1o-9M.
In certain embodiments of the invention, such a binding interaction may be achieved as a
20 result of direct binding of the capture site of the indicator molecule to the capture molecule
present in the capture zone. In this context, direct binding means binding of the indicator
molecule (via the capture site) to the capture molecule without any intermediary.
In some embodiments of the invention, the capture site of the indicator molecule and the
25 capture molecule present in the capture zone are two halves of a binding pair. In this
context, a binding pair consists of two molecules or entities capable of binding to each other.
In certain embodiments of the invention, the binding interaction is specific such that each
member of the binding pair is only able to bind its respective partner, or a limited number of
binding partners. Moreover, as detailed above, it is preferable for the binding pair to exhibit
30 relatively high affinity. The binding pair may be a binding pair found in nature or an artificially
generated pair of interacting molecules or entities.
In some embodiments of the invention, the capture site of the indicator molecule and the
capture molecule are two halves of a binding pair wherein the binding pair is selected from
35 the following:- an antigen and an antibody or antigen binding fragment thereof; biotin and
avidin, streptavidin, neutravidin or captavidin; an immunoglobulin (or appropriate domain
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thereof) and protein A or G; a carbohydrate and a lectin; complementary nucleotide
sequences; a ligand and a receptor molecule; a hormone and hormone binding protein; an
enzyme cofactor and an enzyme; an enzyme inhibitor and an enzyme; a cellulose binding
domain and cellulose fibres; immobilised aminophenyl boronic acid and cis-dial bearing
5 molecules; and xyloglucan and cellulose fibres and analogues, derivatives and fragments
thereof.
In particular embodiments of the invention, the binding pair consists of biotin and streptavidin.
In a further embodiment of the invention, the capture site of the indicator molecule comprises
10 an epitope and the capture molecule comprises an antibody, which specifically binds to the
epitope present at the first capture site. In the context of the present invention, the term
antibody covers native immunoglobulins from any species, chimeric antibodies, humanised
antibodies, F(ab'h fragments, Fab fragments, Fv fragments, sFv fragments and highly related
molecules such as those based upon antibody domains which retain specific binding affinity
15 (for example, single domain antibodies). The antibodies may be monoclonal or polyclonal.
Thus, in specific embodiments, the capture molecule comprises an antibody. In other
embodiments, the capture site comprises a biotin molecule and the capture zone comprises
a streptavidin molecule.
20 In certain embodiments of the invention, binding of the capture site of the indicator molecule
to the capture molecule of the device may be indirect. In the context of the present invention,
"indirect binding" means binding mediated by some intermediate entity capable of bridging
the capture site of the indicator molecule and the capture molecule, for example an "adaptor"
capable of simultaneously binding the capture site of the indicator molecule and the capture
25 molecule.
Wherein binding of the indicator molecule to the capture molecule is indirect and mediated by
an adaptor, it may be possible for a plurality of indicator molecules to bind to each capture
molecule. In this context, a plurality means at least two, at least three, at least four, and so
30 forth. This may be achieved by the incorporation of a multivalent adaptor molecule, for
example, a streptavidin molecule capable of simultaneous binding to multiple biotincontaining
indicator molecules in addition to a capture molecule consisting of or comprising
biotin.
35 Embodiments of the device wherein a plurality of indicator molecules bind to each capture
molecule, may be used to achieve improved assay accuracy as described in greater detail
herein.
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Another key molecule to the invention is the binding molecule. The invention relies upon
binding molecules capable of binding to the novel binding site produced on cleavage, or the
part of the indicator molecule containing the capture site following cleavage, wherein the
5 binding molecules are incapable of binding to the indicator molecule unless and until
cleavage has occurred. Thus, in specific embodiments, the binding molecule comprises an
antibody. For the avoidance of doubt, the term antibody covers native immunoglobulins from
any species, chimeric antibodies, humanised antibodies, F(ab'h fragments, Fab fragments,
Fv fragments, sFv fragments and highly related molecules such as those based upon
1 0 antibody domains which retain specific binding affinity (for example, single domain
antibodies). The antibodies may be monoclonal or polyclonal. The inventors have
produced antibodies which recognise the cleavage region only after cleavage and will
therefore not bind to the indicator molecule (to any significant degree) unless and until
cleavage at the cleavage site has occurred. Antibodies may be produced according to
15 techniques known in the art. This may rely upon immunisation of an animal, such as a
sheep, rabbit or goat, with the cleavage products. For example immunisation may be
performed using the part of the cleavage region which remains connected to the capture site
after cleavage, optionally including the capture site itself. Polyclonal antibodies may be
isolated from serum and affinity purified. Monoclonal antibodies may be produced using well-
20 known and characterised hybridoma technology.
Thus, the invention also provides a binding molecule, typically an antibody, which binds to an
indicator molecule as defined herein after cleavage. The invention provides a binding
molecule, typically an antibody, which binds to a novel binding site in the indicator molecule
25 produced as a result of cleavage wherein the binding molecule is incapable of binding to the
indicator molecule unless and until cleavage has occurred. In some embodiments, the
binding molecule binds in the cleavage region. In specific embodiments, cleavage of the at
least one cleavage site produces at least two parts of the cleavage region of the indicator
molecule, at least one part of which remains connected to the capture site and as a
30 consequence of cleavage contains a binding site for binding molecules and wherein the
binding molecules are incapable of binding to the binding site unless and until cleavage has
occurred. In some embodiments, cleavage of the at least one cleavage site produces two
separate parts of the indicator molecule and thus the binding molecule binds to one or both
of the separate parts following cleavage. In agreement with this, the invention provides a
35 binding molecule, optionally an antibody, which binds to an indicator molecule comprising the
amino acid sequence GPQG but not to an indicator molecule comprising the amino acid
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sequence GPQGIFGQ (SEQ ID NO: 1) (as the cleavage region). Similarly, the invention
provides a binding molecule, optionally an antibody, which binds to an indicator molecule
comprising the amino acid sequence IFGQ but not to an indicator molecule comprising the
amino acid sequence GPQGIFGQ (SEQ ID NO: 1) (as the cleavage region). The invention
5 further provides a binding molecule, optionally an antibody, which binds to an indicator
molecule comprising the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 4 only when
cleaved, in particular cleaved between the isoleucine and arginine residue (i.e. not to the
amino acid sequence prior to cleavage). The indicator molecule may be constrained by
attachment to a scaffold molecule as discussed herein. An example is shown in Figure 23.
10
In those embodiments of the invention in which the indicator molecule is structurally
constrained and in which cleavage of the at least one cleavage site produces at least two
parts of the cleavage region of the indicator molecule which remain connected to one
another, the binding molecules may bind to the cleavage region following cleavage. In
15 specific embodiments, the binding molecules bind to both parts of the cleavage region of the
indicator molecule following cleavage. Thus, the binding molecules may bind a region that
effectively spans the cleavage site following cleavage. Structural constraint of the indicator
molecule, for example using the scaffold molecules as discussed herein, provides a welldefined
and stable binding site for the binding molecules following cleavage. In specific
20 embodiments, the binding site to which the binding molecule binds represents a novel
structural conformation of the indicator molecule. Cleavage may produce at least one new
conformational epitope. The binding site for the binding molecule may comprise any part of
the indicator molecule. This may be with the proviso that enzyme cleavage activity and/or
capture of the indicator molecule are not substantially impeded by binding of the binding
25 molecule. In certain embodiments, the binding site comprises at least a portion of the
cleavage region and/or at least a portion of the linker or spacer region to which the scaffold
molecule is attached and which separates the scaffold molecule from the cleavage region. In
other embodiments, the binding molecule may bind to a novel binding site that comprises at
least a portion of the scaffold molecule.
30
The binding molecule may be directly or indirectly labelled with a reporter molecule to permit
detection of binding of the binding molecule to the indicator molecule. The reporter molecule
may be any substance or moiety suitable for detection by any means available to those
skilled in the art. Thus, the reporter molecule is typically capable of signal generation or
35 production. In certain embodiments of the invention, the reporter molecule is selected from
the following:- a gold particle; a chromogen; a luminescent compound; a fluorescent
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molecule; a radioactive compound; a visible compound; a liposome or other vesicle
containing signal producing substances; an electroactive species; or a combination of
enzyme and its substrate. A suitable enzyme-substrate combination for use as a reporter
moiety may be the enzyme alkaline phosphatase and the substrate nitro blue tetrazolium-5-
5 bromo-4-chloro-3-indolyl phosphate. In a particular embodiment of the invention, the reporter
moiety is a gold particle.
Indirect labelling of the binding molecules with a reporter molecule is also envisaged within
the present invention. Thus, the reporter molecule may be attached to a further binding
1 0 molecule which in turn binds to the binding molecule to provide the label. This indirect
binding may be mediated by an adaptor capable of simultaneously binding the binding
molecule and the reporter molecule. As an illustrative embodiment, where the binding
molecule is an antibody, indirect labelling could be mediated by a further antibody that binds
to the antibody binding molecule in specific fashion. The further antibody may be directly
15 labelled with a reporter molecule such as a gold particle; a chromogen; a luminescent
compound; a fluorescent molecule; a radioactive compound; a visible compound; a liposome
or other vesicle containing signal producing substances; an electroactive species; or a
combination of enzyme and its substrate. A suitable enzyme-substrate combination for use
as a reporter moiety may be the enzyme alkaline phosphatase and the substrate nitro blue
20 tetrazolium-5-bromo-4-chloro-3-indolyl phosphate. In a particular embodiment of the
invention, the reporter moiety is a gold particle.
In embodiments of the invention wherein the reporter molecule binds to the binding molecule
by virtue of an adaptor molecule, the adaptor may be pre-complexed with the binding
25 molecule prior to the addition of the test sample to the indicator molecule, provided that the
adaptor does not prevent binding of the binding molecule to the cleaved indicator molecule.
The adaptor may be any material or molecule capable of mediating the indirect interaction of
the binding molecule with the reporter molecule. In some embodiments, the adaptor is
30 streptavidin and the binding molecule comprises a biotin molecule. The adaptor may also be
an "adaptor binding pair" wherein said binding pair comprises:
(i) a first member capable of binding to the binding molecule; and
(ii) a second member capable of binding to the first member of the pair and to the reporter
molecule. In certain embodiments of the invention, the detection region of the indicator
35 molecule comprises biotin, the first member of the adaptor binding pair is avidin or
streptavidin, the second member of the adaptor binding pair is biotin, and the reporter
molecule comprises a moiety capable of binding biotin.
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The inclusion of an adaptor molecule or an adaptor binding pair may facilitate the binding of
multiple reporter molecules to each binding molecule. For example, the use of multivalent
streptavidin as the adaptor will allow for simultaneous binding of both a biotin-containing
5 binding molecule in addition to multiple biotin-containing reporter molecules.
The invention may be performed in lateral flow or vertical flow devices in certain
embodiments. Generally, therefore, the invention relies upon some form of solid support.
The solid support may define a liquid flow path for the sample. In specific embodiments, the
1 0 solid support comprises a chromatographic medium or a capillary flow device. The invention
may be provided in a test strip format in some embodiments. A representative example is
shown in figure 2 and described in further detail herein.
In specific embodiments of the invention, the capture zone is formed on a solid support. Any
15 support to which the capture molecules may be attached to form a capture zone is intended
to be encompassed. The solid support may take the form of a bead (e.g. a sepharose or
agarose bead) or a well (e.g. in a microplate) for example. Thus, in certain embodiments the
device comprises a solid support to which the capture molecules are attached to form the
capture zone. In the case of the kits of the invention, the solid support may be provided
20 without the capture molecules attached. In those embodiments, the user of the kit may
immobilize the capture molecules on the solid support to form the capture zone prior to use
of the device with a test sample. The kit may, therefore, also comprise means for
immobilizing the capture molecules on the solid support. The immobilizing means may
comprise any suitable reagents to permit the capture zone to be formed. The solid support
25 may be pre-formed with suitable immobilizing means. For example, the solid support may
comprise biotin molecules arranged to interact with avidin (e.g. streptavidin) molecules that
form (part of) the capture molecules. Of course, other binding pair interactions may be used
to immobilize the capture molecules on the solid support to form a capture zone, as
discussed herein and as would be readily understood by one skilled in the art.
30
The capture zone may be defined by the immobilization therein or thereon of capture
molecules capable of binding to the capture site of indicator molecules. Immobilization of
capture molecules may be achieved by any suitable means. Wherein the device is a flow
device comprising a chromatographic medium, the capture molecules may be immobilized by
35 directly binding to the medium or immobilized indirectly via binding to a carrier molecule,
such as a protein, associated with, or bound to, the medium.
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In further embodiments, the solid support further comprises a sample application zone to
which the sample is applied. The sample application zone may be pre-loaded with the
indicator molecule, such that when the test sample is applied any enzyme in the sample acts
upon the cleavage site of the indicator molecule within the sample application zone. The
5 sample application zone may contain a barrier, which holds the sample in the sample
application zone for a pre-determined period of time. This permits the sample to interact with
the indicator molecule for a sufficient period to achieve measurable levels of cleavage. This
may be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 60 minutes or more depending upon the
enzyme to be detected, as would be readily understood by one skilled in the art. The barrier
10 may be degraded by the sample, or otherwise removed, after this period of time thus allowing
the sample to continue to flow through the device. Alternatively, the test sample and
indicator molecule may be pre-mixed or pre-incubated prior to adding the mixture to the
device, such as to the sample application zone. However, where the test sample and
indicator molecule may be pre-mixed or pre-incubated it is possible to omit the sample
15 application zone. Here, it may be possible to add the mixture directly to the capture zone to
permit immobilization of the indicator molecules through interaction with the capture
molecules. In some embodiments, the test sample may be applied to the chromatographic
medium at a site upstream from the capture zone such that it is drawn, for example by
capillary action, through the capture zone. The chromatographic medium may be made from
20 any material through which a fluid is capable of passing, such as a fluidic channel or porous
membrane. In certain embodiments of the invention, the chromatographic medium
comprises a strip or membrane, for example a nitrocellulose strip or membrane.
The binding molecules must be provided in the device in a manner that permits interaction
25 with the indicator molecule, if cleaved at the cleavage site. The binding molecules may,
therefore, be pre-mixed with the indicator molecules prior to application to the device. This
may be before or after the indicator molecules have been mixed with the test sample. It is
preferably after to avoid any effect the binding molecules may have on enzyme activity (in the
test sample) at the cleavage site of the indicator molecule. The binding molecules can also
30 be provided on or in the device at any point upstream of the capture zone, such that the
binding molecules encounter the test sample and indicator molecules before the indicator
molecules are immobilised (via interaction between the capture site of the indicator molecule
and capture molecules defining the capture zone). Alternatively, the binding molecule may
be added to the capture zone after the test sample and indicator molecules have been added
35 to the capture zone. This ensures that any indicator molecule will already be immobilized at
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the capture zone, providing (in the case of cleaved indicator molecule) a binding site for the
binding molecules to produce a signal.
Depending upon the particular enzyme cleavage activity that is being detected, it may be
5 necessary to incorporate suitable enzyme inhibitors into the devices or methods. This may
be important to prevent the enzyme from acting upon other components of the device or
method, such as the binding molecules or capture molecules. Where the test sample is preincubated
with the indicator molecule, it may be advantageous to add an inhibitor of the
enzyme activity at the end of the incubation period. This is preferably before the binding
10 molecules come into contact with the test sample. Alternatively, the enzyme activity inhibitor
or inhibitors may be included in the device at any point upstream of the binding molecules,
where the binding molecules are provided on or in the device. This is upstream of the
capture zone (per the discussion herein above). The inhibitor may be simply dried or
passively adsorbed onto the device such that the test sample mobilises the inhibitor as it
15 passes through the device. It should be noted that use of an inhibitor is not essential. For
example, some of the enzyme activities detected according to the invention such as specific
protease activity may be sufficiently specific that the protease will not act on any other
components of the device or method than the substrate. The cleavage sites of particular
enzymes are well known in the art and can be used to design the various components of the
20 devices and methods. For example, in silica screening may be performed (e.g. using freely
available tools such as BLAST according to standard settings) to confirm that the cleavage
site of the enzyme to be detected is not contained within any of the relevant molecules; such
as the binding molecules and capture molecules. It is also possible to check for crossreactivity
by incubating the relevant molecules (e.g. binding molecules and capture
25 molecules) with the enzyme activity to be tested and detecting whether cleavage occurs. In
some embodiments, the relevant molecules will not be acted upon due to the nature of the
enzyme cleavage activity to be detected. As an example, if a nuclease activity is being
detected, this should not display any cleavage activity in relation to an antibody binding
molecule or streptavidin or antibody capture molecule.
30
The solid support may further comprise a control zone, downstream of the capture zone in
relation to sample flow, and the sample application zone if present, containing further binding
molecules which bind to the binding molecules to indicate successful completion of an assay
using the device. Alternatively, the further binding molecules may bind to a further molecule
35 added to the sample or to the device and which flows with the sample through the device.
The further molecule may be labelled, either directly or indirectly, with a reporter molecule as
defined herein. Preferably, the reporter molecule is the same reporter molecule as attached
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to the binding molecules, for ease of detection, although it may be different. The control
zone is spatially separated from the capture zone, for example to produce two separate test
lines if the reporter is bound or immobilized in each respective zone. This control zone is
used to confirm that the test sample, including the binding molecules, has passed through
5 the entire device and confirms that the device is operating correctly. A positive signal is
expected at the control zone independent of whether enzyme cleavage activity is present in
the sample or not. The further binding molecules are selected based upon the nature of the
binding molecules which bind to the cleavage site of the indicator molecules or on the nature
of the further molecule added to the sample. The binding molecules and further binding
1 0 molecules or further molecules and further binding molecules may form a binding pair as
defined herein. For example, if the binding molecule is a species specific antibody (e.g. a
sheep antibody), the further binding molecule may be an anti-species antibody (e.g. an antisheep
antibody). Alternatively, if the further molecule is an antibody from a different species,
e.g. a chicken or a goat, the further binding molecule may be an appropriate anti-species
15 antibody. This permits immobilization of the binding molecule or further molecule at the
control zone by virtue of a specific interaction. The further binding molecules may be
immobilized in the control zone by any suitable means, for example by a covalent or noncovalent
interaction.
20 In all aspects of the invention, the test sample may be any material known or suspected to
contain an enzyme with cleavage activity. The test sample may be derived from any source.
In certain embodiments, the test sample may be derived from a biological source including
fluids such as blood (including serum and plasma), saliva, urine, milk, fluid from a wound,
ascites fluid, peritoneal fluid, amniotic fluid and so forth. In some embodiments, the test
25 sample is wound fluid and the device is used to detect enzyme activity, preferably protease
activity, in the wound fluid as a means to assess the status and/or rate of healing of a wound.
In specific embodiments, the test sample is urine and the device is used to detect the activity
of enzymes, in particular proteases, in the urine. The specific diagnostic applications of the
invention as discussed herein may be particularly applicable to certain representative sample
30 types, in particular urine, saliva or sputum. In other embodiments, the sample may be an
environmental sample in which cleavage activity may be desirably tested. For example, the
sample may be a water, food or dust sample. In the context of food and drink samples,
cleavage activity may be detected for example in relation to shelf-life of the product.
Samples may also be laboratory or industrial samples, for example to test for proteases or
35 other cleavage enzymes as contaminants. The contaminants may be found during various
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laboratory processes such as protein purification or in industrial processes such as
fermentations.
The test sample may be collected by any suitable means and presented in any form suitable
5 for use with the present invention, including solid or liquid forms. Moreover, as part of
obtaining the test sample from its original source, the sample may undergo one or more
processing or pre-treatment steps prior to testing using the invention. In one embodiment, a
solid sample may be processed so as to produce a solution or suspension for testing.
Moreover, in certain embodiments, the test sample may be stored, for example frozen at
10 around -20 oc, as a means of preserving the samplefor any given length of time prior to
testing using the invention.
It should be noted that the invention is typically performed in vitro based upon isolated
samples. The methods of the invention may include steps of obtaining a sample for testing in
15 some embodiments.
BRIEF DESCRIPTION OF THE DRAWINGS
The invention will now be described by way of example with respect to the accompanying
20 drawings in which:
Figure 1 is a schematic view of four different formats of the assay in accordance with the
invention. Each format relies upon the same basic components of solid support (1 ), capture
molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and
25 a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has
occurred.
Figure 2 is a schematic view of an enzyme detection device in accordance with the present
invention and shows operation of the device in the absence (Fig. 2A) or presence (Fig. 28) of
30 enzyme cleavage activity.
Figure 3 shows the visual read-out of the assay (shown in Figure 2) as levels of MMP activity
in the test sample are increased.
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Figure 4 is a schematic view of an enzyme detection device in accordance with the present
invention. The figure specifies the exact longitudinal dimensions and position of each of the
card components.
5 Figure 5 shows an example of synthesis of a structurally constrained indicator molecule.
In Fig. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc
chemistry. The peptide may be purified for example by High Performance Liquid
Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between
1 0 thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a
structurally constrained "clipped" peptide (4).
In Fig. 58, the indicator molecule is synthesised to include the capture site (1 ), for example
by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.
15 Figure 6 shows schematically the ability of the binding molecules used in the invention to
bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage
activity, the structurally constrained indicator molecule (1) is not bound by the antibody
binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as
antigen and thus only binds to this "open" form of the molecule.
20
Figure 7 (Fig. ?A and 78) demonstrates the sensitivity of the assay of the invention when run
with spiked MMP-9 buffer samples. The detectable limit for MMP-9 was approximately
4ng/ml with a sample volume of 75~1. Fig. ?A shows reader values across the entire
concentration range of MMP-9, whereas Fig. 78 is an expanded view at MMP-9
25 concentrations between 0 and 15 ng/ml.
Figure 8 demonstrates that the specific version of the assay of the invention uses a cleavable
sequence that is biased towards MMP13, MMP12, MMP9, MMP8 and MMP2. Other
versions of the assays of the invention may use sequences with different targets depending
30 on the application required.
Figure 9 demonstrates that measurable amounts of active proteases (in particular MMPs,
including MMP- 9) can be found in urine samples and that higher levels are present in
samples obtained from patients with a respiratory disease. A significant difference was
35 observed with COPD samples when compared to samples collected from healthy controls
(P=0.03) and CF samples to healthy controls (P=0.01 ).
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Figure 10 is a graph comparing the ability of the assay to detect MMP activity in the presence
or absence of EDT A. The graph shows that addition of EDTA to the wound samples inhibits
the readout, confirming the presence of MMP in the samples and also confirming that the
5 assay is specifically measuring active MMPs.
Figure 11 contains graphs (Fig. 11A and 11 B) comparing the ability of a commercially
available active MMP-9 assay kit and the assay of the invention to detect MMP9. Fig. 11A
shows reader values across the entire concentration range of MMP-9, whereas Fig. 11 B is
10 an expanded view at MMP-9 concentrations between 0 and 50 ng/ml. Both figures
demonstrate that the method of the invention produced a steeper curve. According to both
assays, colour development as shown by the absorbance values was seen at 4ng/ml MMP9,
the lowest standard tested.
15 Figure 12 shows MMP9 standard curves using ELISA and lateral flow embodiments of the
invention.
20
Figure 13 shows a number of scaffold molecules useful in the indicator molecules described
herein.
Figure 14 shows a number of scaffold molecules useful in the indicator molecules described
herein, together with proposed nomenclature.
Figure 15 shows some attachment options for scaffold molecules to the indicator molecules.
25 Fig. 15A shows products of cleavage at a single cleavage site and Fig. 15B shows products
of cleavage at two separate cleavage sites.
Figure 16 shows analytical HPLC of the MOL386 peptide.
30 Figure 17 is a mass spectrum of the MOL386 peptide.
Figure 18 is a mass spectrum of the MOL386 peptide modified with PEG-biotin.
Figure 19 is a mass spectrum analysis of the cyclised MOL386 peptide.
35
5
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Figure 20 is a mass spectrum analysis of the cyclised MOL386 peptide modified with PEGbiotin.
Figure 21 shows MMP9 standard curves for all combinations shown in figure 1.
Figure 22 presents the performance of the best combinations derived from the results shown
in figure 21.
Figure 23 shows a cyclised peptide substrate for Human Neutrophil Elastase (HNE) in both
1 0 pre-digested and digested forms.
Figure 24 shows HPLC analysis of elastase digestion of a cyclised peptide substrate
including the amino acid sequence of SEQ ID NO: 3. Fig. 24A presents the raw plot data.
Fig. 248 presents a time course showing the relative increase in product and decrease in
15 substrate over time.
20
Figure 25 presents mass spectrometric data confirming that the cyclised SEQ ID NO: 3
substrate is cleaved at a single site. Fig. 25A is the substrate plot and Fig. 258 is the
hydrolysed product.
Figure 26 presents one route for generating an immunogen to raise antibodies specific for
the cleaved form of the indicator molecule shown in Figure 23.
Figure 27 presents an alternative route for generating an immunogen to raise antibodies
25 specific for the cleaved form of the indicator molecule shown in Figure 23.
Figure 28 shows mass spectrometric characterisation of the MOL488 peptide.
Figure 29 shows mass spectrometric characterisation of the MOL488 peptide following
30 attachment to the scaffold molecule (derived from 1 ,3-dibromomethylbenzene).
35
Figure 30 shows mass spectrometric characterisation of the cyclised MOL488 peptide (i.e.
attached to the scaffold) following cleavage using HNE.
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DESCRIPTION OF PREFERRED EMBODIMENTS
Figure 1 is a schematic view of four different formats of the assay in accordance with the
invention. Each format relies upon the same basic components of solid support (1 ), capture
5 molecule (2), an indicator molecule containing a capture site (3) and a cleavage site (4) and
a binding molecule (5) that binds to the indicator molecule only after cleavage (6) has
occurred.
In formats 1 and 4, the capture molecule (2) is streptavidin. Here, the capture molecule (2)
1 0 binds to a biotin capture site (3) within the indicator molecule. In formats 2 and 3, the capture
molecule (2) is an antibody. Here, the capture molecule (2) binds to an epitope capture site
(3) within the indicator molecule. The epitope is found in the alternative long peptide (ALP)
which is derived from human chorionic gonadotropin (hCG).
15 Once the indicator molecule of the invention is added to a test sample, any enzyme
specifically recognising the cleavage site (4) present, may cleave the indicator molecule (6).
This cleavage event (6) produces a binding site for the specific antibody binding molecule
(5). The binding molecule (5) is unable to bind to the indicator molecule until cleavage (6)
has occurred. Thus, in formats 1 and 3 the antibody binding molecule (5) binds to the amino
20 acid sequence GPQG produced as a result of cleavage of the GPQGIFGQ sequence. In
formats 2 and 4, on the other hand, the antibody binding molecule (5) binds to the amino acid
sequence QGFI, also produced as a result of cleavage of the GPQGIFGQ sequence. In
each format, the antibody binding molecule (5) does not bind to the GPQGIFGQ sequence
prior to cleavage (not shown).
25
Figure 2 is a schematic view of an enzyme detection device in accordance with the present
invention and shows operation of the device in the absence (Fig. 2A) or presence (Fig. 28) of
enzyme cleavage activity. The test strip includes an adhesive liner (1) upon which the other
components of the device are assembled. From right to left, the sample application zone (2)
30 is in the form of an absorbent pad. This is laid partially overlapping the conjugate pad (3),
which is impregnated with the labelled binding molecules (7). In alternative embodiments,
the labelled binding molecules may be impregnated in the sample application zone and this
removes the need for a separate conjugate pad. The conjugate pad (3) is in fluid connection
with a nitrocellulose membrane (4). The nitrocellulose membrane (4) contains immobilized
35 streptavidin molecules (5) which define a capture zone. The membrane (4) further contains
immobilized further binding molecules (6) downstream of the capture zone which bind to
5
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further labelled molecules (11) which pass through the device with the sample and form a
separate control zone. Alternatively, the immobilised further binding molecules may bind to
labelled binding molecules (7). The device optionally further comprises an absorbent pad (8)
to absorb any test sample and reagents reaching the end of the device.
In use, the indicator molecule (9) is added to the test sample prior to bringing the test sample
into contact with the sample application zone (8) of the device. As shown in Figure 2A, in the
absence of enzyme cleavage activity in the test sample, the indicator molecule (9) remains
uncleaved at the cleavage site. Upon sample flow into the conjugate pad (3), the binding
10 molecules (7) are unable to bind to the indicator molecule (9) because cleavage of the
cleavage site has not occurred. The indicator molecules become bound at the capture zone
via the interaction between streptavidin (5) and the biotin capture site (1 0) of the indicator
molecule (9). The labelled binding molecules (7) are not immobilized at the capture zone
because they cannot bind to the indicator molecules (9). Accordingly, the labelled binding
15 molecules flow through to the control zone and beyond. Further labelled molecules (11) also
pass through the device to the control zone where they are immobilized by binding to the
immobilized further binding molecules (6). Thus, absence of enzyme cleavage activity is
displayed as a signal only at the control zone, but not at the capture zone. Excess sample,
potentially containing labelled binding molecules (7), flows into the absorbent pad (8).
20
As shown in Fig. 28, in the presence of enzyme cleavage activity in the test sample, the
indicator molecule (9) is cleaved at the cleavage site. Upon sample flow into the conjugate
pad (3), the binding molecules (7) are able to bind to the indicator molecule (9) because
cleavage of the cleavage site has occurred. The indicator molecules become bound at the
25 capture zone via the interaction between streptavidin (5) and the biotin capture site (1 0) of
the indicator molecule (9). The labelled binding molecules (7) are immobilized at the capture
zone due to binding to the indicator molecules (9) at the cleavage site. Due to the relative
excess of labelled binding molecule (7) to binding sites at the capture zone some labelled
binding molecules (7) still flow through to the control zone and beyond. Further labelled
30 molecules (11) also pass through the device to the control zone where they are immobilized
by binding to the immobilized further binding molecules (6). Thus, presence of enzyme
cleavage activity is displayed as a signal both at the capture zone and the control zone.
Excess sample, potentially containing cleavage products of the indicator molecule that do not
contain the biotin capture site (1 0), flows into the absorbent pad (8).
35
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It should be noted that the control zone is optional. The presence or absence of enzyme
cleavage activity in the sample can be monitored solely based upon the presence or absence
of a corresponding signal at the capture zone.
5 Figure 3 shows the visual read-out of the assay (shown in Figure 2) as levels of MMP activity
in the test sample are increased. As can readily be seen, the signal at the control zone (1) is
constant as MMP amounts increase. In contrast, as MMP amounts increase, the signal at
the capture zone (2) also increases. This is due to cleavage of the indicator molecule at the
cleavage site by MMP activity. This reveals a binding site, enabling binding of the binding
10 molecules which is detected at the capture zone (2) via interaction between capture
molecules defining the capture zone and the capture site of the indicator molecules.
Figure 4 is a schematic view of one specific enzyme detection device in accordance with the
present invention. The table below provides a legend for the figure and specifies the exact
15 longitudinal dimensions and position of each of the card components in this particular
embodiment. Of course, the dimensions and positions may be varied as would be readily
understood by one skilled in the art.
Component Size Position from Datum point
Backing card (1) 60mm Omm
Nitrocellulose Membrane (2) 25mm 20mm
Conjugate Pad (3) 17mm 5mm
Sample Pad (4) 10mm Omm
Absorbent Pad (5) 22mm 38mm
20 Figure 5 shows an example of synthesis of a structurally constrained indicator molecule. It
should be noted that additional spacer or linker regions may be included between the
cleavge region and the site of attachment of the scaffold molecule.
In Fig. 5A initially, a linear peptide (1) is synthesised, for example using solid phase Fmoc
25 chemistry. The peptide may be purified for example by High Performance Liquid
Chromatography (HPLC). The peptide is then constrained, or cyclised, by reaction between
thiol groups on the peptide (2) and the scaffold molecule (3). This reaction produces a
structurally constrained "clipped" peptide (4).
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In Fig. 58, the indicator molecule is synthesised to include the capture site (1 ), for example
by synthesis of the linear peptide on a pre-loaded Biotin-PEG resin.
Figure 6 shows schematically the ability of the binding molecules used in the invention to
5 bind exclusively to the cleaved indicator molecule. In the absence of enzyme cleavage
activity, the structurally constrained indicator molecule (1) is not bound by the antibody
binding molecule (2). This antibody is generated using the cleaved indicator molecule (3) as
antigen and thus only binds to this "open" form of the molecule.
1 0 Figures 13 and 14 show a range of suitable scaffold molecules for use in the invention.
15
Figure 15 shows, in schematic form, some attachment options for scaffold molecules to the
indicator molecules. Fig. 15A shows products of cleavage at a single cleavage site and Fig.
158 shows products of cleavage at two separate cleavage sites.
A Human Neutrophil Elastase substrate is shown in Figure 23 in the form of an indicator
molecule of the invention. Synthesis of this substrate/indicator molecule is discussed in
Examples 9 and 10 below. The substrate contains the amino acid sequence CQESIRLPGC
(SEQ ID NO: 3) which is cyclised using an appropriate scaffold (in this case 1 ,3-
20 dibromomethylbenzene). Cyclisation utilises the thiol groups provided by the two cysteine
residues to link with the scaffold. Cleavage by HNE at the peptide bond between the
isoleucine and arginine residues opens up the structure to reveal a new binding site (or
"cryptotope"). HNE cleaves the substrate at a single site to produce a reliable binding site.
The additional tyrosine residue (see SEQ ID NO: 4) facilitates attachment to further moieties
25 such as a carrier protein as discussed herein.
30
35
The invention will be further understood with reference to the following experimental
examples.
EXAMPLES
Throughout the examples and figures the invention may be referred to as "Ultimate ELTABA"
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Example 1: A lateral flow Platform of the invention for detection of Matrix
Metalloprotease-9 (MMP-9).
A kit comprises the following components:-
5 1) A device for sample collection (e.g. for urine)
2) A lateral flow test-strip, which is mounted in a plastic case. The test strip has a capture
zone comprising polystreptavidin as a first test line across the flow-path of the test strip. A
second capture zone comprising anti-chicken antibodies adsorbed as a control line across
the flow-path of the test strip, downstream of the test line may be included as a control line.
10 There is an observation window in the plastic case through which to view the test and control
line. There is also an integrated sample-receiving pad, upstream of the first test line. In
addition, the test strip has gold particles bearing sheep antibody (CF1522) dried into the test
strip, downstream of the sample-receiving pad which can be reconstituted by the addition of
the sample.
15 3) A tube, in which the sample collection device may be placed, together with the indicating
molecule.
20
4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ)
which carries a terminal biotin group connected via a polyethylene glycol spacer/linker which
allows it to form a complex with the capture line, polystreptavidin.
The test Strip
A test strip for the detection of protease activity in a sample was constructed in accordance
with the present invention, as described below. The assay was based on the cleavage of the
indicator molecule in the presence of various MMP's to expose an epitope visible to the
25 Sheep antibody (CF1522) conjugated to gold particles.
The methods used were all in accordance with standard procedures well known in the art.
A. Preparation of CF1522:40nm gold conjugate
Affinity purified sheep antibody CF1522 (lg Innovations, CF1522) was conjugated to 40nm
gold particles at a concentration giving an OD of 5 at 520nm (BBIInternational, GC40). The
30 antibody was loaded at a concentration of 15~g/ml in a 20mM BES buffer pH 7.8. 0.2% BSA
(Sigma, A7906) was used as a blocking solution to minimise non-specific binding.
B. Preparation of Gold-Impregnated Conjugate Pads
A glass fibre conjugate pad (Millipore, G041, 17 mmx300mm) was sprayed with
CF1522:40nm gold conjugate (Mologic) at 004, diluted in gold drying buffer (1M Tris,
35 150mM sodium chloride, 20mM sodium Azide, 3% BSA, 5% Sucrose, 1% Tween 20 at pH
9.4) at 0.8~1/mm with the lsoflow dispenser (15mm spray height). The processed conjugate
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band was dried in a tunnel dryer at 60°C at a speed of 5mm/sec. The dried gold conjugateimpregnated
conjugate pads were stored dried in a sealed foil pouch with desiccant at room
temperature.
C. Preparation of antibody-Impregnated Nitrocellulose Membrane
5 All reagents were striped on Unistart CN140 membrane (Sartorius, CN140, 25mm x 300 mm)
at a dispense rate of 0.1 ~1/mm. A test line polystreptavidin (BBI, Polystrep N 01041 048K) at
a concentration of 1 mg/ml was positioned 7mm from base of membrane. Processed
membrane was dried in a tunnel dryer at 60°C at a speed of 1 Omm/sec. The dried antibodyimpregnated
Nitrocellulose Membrane was stored in a sealed foil pouch with desiccant at
1 0 room temperature.
D. Card Assembly
A test card was assembled according to the following procedure and in accordance with
Figure 4 which specifies the exact longitudinal dimensions and position of each of the card
components.
15 • 1. A 60x300 mm piece of clear plastic film with a release liner protected adhesive,
serving as the back laminate, designated 1 in Figure 4, (G&L Precision Die Cutting,
GL-48077) was placed on top of a worktable. The release liner was peeled to expose
the adhesive.
• 2. The reaction membrane (prepared as in section C) was attached on top of the
20 adhesive side of the back cover, 20mm from the lower end.
• 3. The impregnated conjugate pad (prepared as in section B) was attached on top of
the back cover with 2mm overlap on top of the reaction membrane.
• 4. The sample pad (MDI, FR-1, 1 Ox300 mm) was placed on top of the back cover
with 5mm overlap on top of the conjugate pad.
25 • 5. The absorbent pad (Gel blotting paper, Ahlstrom, grade 222, 22x300 mm) was
placed on top of the upper side of the back cover with a 2mm overlap on top of the
reaction membrane.
The card was trimmed to 5mm width strips using an automated die cutter (Kinematic, 2360)
and assembled into plastic housings (Forsite). The devices were closed using a pneumatic
30 device clamp specifically manufactured for these devices at Mologic.
The table lists the strip components and respective positioning on a backing card.
Component Size Position from Datum point
Backing card (1) 60mm Omm
Nitrocellulose Membrane (2) 25mm 20mm
Conjugate Pad (3) 17mm 5mm
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Sample Pad (4) 10mm Omm
Absorbent Pad (5) 22mm 38mm
Buffer standards were produced containing different concentrations of active MMP-9 (Aiere
San Diego) ranging from 1 OOOng/ml down to 1 ng/ml.
5 STEP 1 : Each standard was placed in a collection device with a defined amount of peptide
(25ng/test). The collection device was rotated vigorously in order for the sample to mix
sufficiently with the substrate solution. This reaction mixture was incubated at ambient
temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
10 sample receiving pad which subsequently made contact with the conjugate pad and rehydrated
the dried CF1522 antibody attached to the gold particles. Intact indicator molecule
was not recognised by the gold conjugate and migrated in an uncomplexed state towards the
polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
15 site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
with the cleaved stub.
The lines that were formed were assessed by their relative intensities. The presence of a test
line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
20 between these extremes indicated different levels of protease in the test sample. The
intensity of the developed coloured lines was measured visually and with a Forsite Lateral
flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was
the lowest detectable colour intensity and 1 0 was the highest observed colour intensity was
used for the visual readings.
25
Figure 7 (Fig. ?A and 78) demonstrates the sensitivity of the assay when run with spiked
MMP-9 buffer samples. The detectable limit for MMP-9 was approximately 4ng/ml with a
sample volume of 75~1. Fig. ?A shows reader values across the entire concentration range
of MMP-9, whereas Fig. 78 is an expanded view at MMP-9 concentrations between 0 and 15
30 ng/ml.
The reader units are displayed in the table below where a value above 400 was deemed a
positive result:
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ll'OO 677Q
5{::0 6729
25.0 622.5
125 5581
fiLS 3815
31.:25 2029
1.5.625 8:82.
7.8125 .524
3.%525 41.3
1 .. 95312:5 343
03765625 3·3:8·
0 312
Example 2 Matrix Metalloprotease (MMP) specificity of a lateral flow format of the
5 invention
The kit and test strip synthesis were performed as for Example 1 .
Various MMP's (Enzo) were prepared in buffer (Aq. Solution of 50mM Tris, 150mM sodium
1 0 chloride, 20mM sodium azide, 1% vol/vol Tween 20, at pH 8.0) at 0.5~g/ml.
STEP 1: Each MMP solution was placed in a collection device with a defined amount of
peptide (25ng/test). The collection device was rotated vigorously in order for the sample to
mix sufficiently with the substrate solution. This reaction mixture was incubated at ambient
15 temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
sample receiving pad which subsequently made contact with the conjugate pad and rehydrated
the dried CF1522 antibody attached to the gold particles. Intact indicator molecule
was not recognised by the gold conjugate and migrated in an uncomplexed state towards the
20 polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
with the cleaved stub.
The lines that were formed were assessed by their relative intensities. The presence of a test
25 line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
between these extremes indicated different levels of protease in the test sample. The
5
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intensity of the developed coloured lines was measured visually and with a Forsite Lateral
flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was
the lowest detectable colour intensity and 1 0 was the highest observed colour intensity was
used for the visual readings.
Figure 8 demonstrates that this version of the invention uses a cleavable sequence that is
biased towards MMP13, MMP12, MMP9, MMP8 and MMP2. Other versions of this invention
may use sequences with different targets depending on the application required.
10 The table below shows the read-out values for each of the MMPs tested:
&~~~:P R:ea.der value
- 4TJ.S
2 16-0S.S
~
~ 33:6.5
7 373
8 11.4U.S
3 3844
l.JJ 444
11 279.5
'~.:~.. l252.S
13 6348.5
15
Example 3 Detection of enzyme activity in urine
The kit and test strip synthesis were performed as for Example 1 .
20 Samples were collected from healthy volunteers (9) and from patients suffering from a
respiratory disease. Samples were donated from nine patients with Cystic Fibrosis (CF) and
seven patients with Chronic Obstructive Pulmonary Disease (COPD) and stored at -80 o C
until used.
25 STEP 1: Each sample was placed in a collection device with a defined amount of peptide
(25ng/test). The collection device was rotated vigorously in order for the sample to mix
sufficiently with the substrate solution. This reaction mixture was incubated at ambient
temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
30 sample receiving pad which subsequently made contact with the conjugate pad and rewo
2015/059487 PCT/GB2014/053171
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hydrated the dried CF1522 antibody attached to the gold particles. Intact indicator molecule
was not recognised by the gold conjugate and migrated in an uncomplexed state towards the
polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
5 site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
with the cleaved stub.
The lines that were formed were assessed by their relative intensities. The presence of a test
line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
1 0 between these extremes indicated different levels of protease in the test sample. The
intensity of the developed coloured lines was measured visually and with a Forsite Lateral
flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was
the lowest detectable colour intensity and 1 0 was the highest observed colour intensity was
used for the visual readings.
15
Figure 9 demonstrates that measurable amounts of active proteases (in particular MMPs,
including MMP- 9) can be found in urine samples and that higher levels are present in
samples obtained from patients with a respiratory disease. A significant difference was
observed with COPD samples when compared to samples collected from healthy controls
20 (P=0.03) and CF samples to healthy controls (P=0.02).
25
Example 4 Detection of enzyme activity in wound fluid
The kit and test strip synthesis were performed as for Example 1 .
Wound samples from 18 patients were tested on the ultimate EL TABA device to measure
active MMP's in this biologic matrix. The samples were extracted from a swab (Copan,
552C.US) in MMP buffer buffer (Aq. Solution of 50mM Tris, 1 OOmM sodium chloride, 1 OmM
Calcium Chloride, 501-JM 20mM zinc chloride, 0.025% Brij 35, 0.05% sodium azide at pH 8.0)
30 and then frozen at -2ooc until use. The addition d a chelating agent (5mM EDTA)
determined the specificity of the device to calcium dependent enzymes e.g. MMP's.
STEP 1: Each wound sample was diluted 1 in 20 in MMP buffer and 751JI was placed in a
collection device with a defined amount of peptide (25ng/test). The collection device was
35 rotated vigorously in order for the sample to mix sufficiently with the substrate solution. This
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reaction mixture was incubated at ambient temperature for a defined period of time (e.g. 10
minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
sample receiving pad which subsequently made contact with the conjugate pad and re-
5 hydrated the dried biotin attached to the gold particles. Intact indicator molecule was not
recognised by the gold conjugate and migrated in an uncomplexed state towards the
Polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
1 0 with the cleaved stub.
The lines that were formed were assessed by their relative intensities. The presence of a test
line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
between these extremes indicated different levels of protease in the test sample. The
15 intensity of the developed coloured lines was measured visually and with a Forsite Lateral
flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was
the lowest detectable colour intensity and 1 0 was the highest observed colour intensity was
used for the visual readings.
20 Figure 10 shows that addition of EDTA to the wound samples inhibits the readout, confirming
the presence of MMP in the samples and also confirms that the assay is specifically
measuring active MMPs.
25 Example 5 Comparison of sensitivity of the invention to a commercial MMP-9 activity
assay kit
The commercial kit is designed for specifically detecting MMP-9 in biologic samples such as
culture medium, serum, plasma, synovial fluid, and tissue homogenate. A monoclonal anti-
30 human MMP is used to pull down both pro and active forms of MMP from the mixture first,
and then the activity of MMP9 is quantified using fluorescence resonance energy transfer
(FRET) peptide. An MMP-9 standard AMPA activated in-house was run on both the kit and a
lateral flow format of the invention at a range of 250ng/ml- 4ng/ml. For the commercial
assay the MMP-9 was diluted in an MMP buffer supplied in the kit and a Tris buffer saline 1%
35 Tween20 for lateral flow devices.
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The lateral flow kit and test strip synthesis were performed as for Example 1.
Buffer standards were produced containing different concentrations of active MMP-9 (Aiere
San Diego) ranging from 250ng/ml down to 4ng/ml in a Tris buffer saline 1% Tween (Aq.
5 Solution of 50mM Tris, 150mM sodium chloride, 20mM sodium azide, 1% vol/vol Tween 20,
at pH 8.0).
STEP 1 : Each standard was placed in a collection device with a defined amount of peptide
(25ng/test). The collection device was rotated vigorously in order for the sample to mix
10 sufficiently with the substrate solution. This reaction mixture was incubated at ambient
temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
sample receiving pad which subsequently made contact with the conjugate pad and rehydrated
the dried CF1522 antibody attached to the gold particles. Intact indicator molecule
15 was not recognised by the gold conjugate and migrated in an uncomplexed state towards the
polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
with the cleaved stub.
20
The lines that were formed were assessed by their relative intensities. The presence of a test
line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
between these extremes indicated different levels of protease in the test sample. The
25 intensity of the developed coloured lines was measured visually and with a Forsite Lateral
flow device reader. A semi-quantitative scoring system with a scale of 0-10, in which 1 was
the lowest detectable colour intensity and 1 0 was the highest observed colour intensity was
used for the visual readings.
30 Figure 11 (Fig. 11A and 11 B) is a graph comparing the ability of a commercially available
active MMP-9 assay kit and the assay of the invention to detect MMP9. Fig. 11A shows
reader values across the entire concentration range of MMP-9, whereas Fig. 11 B is an
expanded view at MMP-9 concentrations between 0 and 50 ng/ml. Both figures demonstrate
that the assay of the invention produced a steeper curve. According to both assays, colour
35 development as shown by the absorbance values was seen at 4ng/ml MMP9, the lowest
standard tested.
wo 2015/059487 PCT/GB2014/053171
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Numerical read-outs for each assay are shown in the table below:
~g/m~ ~·HV1PS Reference 3ssav U!tima1:e HTABA
250 2flM6t'i5 6225
125 16.2622 5.5.81
625 1127%5 3.826
3L25 o23DL5 2029
15j:i25 3125'5 S82.
7.&125 1314(L5 524
:t9062S 7(}()1 413
D 3Sl8.5 312.
5
Example 6 Testing of substrate in both ELISA and LF format
1 0 ELISA format
1) A device for sample collection (e.g. for urine)
2) A 96 well plate coated with polystreptavidin
3) A tube, in which the sample collection device may be placed, together with the indicating
molecule.
15 4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ)
which carries a terminal biotin group connected via a polyethylene glycol spacer/linker which
allows it to form a complex with the capture line, polystreptavidin.
5) A sheep antibody CF1522 conjugated to alkaline phosphatase (AP)
6) An Alkaline phosphatase substrate p-nitrophenylphosphate (pNPP) that enables the
20 development of a soluble yellow reaction product that may be read at 405nm.
Samples were collected from healthy volunteers (9) and from patients suffering from a
respiratory disease. Samples were donated from nine patients with Cystic Fibrosis (CF) and
seven patients with Chronic Obstructive Pulmonary Disease (COPD) and stored at -80 o C
until used.
25 STEP 1: Each sample was placed in a collection device with a defined amount of peptide
(25ng/test). The collection device was rotated vigorously in order for the sample to mix
sufficiently with the substrate solution. This reaction mixture was incubated at ambient
temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of sample was added to the
30 streptavidin plate (Nunc, 442404) and incubated for a further 1 hr at ambient where the biotin
labelled indicator molecule becomes immobilized by the streptavidin bound to the plate.
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STEP 3: The plate was washed 3 times with 1 00~1 in a wash buffer, Tris buffer saline 0.1%
Tween (Aq. Solution of 50mM Tris, 150mM sodium chloride, 20mM sodium azide, 0.1%
vol/vol Tween 20, at pH 8.0).
STEP 4: CF1522-AP (Mologic) was diluted 1/500 in 1% BSA in PBST and incubated on the
5 plate for 1 hr at ambient. The antibody will form a complex with the cleaved stubs exposed by
any MMP present in the sample and in the absence of the cleaved stub there will be no
binding of the antibody.
STEP 5: The plate was washed 3 times with 1 00~1 in a wash buffer, Tris buffer saline 0.1%
Tween (Aq. Solution of 50mM Tris, 150mM sodium chloride, 20mM sodium azide, 0.1%
10 vol/vol Tween 20, at pH 8.0).
15
STEP 6: The plate was incubated with pNPP substrate and then read at 405nm after 30
minute incubation at 3JDC. An MMP9 standard curve 6 represented in figure 14b used as a
reference. The colour of the wells indicate different levels of protease in the test sample
represented by the OD 405nm in figure 14b,
Lateral flow format
The kit and test strip synthesis were performed as for Example 1 .
Buffer standards were produced containing different concentrations of active MMP-9 (Aiere
20 San Diego) ranging from 50ng/ml down to 0.39ng/ml and 62.5ng/ml down to 0.97ng/ml for
the ELISA and lateral flow device respectively.
STEP 1: Each sample was placed in a collection device with a defined amount of peptide
(25ng/test). The collection device was rotated vigorously in order for the sample to mix
25 sufficiently with the substrate solution. This reaction mixture was incubated at ambient
temperature for a defined period of time (e.g. 1 0 minutes).
STEP 2: At the end of the incubation period, a defined volume of liquid was dropped onto the
sample receiving pad which subsequently made contact with the conjugate pad and rehydrated
the dried CF1522 antibody attached to the gold particles. Intact indicator molecule
30 was not recognised by the gold conjugate and migrated in an uncomplexed state towards the
polystreptavidin test line where it was immobilised via the biotin attached to the indicator
molecule. Any MMP-9 present in the sample cleaved the indicator molecule at the cleavage
site, exposing the recognisable epitope thus allowing the gold conjugate to form a complex
with the cleaved stub.
35
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The lines that were formed were assessed by their relative intensities. The presence of a test
line indicated that there was protease present in the test sample. A negative test line
indicated a zero or low level of protease that was below the detectable limit. Stages in
between these extremes indicated different levels of protease in the test sample. The
5 intensity of the developed coloured lines was measured visually and with an NES Lateral flow
device reader.
The results of an MMP9 standard curve can be seen in Figure 12. Figure 12 demonstrates
that the two MMP9 standard curves produced by the ELISA and the Lateral Flow are similar
10 with sensitivity down to 4ng/ml.
15
The numerical read-outs from the two assays are also shown in the table below:
Lateral Flow standard
ELISA standard curve curve
ng/ml Reader
ng/ml MMP9 00405 MMP9 value
50.00 0.50 62.50 3826.00
25.00 0.27 31.25 2029.00
12.50 0.18 15.63 882.00
6.25 0.17 7.81 524.00
3.13 0.13 3.91 413.00
1.56 0.14 1.95 343.00
0.78 0.14 0.98 338.00
0.39 0.14 0.00 312.00
Example 7 - Synthesis of an example indicator molecule
A peptide termed MOL386 (amino acid sequence: CGPQGIFGQC) was synthesised on solid
phase using Fmoc- chemistry. Briefly, synthesis was performed on a microwave assisted
20 automated synthesiser (CEM Liberty).Coupling steps were carried out on PEG-polystyrene
resin preloaded with Fmoc-Cys(Trt) in DMF solvent with a fivefold excess of amino acid
building block, HBTU activator and a tenfold excess of DIPEA base. Deprotection steps were
carried out in 5% Piperazine/DMF. Completed peptide resin was dried and then cleaved
using 95% TFA, 2.5% TIPS and 2.5% water for 2 hours. TFA liquors were dried in vacuo and
25 precipitated in ether to afford colourless peptide solid. Recovered peptide was freeze dried
from 50% acetonitrile and purified by HPLC (Fig. 16) using a C18 reverse phase column and
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a gradient of 5% acetonitrile/water (0.1% TFA) to 1 00% acetonitrile (0.1% TFA). Isolated
fractions were combined and freeze dried and analysed by electrospray mass spectrometry
(Fig. 17) to identify target peptide (expected MH+ 1010.17, measured 101 0.3). The
biotinylated form (CGPQGIFGQC-PEG-biotin) was synthesised from preloaded Biotin-PEG-
5 NovaTag Resin (Merck) (expected MH+ 1438.76, measured 1439.7, Fig. 18). The biotin
provides a capture site for immobilization of the indicator molecule.
Attachment of the scaffold molecule (synthesis of cyclised peptide)
Peptide (1 mg) was dissolved in PBS 250ul along with 1 mg of 1 ,3-dibromomethylbenzene
10 and agitated gently overnight. The reaction was then diluted with 1 ml of water and injected
directly on to HPLC for purification using a C18 reverse phase column and a gradient of 5%
acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Product peak was isolated
and freeze dried to afford a colourless solid (expected MH+ 1112.30, measured 1112.8, Fig.
19). The same procedure was used for the biotinylated peptide (expected MH+ 1540.89,
15 measured 1539.8, Fig. 20).
Example 8 - Test format generation
Antibodies were generated to recognise a cleaved peptide sequence. In this example
20 (GPQGIFGQ), a target for MMP digestion, is used in an immunoassay to measure the
enzyme activity in a clinical sample. The antibodies were raised to peptide KLH conjugates
using methods known to those skilled in the art. Sheep antibodies CF1522 and CF1523
were generated to recognise cleaved stub 'IFGQ' whereas sheep antibodies CF1524 and
CF1525 were generated to recognise cleaved stub 'GPQG'. The antibodies were affinity
25 purified using the specific peptides they were raised against and then analysed by ELISA to
determine the most appropriate assay format to give the best sensitivity.
30
Peptides containing the cleavable sequence (GPQGIFGQ) were synthesised with a biotin or
Pegylated biotin attached to either the C-terminus (MOL038 and PCL008-A2 respectively) or
the N-terminus (MOL31 0 and MOL378 respectively).
Peptide Sequence
MOL038 Biotin-G PQG I FGQES I RLPGCPRGVN PVVS
PCL008-A2 Biotin-PEG-Asp -AEEAc-AEEAc- GPQGIFGQESIRLPGCPRGVNPVVS
MOL31 0 SIRLPGCPRGVNPVVSGPQGIFGQ- Biotin
MOL378 SIRLPGCPRGVNPVVSGPQGIFGQ-AEEAc-AEEAc- PEG-Asp Biotin
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The peptide can be anchored to either streptavidin capture via the biotin or to sheep antibody
CF1 060 capture via the ALP sequence. The proposed formats shown schematically in figure
1 were evaluated.
5 ELISA format
1) A device for sample collection (e.g. for urine)
2) A 96 well plate coated with polystreptavidin (Nunc, 442404) or CF1 060 overnight at
ambient (Nunc, Maxisorb)
3) A tube, in which the sample collection device may be placed, together with the indicating
1 0 molecule.
4) An indicator molecule containing the cleavable sequence, in this example, (GPQGIFGQ)
which carries a terminal biotin group which may be connected via a polyethylene glycol
spacer/linker on the N or the C-terminus.
5) Sheep antibodies CF1522, CF1523, CF1524 and CF1525 conjugated to alkaline
15 phosphatase (AP)
6) An Alkaline phosphatase substrate p-nitrophenylphosphate (pNPP) that enables the
development of a soluble yellow reaction product that may be read at 405nm.
Active MMP9 (Aiere San Diego) was diluted to 2, 0.25, 0.062, 0.0156 and 0.039~g/ml in
MMP buffer (Aq. Solution of 50mM Tris, 1 OOmM sodium chloride, 1 OmM Calcium Chloride,
20 501-JM 20mM zinc chloride, 0.025% Brij 35, 0.05% sodium azide at pH 8.0)
STEP 1: Each MMP9 standard was placed in a collection device with a defined amount of
each peptide (20ng/test). The collection device was rotated vigorously in order for the
sample to mix sufficiently with the substrate solution. This reaction mixture was incubated at
ambient temperature for a defined period of time (e.g. 30 minutes).
25 STEP 2: At the end of the incubation period, a defined volume of sample was added to the
streptavidin plate and CF1 060 sensitised plate and incubated for a further 1 hr at ambient
where the peptides becomes immobilized by the streptavidin or CF1 060 bound to the plate.
STEP 3: The plate was washed 3 times with 1 00~1 in a wash buffer, Tris buffer saline 0.1%
Tween (Aq. Solution of 50mM Tris, 150mM sodium chloride, 20mM sodium azide, 0.1%
30 vol/vol Tween 20, at pH 8.0).
STEP 4: sheep antibodies conjugated to Alkaline Phosphatase (Mologic) were diluted 1/500
in 1% BSA in PBST and incubated on the plate for 1 hr at ambient. The antibody will form a
complex with the cleaved stubs exposed by any MMP9 present in the sample, in the absence
of the cleaved stub there will be no binding of the antibody.
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STEP 5: The plate was washed 3 times with 1 00~1 in a wash buffer, Tris buffer saline 0.1%
Tween (Aq. Solution of 50mM Tris, 150mM sodium chloride, 20mM sodium azide, 0.1%
vol/vol Tween 20, at pH 8.0).
STEP 6: The plate was incubated with pNPP substrate and then read at 405nm after 30
5 minute incubation at 3JDC. MMP9 standard curves are represented in figure 21 for all
combinations. A difference in colour of the wells indicates different levels of protease in the
test sample represented by the OD 405nm.
Figure 21 shows the results of testing each format. With a streptavidin capture line, the
1 0 selected peptide is MOL378 with sheep antibody CF1522 and PCL008-A2 with sheep
antibody CF1525 as predicted. Both peptides contained a PEG-Asp -AEEAc-AEEAc
required to reduce any steric hindrance. With a CF1 060 capture line, the selected peptide is
MOL038 or PCL008-A2 with sheep antibody CF1522 and MOL378 with sheep antibody
CF1525 as predicted. The performance of the best combinations is shown in figure 22.
15 Here, format 4 using sheep antibody CF1522 with peptide MOL378 shows the most promise.
Example 9 - Synthesis of a Human Neutrophil Elastase Sensitive Indicator molecule
A peptide termed MOL 488 (amino acid sequence YCQESIRLPGC- SEQ ID NO: 4) was
20 synthesised on solid phase using Fmoc- chemistry. Briefly, synthesis was performed on a
microwave assisted automated synthesiser (CEM Liberty).Coupling steps were carried out on
PEG polystyrene resin with a fivefold excess of amino acid building block, DIC and Oxyma.
Deprotection steps were carried out in 20% Piperidine/DMF. Completed peptide resin was
dried and then cleaved using 95% TFA, 2.5% TIPS and 2.5% water for 2 hours. TFA liquors
25 were dried in vacuo and precipitated in ether to afford colourless peptide solid. Recovered
peptide was freeze dried from 50% acetonitrile and purified by HPLC using a C18 reverse
phase column and a gradient of 5% acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1%
TFA). Isolated fractions were combined and freeze dried and analysed by electrospray mass
spectrometry (expected MH+ 1268.5, measured 1267.86 ±1.39)- see Figure 28
30
Attachment of the scaffold molecule (synthesis of cyclised peptide)
Peptide (1 mg) was dissolved in PBS 250ul along with 1 mg of 1 ,3-dibromomethylbenzene
and agitated gently overnight. The reaction was then diluted with 1 ml of water and injected
35 directly on to HPLC for purification using a C18 reverse phase column and a gradient of 5%
acetonitrile/water (0.1% TFA) to 100% acetonitrile (0.1% TFA). Product peak was isolated
wo 2015/059487 PCT/GB2014/053171
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and freeze dried to afford a colourless solid (expected MH+ 1370.6, measured 1371.9 ± 3.8);
see Figure 29.
Preparative Enzymatic cleavage of cyclised MOL488
5 Cyclised Peptide (1 mg) was dissolved in Cleavage Buffer (i 00 rnM Tris pH 8.0, 0.05% Brij)
at a final concentration of 5 mg/ml. Enzyme Human Neutrophil elastase (Leebio Solutions
!r:c.) was added to a final concentration of U/ml. To follow reaction progress timed aliquots
were quenched in 5 volumes of starting buffer (5% acetonitrile, 0.1% TFA) and checked on
HPLC. A new product peak evolved over time and after approximately three hours the
10 reaction was stopped and the product fraction purified by HPLC (expected MH+ 1388.6,
measured 1388.8 ± 2.5)- see Figure 30.
15
The same procedure was also followed in respect of a similar substrate but in this case
lacking the tyrosine residue (SEQ ID NO: 3).
HPLC results are summarised in Figure 24. It can be seen from Fig. 24A that a single
cleavage product results from HNE activity on the peptide. Fig. 24B presents a time course
showing the relative increase in product and decrease in substrate over time.
20 Figure 25 presents mass spectrometric data confirming that the substrate is cleaved at a
single site and that the substrate otherwise remains intact. Fig. 25A is the substrate plot and
Fig. 25B is the hydrolysed product.
Example 10 - Conjugation Methods
25 To conjugate the protease-digested peptide product to a carrier protein to immunise and
develop antibodies, a chemistry orthogonal to the Clip thiol alkylation route needs to be
applied. A combination of three different chemistries (diazo, oxime and triazole) are
considered in this instance to achieve conjugation. In the first option (Fig. 26) the peptide can
be synthesised with a pendant tyrosine residue. The heterobifunctional reagent FBDP
30 (Sigma) is used to conjugate an aminooxy linker (Berry Associates) on to the phenol group of
the tyrosine creating a pendant azide tail. This in turn can be conjugated to a carrier protein
labelled with an alkyne reagent. Alternatively, the peptide can be synthesised with an
aminooxy terminus (Fig. 27) and this can then be crosslinked directly to tyrosine residues on
the carrier protein using the FBDP reagent.
35
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The present invention is not to be limited in scope by the specific embodiments described
herein. Indeed, various modifications of the invention in addition to those described herein
will become apparent to those skilled in the art from the foregoing description and
accompanying figures. Such modifications are intended to fall within the scope of the
5 appended claims. Moreover, all aspects and embodiments of the invention described herein
are considered to be broadly applicable and combinable with any and all other consistent
embodiments, including those taken from other aspects of the invention (including in
isolation) as appropriate. Various publications are cited herein, the disclosures of which are
incorporated by reference in their entireties.
10
Sequence Listing.txt
SEQUENCE LISTING
<110> Mologic Limited
<120> DETECTION OF CLEAVAGE ACTIVITY OF AN ENZYME
<130> P127058WO00
<150> GB1318728.1
<151> 2013-10-23
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 8
<212> PRT
<213> Ar􀆟ficial sequence
<220>
<223> Synthe c pep de
<400> 1
Gly Pro Gln Gly Ile Phe Gly Gln
1 5
<210> 2
<211> 10
<212> PRT
<213> Ar􀆟ficial sequence
<220>
<223> Synthe c pep de
<400> 2
Cys Gly Pro Gln Gly Ile Phe Gly Gln Cys
1 5 10
<210> 3
<211> 10
Page 1
Sequence Listing.txt
<212> PRT
<213> Ar􀆟ficial sequence
<220>
<223> Synthe c pep de
<400> 3
Cys Gln Glu Ser Ile Arg Leu Pro Gly Cys
1 5 10
<210> 4
<211> 11
<212> PRT
<213> Ar􀆟ficial sequence
<220>
<223> Synthe c pep de
<400> 4
Tyr Cys Gln Glu Ser Ile Arg Leu Pro Gly Cys

CLAIMS
1 . An enzyme detection device for detecting the presence in a test sample of cleavage
5 activity of an enzyme capable of cleaving a substrate, the device comprising:
(i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
1 0 wherein cleavage of the at least one cleavage site produces a novel binding site;
(ii) a capture zone to receive the test sample, wherein the capture zone comprises capture
molecules capable of binding to the capture site of the indicator molecule in order to
immobilise the indicator molecule including the novel binding site; and
(iii) binding molecules capable of binding to the novel binding site, wherein the binding
15 molecules are incapable of binding to the indicator molecule unless and until cleavage has
occurred.
2. The enzyme detection device of claim 1 wherein cleavage of the at least one
cleavage site produces two parts of the cleavage region, at least one part of which remains
20 connected to the capture site and wherein the binding molecules are capable of binding to
the part of the indicator molecule containing the at least one part of the cleavage region
connected to the capture site.
3. The enzyme detection device of claim 1 or 2 wherein cleavage of the at least one
25 cleavage site produces two separate parts of the indicator molecule.
4. The enzyme detection device of claim 1 or 2 wherein the indicator molecule is
structurally constrained such that cleavage of the at least one cleavage site produces two
parts of the cleavage region of the indicator molecule which remain connected to one
30 another.
5. The enzyme detection device of any one of claims 1 to 4 wherein the binding
molecules bind to the cleavage region following cleavage.
35 6. The enzyme detection device of claim 5 wherein the binding molecules bind to both
parts of the cleavage region of the indicator molecule following cleavage.
wo 2015/059487 PCT/GB2014/053171
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7. The enzyme detection device of any one of claims 1 to 6 wherein the at least one
cleavage site comprises a peptide bond located between two amino acid residues.
5 8. The enzyme detection device of any one of claims 1 to 7 wherein the enzyme
capable of cleaving a substrate comprises a protease.
9. The enzyme detection device of claim 8 wherein the protease is a matrix
metalloproteinase.
10
1 0. The enzyme detection device of any one of claims 1 to 9 wherein the at least one
cleavage site is biased for cleavage by specific proteases.
11. The enzyme detection device of any one of claims 1 to 1 0 wherein the at least one
15 cleavage site is biased for cleavage by specific matrix metalloproteinases.
12. The enzyme detection device of any one of claims 1 to 11 wherein the at least one
cleavage site is biased for cleavage by MMP-13 and/or MMP- 9.
20 13. The enzyme detection device of any one of claims 1 to 12 wherein the at least one
cleavage site is biased for cleavage by MMP-13, 9, 2, 12 and 8, optionally in that order of
preference.
14. The enzyme detection device of any one of claims 1 to 13 wherein the cleavage site
25 is within the amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ), producing a part containing
the amino acid sequence GPQG and a part containing the amino acid sequence IFGQ
following cleavage.
15. The enzyme detection device of any one of claims 1 to 14 wherein the binding
30 molecule comprises an antibody.
16. The enzyme detection device of any one of claims 1 to 15 wherein the capture site
comprises a biotin molecule and the capture zone comprises a streptavidin molecule.
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17. The enzyme detection device of any one of claims 1 to 16 wherein the device
comprises a solid support to which the capture molecules are attached to form the capture
zone.
5 18. The enzyme detection device of claim 17 wherein the solid support further comprises
a sample application zone to which the sample is applied.
19. The enzyme detection device of claim 17 or 18 wherein the solid support further
comprises a control zone, downstream of the capture zone in relation to the sample
1 0 application zone, containing further binding molecules which bind to the binding molecules to
indicate successful completion of an assay using the device.
20. The enzyme detection device of any one of claims 17 to 19 wherein the solid support
comprises a chromatographic medium
15
21. A method for detecting the presence or absence in a test sample of cleavage activity
of an enzyme capable of cleaving a substrate, the method comprising:
(i) bringing an indicator molecule into contact with the test sample, said indicator molecule
20 comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
wherein cleavage of the at least one cleavage site produces a novel binding site;
25 (ii) adding to the test sample binding molecules capable of binding to the novel binding site,
wherein the binding molecules are incapable of binding to the indicator molecule unless and
until cleavage has occurred;
(iii) capturing the part of the indicator molecule containing the novel binding site at a capture
zone through binding of capture molecules in the capture zone to the capture site; and
30 (iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the novel binding site of the indicator molecule captured in the capture zone.
22. A method for diagnosing a respiratory condition in a test sample by detecting
cleavage activity of an enzyme capable of cleaving a substrate, the method comprising:
35
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(i) bringing an indicator molecule into contact with the test sample, said indicator molecule
comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by said
enzyme if said enzyme cleavage activity is present; and
5 (b) a capture site;
wherein cleavage of the at least one cleavage site produces a novel binding site;
(ii) adding to the test sample binding molecules capable of binding to the novel binding site,
wherein the binding molecules are incapable of binding to the indicator molecule unless and
until cleavage has occurred;
1 0 (iii) capturing the part of the indicator molecule containing the novel binding site at a capture
zone through binding of capture molecules in the capture zone to the capture site; and
(iv) detecting cleavage of the at least one cleavage site by determining binding of the binding
molecules to the novel binding site of the indicator molecule captured in the capture zone
wherein an increased level of cleavage compared to a control diagnoses the respiratory
15 condition.
23. The method of claim 22 wherein the respiratory condition is chronic obstructive
pulmonary disease or inflammation of the respiratory tract as a result of cystic fibrosis.
20 24. The method of any one of claims 21 to 23 wherein cleavage of the at least one
25
cleavage site produces two parts of the cleavage region, at least one part of which
remains connected to the capture site and wherein the binding molecules are capable of
binding to the part of the indicator molecule containing the at least one part of the cleavage
region connected to the capture site.
25. The method of any one of claims 21 to 24 wherein cleavage of the at least one
cleavage site produces two separate parts of the indicator molecule.
26. The method of any one of claims 21 to 24 wherein the indicator molecule is
30 structurally constrained such that cleavage of the at least one cleavage site produces two
parts of the cleavage region of the indicator molecule which remain connected to one
another.
27. The method of any one of claims 21 to 26 wherein the binding molecules bind to the
35 cleavage region following cleavage.
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28. The method of claim 27 wherein the binding molecules bind to both parts of the
cleavage region of the indicator molecule following cleavage.
29. The method of any one of claims 21 to 28 wherein the at least one cleavage site
5 comprises a peptide bond located between two amino acid residues.
30. The method of any one of claims 21 to 29 wherein the enzyme capable of cleaving a
substrate comprises a protease.
10 31. The method of claim 30 wherein the protease is a matrix metalloproteinase.
32. The method of any one of claims 21 to 31 wherein the at least one cleavage site is
biased for cleavage by specific proteases.
15 33. The method of any one of claims 21 to 32 wherein the at least one cleavage site is
biased for cleavage by specific matrix metalloproteinases.
34. The method of any one of claims 21 to 33 wherein the at least one cleavage site is
biased for cleavage by MM P-13 and/or MM P- 9.
20
35. The method of any one of claims 21 to 34 wherein the at least one cleavage site is
biased for cleavage by MMP-13, 9, 2, 8 and 12, optionally in that order of preference.
36. The method of any one of claims 21 to 38 wherein the cleavage site is within the
25 amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ), producing a part containing the amino
acid sequence GPQG and a part containing the amino acid sequence IFGQ following
cleavage.
37. The method of any one of claims 21 to 36 wherein the binding molecule comprises
30 an antibody
38. The method of any one of claims 21 to 37 wherein the capture site comprises a biotin
molecule and the capture zone comprises a streptavidin molecule.
35 39. The method of any one of claims 21 to 38 wherein the device comprises a solid
support to which the capture molecules are attached to form the capture zone
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40. The method of claim 39 wherein the solid support further comprises a sample
application zone to which the sample is applied.
5 41. The method of claims 39 or 40 wherein the solid support further comprises a control
zone, downstream of the capture zone in relation to the sample application zone, containing
further binding molecules which bind to the binding molecules to indicate successful
completion of an assay using the device.
10 42. The method of any one of claims 35 to 37 wherein the solid support comprises a
chromatographic medium.
43. The method of any one of claims 35 to 38 wherein the test sample and indicator
molecule are mixed prior to adding the test sample to the solid support.
15
44. An antibody which binds to the amino acid sequence GPQG but not to the amino acid
sequence GPQGIFGQ (SEQ ID NO: 1 ).
45. An antibody which binds to the amino acid sequence IFGQ but not to the amino acid
20 sequence GPQGIFGQ (SEQ ID NO: 1 ).
46. An enzyme detection kit for detecting the presence in a test sample of cleavage
activity of an enzyme capable of cleaving a substrate, the kit comprising:
25 (i) an indicator molecule for adding to the test sample, said indicator molecule comprising
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present; and
(b) a capture site;
wherein cleavage of the at least one cleavage site produces a novel binding site;
30 (ii) capture molecules capable of binding to the capture site of the indicator molecule
(iii) a solid support to which the capture molecules can be attached (i.e. are attachable or
attached) to form a capture zone to receive the test sample; and
(iv) binding molecules capable of binding to the novel binding site, wherein the binding
molecules are incapable of binding to the indicator molecule unless and until cleavage has
35 occurred.
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47. The enzyme detection kit of claim 46 wherein cleavage of the at least one cleavage
site produces two parts of the cleavage region, at least one part of which
remains connected to the capture site and wherein the binding molecules are capable of
binding to the part of the indicator molecule containing the at least one part of the cleavage
5 region connected to the capture site.
48. The enzyme detection kit of claim 46 or 47 wherein cleavage of the at least one
cleavage site produces two separate parts of the indicator molecule.
10 49. The enzyme detection kit of claim 46 or 47 wherein the indicator molecule is
structurally constrained such that cleavage of the at least one cleavage site produces two
parts of the cleavage region of the indicator molecule which remain connected to one
another.
15 50. The enzyme detection kit of any one of claims 46 to 49 wherein the binding
molecules bind to the cleavage region following cleavage.
51. The enzyme detection kit of claim 50 wherein the binding molecules bind to both
parts of the cleavage region of the indicator molecule following cleavage.
20
52. The enzyme detection kit of any one of claims 46 to 51 wherein the at least one
cleavage site comprises a peptide bond located between two amino acids.
53. The enzyme detection kit of any one of claims 46 to 52 wherein the enzyme capable
25 of cleaving a substrate comprises a protease.
54. The enzyme detection kit of claim 53 wherein the protease is a matrix
metalloproteinase.
30 55. The enzyme detection kit of any one of claims 46 to 54 wherein the at least one
cleavage site is biased for cleavage by specific proteases.
56. The enzyme detection kit of any one of claims 46 to 55 wherein the at least one
cleavage site is biased for cleavage by specific matrix metalloproteinases.
35
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57. The enzyme detection kit of any one of claims 46 to 56 wherein the at least one
cleavage site is biased for cleavage by MMP-13 and/or MMP- 9.
58. The enzyme detection kit of any one of claims 46 to 57 wherein the at least one
5 cleavage site is biased for cleavage by MMP-13, 9, 2, 8 and 12, optionally in that order of
preference.
59. The enzyme detection kit of any one of claims 46 to 58 wherein the cleavage site is
within the amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ), producing a part containing the
10 amino acid sequence GPQG and a part containing the amino acid sequence IFGQ following
cleavage.
60. The enzyme detection kit of any one of claims 46 to 59 wherein the binding molecule
comprises an antibody.
15
61. The enzyme detection kit of any one of claims 46 to 60 wherein the capture site
comprises a biotin molecule
62. The enzyme detection kit of any one of claims 46 to 61 wherein the capture molecule
20 comprises a streptavidin molecule.
63. The enzyme detection kit of any one of claims 46 to 62 wherein the kit comprises a
solid support to which the capture molecules are attached to form the capture zone
25 64. The enzyme detection kit of any one of claims 46 to 63 wherein the solid support
30
further comprises a sample application zone to which the sample is applied.
65. The enzyme detection kit of any one of claims 46 to 64 wherein the kit further
comprises further binding molecules capable of binding to the binding molecules.
66. The enzyme detection kit of claim 65 wherein the solid support further comprises a
control zone, downstream of the capture zone in relation to the sample application zone, to
which the further binding molecules can be attached.
35 67. The enzyme detection kit of any one of claims 46 to 66 wherein the solid support
comprises a chromatographic medium.
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68. Use of an enzyme detection device according to any one of claims 1 to 20 for
diagnosing a respiratory condition in a test sample.
5 69. Use of a method according to any one of claims 21 to 43 for diagnosing a respiratory
10
condition in a test sample.
70. Use of an enzyme detection kit according to any one of claims 46 to 62 for
diagnosing a respiratory condition in a test sample.
71. The use according to any one of claims 68 to 70 wherein the respiratory condition is
chronic obstructive pulmonary disease or inflammation of the respiratory tract as a result of
cystic fibrosis.
15 72. An indicator molecule for use in detecting the presence in a test sample of cleavage
20
activity of an enzyme capable of cleaving a substrate, the indicator molecule comprising:
(a) a cleavage region comprising at least one cleavage site, which can be cleaved by
said enzyme if said enzyme cleavage activity is present,
(b) a capture site; and
(c) a scaffold molecule which acts to connect at least two parts of the indicator
molecule outside of the at least one cleavage site, optionally outside of the cleavage
region
wherein the scaffold further acts to structurally constrain the indicator molecule in a manner
such that cleavage of the at least one cleavage site produces a novel binding site to which
25 binding molecules bind, but wherein the binding molecules are incapable of binding to the
indicator molecule unless and until cleavage has occurred.
73. The indicator molecule of claim 72 wherein the novel binding site represents a novel
structural conformation of the indicator molecule.
30
74. The indicator molecule of claim 72 or 73 wherein the novel binding site comprises at
least a portion of the cleavage region.
75. The indicator molecule of anyone of claims 72 to 74 wherein the novel binding site
35 comprises at least a portion of the scaffold molecule.
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76. The indicator molecule of any one of claims 72 to 75 wherein the novel binding site
contains a portion of both of the at least two parts of the indicator molecule produced by
cleavage.
5 77. The indicator molecule of any one of claims 72 to 76 wherein the novel binding site
includes the cleavage site.
78. The indicator molecule of any one of claims 72 to 77 wherein the cleavage site is
specific for cleavage by a protease.
10
79. The indicator molecule of claim 78 wherein the protease is a matrix
metalloproteinase.
80. The indicator molecule of any one of claims 72 to 79 wherein the at least one
15 cleavage site is biased for cleavage by specific proteases.
81. The indicator molecule of any one of claims 72 to 80 wherein the at least one
cleavage site is biased for cleavage by specific matrix metalloproteinases.
20 82. The indicator molecule of any one of claims 72 to 81 wherein the at least one
cleavage site is biased for cleavage by MMP-13 and/or MMP- 9.
83. The indicator molecule of any one of claims 72 to 82 wherein the at least one
cleavage site is biased for cleavage by MMP-13, 9, 2, 12 and 8, optionally in that order of
25 preference.
30
35
84. The indicator molecule of any one of claims 72 to 83 wherein the cleavage site is
within the amino acid sequence GPQGIFGQ (SEQ ID NO: 1 ), producing a part containing the
amino acid sequence GPQG and a part containing the amino acid sequence IFQG.
85. The indicator molecule of any one of claims 72 to 81 which comprises a scaffold
molecule selected from the scaffold molecules shown in figure 14 or 15.
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86. The indicator molecule of any one of claims 72 to 85 wherein the binding molecule
comprises an antibody.
87. The indicator molecule of any one of claims 72 to 86 wherein the capture site
5 comprises a biotin molecule.
88. The indicator molecule of any one of claims 72 to 87 wherein the scaffold molecule
comprises a (hetero)aromatic molecule.
1 0 89. The indicator molecule of claim 86 wherein the (hetero)aromatic molecule comprises
at least two benzylic halogen substitutents.
15
90. The indicator molecule of any one of claims 72 to 89 wherein said scaffold is a
halomethylarene.
91. The indicator molecule of claim 90 wherein said halomethylarene is selected from the
group consisting of bis(bromomethyl)benzene, tris(bromomethyl)benzene and
tetra(bromomethyl)benzene, or a derivative thereof.
20 92. The indicator molecule of any one of claims 72 to 91 wherein said scaffold is selected
from the group consisting of ortho, meta and para bis(bromomethyl) benzenes, optionally
from 1 ,2-bis(bromomethyl) benzene, 1 ,3-bis(bromomethyl) benzene and 1 ,4-
bis(bromomethyl) benzene, or from further substituted halomethylarenes such as 1 ,3,5-
tris(bromomethyl)benzene, 1 ,2,4,5-tetrakis(bromomethyl)benzene and 1 ,2,3,4,5,6-
25 hexakis(bromomethyl)benzene, or from polycyclic halomethylarenes such as 2,7-
bis(bromomethyl)-naphthalene, 1 ,4-bis(bromomethyl)-naphthalene, 1 ,8-bis(bromomethyl)naphthalene,
1 ,3-bis(bromomethyl)-naphthalene, 1 ,2-bis(bromomethyl)-naphthalene, 2,3-
bis(bromomethyl)-naphthalene, 2,6-bis(bromomethyl)-naphthalene, 1 ,2,3,4-
tetrakis(bromomethyl)-naphthalene, 9,1 0-bis(bromomethyl)-phenanthrene, 5,10-
30 bis(bromomethyl)-anthracene, and 1-(bromomethyl)-3-[3-(bromomethyl)benzyl]benzene, or
from methyl substituted halomethylarenes such as 1 ,3-bis(bromomethyl)-5-methylbenzene,
2,5-bis(bromomethyl)-1 ,3-dimethylbenzene, 2,5-bis(bromomethyl)-1 ,4-dimethylbenzene, 2,4-
bis(bromomethyl)-1 ,3,5-trimethylbenzene and 3,6-bis(bromomethyl)durene, or from nitro
substituted halomethylarenes such as 3,4-bis(bromomethyl)-nitrobenzene and 2,3-
35 bis(bromomethyl)-nitrobenzene, or from hydroxy substituted halomethylarenes such as 1 ,3-
bis(bromomethyl)-5-hydroxybenzene, or from cyano substituted halomethylarenes such as
5
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2,6-bis(bromomethyl)-benzonitrile or from methoxy substituted halomethylarenes such as
1 ,3-bis(bromomethyl)-5-methoxybenzene, 1 ,3-bis(bromomethyl)-2-methoxy-5-
methylbenzene, 1 ,3-bis(bromomethyl)-5-hydroxybenzene, 2,3-bis(bromomethyl)-1 ,4-
dimethoxybenzene, and 2,5-bis(bromomethyl)-1 ,4-dimethoxybenzene.
93. The enzyme detection device of anyone of claims 1 to 20 incorporating an indicator
molecule as defined in any one of claims 72 to 92.
94. The method of any one of claims 21 to 43 incorporating an indicator molecule as
1 0 defined in any one of claims 72 to 92.
95. The enzyme detection kit of any one of claims 46 to 67 incorporating an indicator
molecule as defined in any one of claims 72 to 92.
15 96. Use of an indicator molecule as define in any one of claims 72 to 92 for detecting the
presence in a test sample of cleavage activity of an enzyme.
97. An enzyme detection device as defined herein with reference to the figures.
20 98. A method as defined herein with reference to the figures.
99. An enzyme detection kit as defined herein with reference to the figures.
100. An indicator molecule of formula I or II (optionally lacking the tyrosine residue) or as
25 defined herein with reference to the figures.
101. A binding molecule as defined herein with reference to the figures.