U.S. patent application number 15/358560 was filed with the patent office on 2017-10-12 for non-invasive enzyme screen for tissue remodelling-associated conditions.
The applicant listed for this patent is Children's Medical Center Corporation. Invention is credited to Marsha A. Moses, Li Yan.
Application Number | 20170292953 15/358560 |
Document ID | / |
Family ID | 22906730 |
Filed Date | 2017-10-12 |
United States Patent
Application |
20170292953 |
Kind Code |
A1 |
Moses; Marsha A. ; et
al. |
October 12, 2017 |
NON-INVASIVE ENZYME SCREEN FOR TISSUE REMODELLING-ASSOCIATED
CONDITIONS
Abstract
Methods and kits for diagnosing the presence of and prognosing
the appearance of tissue remodelling-associated conditions,
involving the presence of enzyme complexes in a biological sample,
are disclosed. In particular, the method pertains to diagnosing the
presence of or prognosing appearance of metastatic cancer by the
identification of high molecular weight enzyme complexes comprising
MMPs.
Inventors: |
Moses; Marsha A.;
(Brookline, MA) ; Yan; Li; (Berwyn, PA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Children's Medical Center Corporation |
Boston |
MA |
US |
|
|
Family ID: |
22906730 |
Appl. No.: |
15/358560 |
Filed: |
November 22, 2016 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
14855718 |
Sep 16, 2015 |
|
|
|
15358560 |
|
|
|
|
14146084 |
Jan 2, 2014 |
|
|
|
14855718 |
|
|
|
|
12246085 |
Oct 6, 2008 |
|
|
|
14146084 |
|
|
|
|
11585354 |
Oct 23, 2006 |
7432066 |
|
|
12246085 |
|
|
|
|
09977878 |
Oct 15, 2001 |
7153660 |
|
|
11585354 |
|
|
|
|
60240489 |
Oct 13, 2000 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
G01N 33/574 20130101;
G01N 33/573 20130101; G01N 33/57434 20130101; C12Q 1/37 20130101;
G01N 2333/99 20130101; G01N 33/57488 20130101; G01N 2333/96486
20130101; G01N 2550/00 20130101; G01N 2333/96494 20130101 |
International
Class: |
G01N 33/573 20060101
G01N033/573; G01N 33/574 20060101 G01N033/574 |
Claims
1. A non-invasive method for facilitating the diagnosis of a
subject for a tissue remodelling-associated condition, comprising:
obtaining a biological sample from a subject; detecting a high
molecular weight enzyme complex: in the biological sample; and
correlating the presence or absence of the high molecular weight
enzyme complex with the presence or absence of a tissue
remodelling-associated condition, thereby facilitating the
diagnosis of the subject for a tissue remodelling-associated
condition.
2. The method of claim 1, wherein the tissue remodelling-associated
condition is cancer.
3. The method of claim 1, wherein the tissue remodelling-associated
condition is an arthritic condition, an obstructive condition, or a
degenerative condition.
4-6. (canceled)
7. The method of claim 2, wherein the cancer is selected from the
group consisting of cancers of the nervous system, breast, retina,
lung, skin, kidney, liver, pancreas, genito-urinary tract, and
gastrointestinal tract.
8. The method of claim 2, wherein the cancer appears in cells of
mesodermal origin.
9. The method of claim 2, wherein the cancer appears in cells of
endodermal origin.
10. The method of claim 2, wherein the cancer affects cells of bone
or of hematopoietic origin.
11-26. (canceled)
27. A non-invasive method for facilitating the diagnosis of a
subject for a tissue remodelling-associated condition, comprising:
obtaining a biological sample from a subject; detecting lipocalin
in the biological sample; and correlating the presence or absence
of the lipocalin with the presence or absence of a tissue
remodelling-associated condition, thereby facilitating the
diagnosis of the subject for a tissue remodelling-associated
condition.
28. The method of claim 27, wherein the tissue
remodelling-associated condition is cancer.
29. The method of claim 27, wherein the tissue
remodelling-associated condition is an arthritic condition, an
obstructive condition, or a degenerative condition.
30-32. (canceled)
33. The method of claim 28, wherein the cancer is selected from the
group consisting of cancers of the nervous system, breast, retina,
lung, skin, kidney, liver, pancreas, to genito-urinary tract, and
gastrointestinal tract.
34. The method of claim 28, wherein the cancer appears in cells of
mesodermal origin.
35. The method of claim 28, wherein the cancer appears in cells of
endodermal origin.
36. The method of claim 28, wherein the cancer affects cells of
bone or of hematopoietic origin.
37-48. (canceled)
49. A kit for facilitating the diagnosis and prognosis of a tissue
remodelling-associated condition, comprising: a container having a
reagent for detecting a high molecular weight enzyme complex in a
biological sample, and instructions for using said reagent for
detecting the high molecular weight enzyme complex for facilitating
the diagnosis and prognosis of a tissue remodelling associated
condition.
50. The kit of claim 49, wherein the tissue remodelling-associated
condition is cancer.
51. The kit of claim 49, wherein the tissue remodelling-associated
condition is an arthritic condition, an obstructive condition, or a
degenerative condition.
52-54. (canceled)
55. The kit of claim 49, wherein the cancer is selected from the
group consisting of cancers of the nervous system, breast, retina,
lung, skin, kidney, liver, pancreas, genito-urinary tract, and
gastrointestinal tract.
56-80. (canceled)
Description
RELATED APPLICATIONS
[0001] The present application claims priority to U.S. provisional
application Ser. No. 60/240,489 filed on Oct. 13, 2000, the
contents of which are expressly incorporated herein by reference.
This application is also related to Ser. No. 08/639,373 filed on
Apr. 26, 1996, (abandoned), U.S. Pat. No. 6,037,138, and Ser. No.
09/469,637 (pending), the entire contents of each are expressly
incorporated herein by reference. The contents of Yan, L. et al.
(2001) J. Biol.Chem. 276: 37258-37265 are expressly incorporated
herein by reference.
BACKGROUND OF THE INVENTION
[0002] Matrix metalloproteinases (MMP) are a family of
endopeptidases whose activities depend on metal ions, such as
Zn.sup.+- and Ca.sup.++. Collectively, MMPs are capable of
degrading all the molecular components of extracellular matrix
(ECM), the barrier separating the tumor cells from the normal
surrounding tissues, which is disassembled as part of the
metastatic process (Lochter, A., et al. (1998) Ann NY Acad Sci.
857: 180-93). MMPs have been shown to play important roles in a
variety of biological as well as pathological processes, especially
in tumor cell invasion and metastasis (Kleiner, D. E. and
Stetler-Stevenson, W. G. (1999) Cancer Chemother Pharmacol. 43:
S42-51). Overproduction of MMPs by tumor cells or surrounding
stromal cells has been correlated with the metastatic phenotype. In
particular, U.S. Ser. No. 09/469,637, the contents of which are
herein incorporated by reference in their entirety, teaches that
intact and biologically active MMPs can be detected in biological
samples of cancer patients and are independent predictors of
disease status. The MMP activities detected in U.S. Ser. No.
09/469,637 include, for example, MMP-9 (92 kDa, gelatinase B, type
IV collagenase, EC3.4.24.35) and MMP-2 (72 kDa, gelatinase A, type
IV collagenase, EC3.4.24.24). Both of these MMPs have been shown to
be independent predictors of tissue remodeling-associated
conditions, e.g., cancer. In addition to these two major gelatinase
species, several MMP activities with molecular sizes of equal to,
or greater than, 150 kDa were observed and were referred to as high
molecular weight (hMW) MMPs. Elevated MMP levels in biological
fluids, including serum, plasma, and urine from animals bearing
experimental tumors or from cancer patients have also been reported
in several other studies (Nakajima, M., et al., (1993) Cancer Res.
53: 5802-7; Zucker, S., et al. (1994) Ann NY Acad Sci. 732: 248-62;
Baker, T., et al. (1994) Br J Cancer. 70: 506-12; Garbisa, S., et
al. (1992) Cancer Res. 52: 4548-9, 1992).
SUMMARY OF THE INVENTION
[0003] With the advances in cancer therapies, early diagnosis
and/or prognosis are becoming increasingly important for the
disease outcome. Accordingly, the present invention characterizes
the molecular identity of hMW MMPs found in biological samples of
subjects diagnosed with tissue remodelling-associated diseases,
e.g., cancer, and provides early diagnosis/prognosis of such
diseases. With the identification of these hMW MMPS, e.g., high
molecular weight enzyme complexes, the present invention
facilitates the development of non-invasive diagnostic and/or
prognostic methods to predict tissue remodelling-associated
diseases, such as cancer.
[0004] The present invention provides methods and kits for
detecting biological markers, e.g., high molecular weight enzyme
complexes, to non-invasively monitor the diagnosis and prognosis of
tissue remodelling-associated conditions, e.g., cancers. Tissue
remodelling-associated conditions encompassed by such methods
include diseases such as prostate cancer, breast cancer, ovarian
cancer, brain tumors, arthritic conditions, obstructive conditions,
and ulcerative conditions. The methods of the instant invention use
biological fluid samples, e.g., urine samples, that may be obtained
by personnel without medical training, and do not require visiting
a clinic or hospital. The statistical association between positive
results and occurrence of tissue remodelling-associated conditions
are applied to early diagnoses of the appearance of these
conditions, and to prognoses of changes in these conditions.
[0005] In one embodiment, the present invention provides
non-invasive methods for facilitating the diagnosis of a subject
for a tissue remodelling-associated condition. Such methods include
obtaining a biological sample from a subject, and detecting a high
molecular weight enzyme complex in the biological sample. The
methods further include correlating the presence or absence of the
high molecular weight enzyme complex with the presence or absence
of a tissue remodelling-associated condition, thereby facilitating
the diagnosis of the subject for a tissue remodelling-associated
condition.
[0006] In another embodiment, the tissue remodelling-associated
condition is cancer, e.g., organ-confined prostate cancer,
metastatic prostate cancer, cancer found in cells of epithelial
origin, mesodermal origin, endodermal origin or hematopoietic
origin, and cancer selected from the group consisting of cancers of
the nervous system, breast, retina, lung, skin, kidney, liver,
pancreas, genito-urinary tract, and gastrointestinal tract. In
another embodiment, the tissue remodelling-associated condition is
an arthritic condition, an obstructive condition, or a degenerative
condition.
[0007] In still another embodiment, the high molecular weight
enzyme complex comprises a protease, e.g., a serine protease, e.g.,
a matrix metalloproteinase, e.g., an MMP-9.
[0008] In yet another embodiment, the high molecular weight enzyme
complex further comprises a lipocalin, e.g., NGAL, and/or a TIMP,
e.g., TIMP-1.
[0009] In still another embodiment, the high molecular weight
enzyme complex comprises an enzyme complexed with itself to form a
multimer, e.g., a dimer or a trimer. Such a multimer can further be
complexed with a lipocalin, e.g., NGAL, and/or a TIMP, e.g.,
TIMP-1.
[0010] In still yet another embodiment, the molecular weight of the
high molecular weight enzyme complex is at least about 115 kDa to
at least about 125 kDa. In another embodiment, the molecular weight
of the high molecular weight enzyme complex is at least about 150
kDa.
[0011] In another embodiment, the methods of the present invention
include obtaining a biological sample from a subject and detecting
lipocalin in the biological sample. Such methods further include
correlating the presence or absence of the lipocalin with the
presence or absence of a tissue remodelling-associated condition,
thereby facilitating the diagnosis of the subject for a tissue
remodelling-associated condition.
[0012] In still another embodiment, the present invention provides
kits for facilitating the diagnosis and prognosis of a tissue
remodelling-associated condition. Such kits include a container
having a reagent for detecting a high molecular weight enzyme
complex in a biological sample and instructions for using the
reagent for detecting the high molecular weight enzyme complex
which facilitates the diagnosis and prognosis of a tissue
remodelling-associated condition.
DESCRIPTION OF THE FIGURES
[0013] FIG. 1 shows a substrate gel electrophoresis and NGAL
Western blot analysis of urine samples. A. Substrate gel
electrophoresis of MMPs in urine samples: 50 .mu.l of untreated
urine samples were analyzed for MMP activities. Four major
gelatinase activities were detected with apparent molecular masses
of approximately 200,000, 125,000, 92,000, and 72,000. Their
identities are marked with arrows on right. The molecular size
markers are Perfect Protein Markers (Novagen, Madison, Wis.) with
sizes of 150 kDa, 100 kDa, 75 kDa, and 50 kDa (arrows on left). B.
20 .mu.g of concentrated urine samples were separated on a 4-15%
SDS-PAGE gel under non-reducing conditions. Western blot analysis
was carried out using a polyclonal antibody against human NGAL. The
molecular size markers are Kaleidoscope Prestained Standards
(Bio-Rad, Hercules, Calif.) with sizes of 126 kDa, 90 kDa, 44 kDa,
34 kDa, and 17 kDa (arrows on left).
[0014] FIG. 2 shows a Western blot and substrate gel
electrophoresis of urine samples and purified human neutrophil
MMP-9/NGAL complex. A. NGAL Western blot analysis: A concentrated
urine sample containing the 125 kDa MMP activity, together with
purified human neutrophil MMP-9/NGAL, were separated by 4-15%
SDS-gel electrophoresis under non-reducing conditions, and
subsequently subjected to Western blot analysis using a polyclonal
antibody against human NGAL. The 125 kDa MMP-9/NGAL complex is
marked (arrow on right). B. Substrate gel electrophoresis: The same
urine sample and purified human neutrophil MMP-9/NGAL complex were
analyzed with substrate gel electrophoresis. The positions of MMP-9
dimer (200 kDa), MMP-9/NGAL (125 kDa), MMP-9 (92 kDa), and MMP-2
(72 kDa) are denoted with arrows on right. The molecular size
markers are Perfect Protein Markers (Novagen, Madison, Wis.) with
sizes of 150 kDa, 100 kDa, 75 kDa, and 50 kDa (arrows on left).
[0015] FIG. 3 shows an immunoprecipitation of the 125 kDa MMP
activity using anti-NGAL antibody. 50 .mu.l of urine samples (1:1
v/v diluted with RIPA) containing the 125 kDa MMP activity were
mixed with 1.0, 0.1 or 0.01 .mu.l of anti-NGAL antibody or a
control antibody. After incubating on ice for thirty minutes, the
antibody-antigen complexes were removed using Zysorbin. The
supernatants were subjected to substrate gel electrophoresis to
detect the remaining MMP activities. The increased MMP-2 activity
observed in the sample treated with 1.0 .mu.l of the control
antibody was the endogenous MMP-2 activity from the serum.
[0016] FIG. 4 shows reconstitution of MMP-9/NGAL complexes in
vitro. A. Recombinant human MMP-9 and NGAL were diluted in
gelatinase buffers with different pH values and were subsequently
mixed in a molar ratio of 1:10 (proMMP-9 to NGAL). In vitro
reconstitution was carried out at 37.degree. C. for one hour. 10
.mu.M proMMP-9 was loaded in each lane. Purified human neutrophil
MMP-9/NGAL was included as a control. B. Recombinant human MMP-9
and NGAL were diluted in normal urine containing no MMP activities
and were subsequently mixed in different molar ratios (proMMP-9 to
NGAL=2:1, 1:5, 1: 1 0, 1:20). After one hour incubation at
37.degree. C., MMP-9/NGAL complex formation was analyzed using
substrate gel electrophoresis. The positions of the 125 kDa and 115
kDa MMP-9/NGAL activity are respectively denoted with the arrow and
the arrowhead on right. The molecular size markers are Perfect
Protein Markers (Novagen, Madison, Wis.) with sizes of 150 kDa, 100
kDa, 75 kDa, and 50 kDa (arrows on left).
DETAILED DESCRIPTION OF THE INVENTION
[0017] The present invention features non-invasive methods for
facilitating the diagnosis of a subject for a tissue
remodelling-associated condition (TRAC), especially cancers,
obstructive and degenerative conditions, and arthritic conditions.
Detection of a pattern of enzyme complexes, e.g., high molecular
weight (hMW) enzyme complexes, in a biological sample from a
subject is used to facilitate diagnosis and prognosis of a
TRAC.
[0018] The language "high molecular weight enzyme complex" includes
an enzyme associated with or bound to another molecule wherein the
complex has a high molecular weight allowing it to be used for its
intended function of the present invention. Examples of enzyme
complexes include, among others, an enzyme bound to another enzyme,
an enzyme bound to an enzyme inhibitor, and an enzyme bound to a
protein binding molecule, e.g., a lipocalin. Enzyme complexes which
comprise enzymes bound to themselves, e.g., multimers, e.g., dimers
and trimers, are also encompassed by the present invention.
[0019] High molecular weight enzyme.sup.-complexes include enzyme
complexes which have a molecular weight of at least about 115 kDa,
e.g., at least about 120 kDa, e.g., at least about 125 kDa, e.g.,
at least about 130 kDa, e.g., at least about 135 kDa, and, e.g., at
least about 140 kDa. High molecular weight enzyme complexes which
have a molecular weight of at least about 145 kDa, e.g., at least
about 150 kDa, and greater than 150 kDa are also included.
[0020] The ranges of high molecular weight values intermediate to
those listed also are intended to be part of this invention, e.g.
at least about 115 kDa to at least about 120 kDa, at least about
120 kDa to at least about 125 kDa, at least about 125 kDa to at
least about 130 kDa, at least about 130 kDa to at least about 135
kDa, at least about 135 kDa to at least about 140 kDa, at least
about 140 kDa to at least about 145 kDa, and at least about 145 kDa
to at least about 150 kDa. For example, ranges of high molecular
weight values using a combination of any of the above values
recited as upper and/or lower limits are intended to be
included.
[0021] In one embodiment of the invention, the high molecular
weight enzyme complex does not have a molecular weight of 115 kDa.
In another embodiment, the high molecular weight enzyme complex
does not include NGAL. In another embodiment, the high molecular
weight enzyme complex does not include a progelatinase B enzyme. In
yet another embodiment of the invention, the high molecular weight
enzyme complex does not include a progelatinase B enzyme associated
with NGAL.
[0022] The term "enzyme" is art recognized and includes protein
catalysts of chemical reactions. Enzymes can be a whole intact
enzyme or portions or fragments thereof. The enzymes encompassed by
the enzyme complexes of the current invention include naturally
occurring enzymes that catalytically degrade proteins, i.e. the
enzymes known as proteases or proteinases. By proteinase is meant a
progressive exopeptidase that digest proteins by removing amino
acid residues from either the N terminal or C terminal which
reaction proceeds to achieve significant degradation, or an
endopeptidase which destroys the amide bond between amino acid
residues with varying degrees of residue specificity. The term
"protease" may also include the highly specific amino acid
peptidases that remove a single amino acid from an N terminus or C
terminus of a protein. Examples are alanine aminopeptidase (EC
3.4.11.2) and leucine aminopeptidase (EC 3.4.11.1), which remove
alanine or leucine, respectively, from the amino terminus of a
protein that may have alanine and leucine, respectfully, at the
amino terminus. The molecular weights of the enzymes comprising the
enzyme complexes of the invention include, but not limited to,
molecular weights in the range of approximately 72 kDa,
approximately 92 kDa, approximately 115 kDa to approximately 125
kDa and approximately 150 kDa or greater. The term "enzyme"
includes polymorphic variants that are silent mutations naturally
found within the human population.
[0023] In one embodiment, the enzyme complexes of the present
invention comprise proteases or proteinases. The term proteases
(and its equivalent term proteinases) is intended to include those
endopeptidases and progressive exopeptidases that are capable of
substantially reducing the molecular weight of the substrate and
destroying its biological function, especially if that biological
function of the substrate is to be a structural component of a
matrix barrier. Amino acid peptidases such as alanine
aminopeptidase and leucine aminopeptidase are also broadly included
among proteases, however do not share the property of significantly
reducing the molecular weight of the substrate protein.
[0024] Many thousands of proteases occur naturally, and each may
appear at different times of development and in different locations
in an organism. The invention herein features enzymes of the class
of the matrix metalloproteinases (MMPs, class EC 3.4.24). These
enzymes, which require a divalent cation for activity, are normally
expressed early in the development of the embryo, for example,
during hatching of an zygote from the zona pellucida, and again
during the process of attachment of the developing embryo to the
inside of the uterine wall. Enzyme activities such as
N-acetylglucosaminidase (EC 3.2.1.50) appear in urine in the case
of renal tubular damage, for example, due to diabetes (Can, M.
(1994) J. Urol. 151(2):442-445; Jones, A., et. al. (1995) Annals.
Clin. Biochem., 32:68-62). That these activities appear in urine as
a result of renal tubular damage is irrelevant to the present
invention as described herein.
[0025] The term "matrix-digesting enzyme" includes an enzyme
capable of digesting or degrading a matrix, e.g., a mixture of
proteins and proteoglycans that comprise a layer in a tissue on
which certain types of cells are found. Matrix-digesting enzymes
are expressed during stages of normal embryogenesis, pregnancy and
other processes involving tissue remodelling. In addition, some of
these enzymes, for example some matrix metalloproteinases (MMPs),
degrade the large extracellular matrix proteins of the parenchymal
and vascular basement membranes that serve as mechanical barriers
to tumor cell migration. These MMPs are produced in certain cancers
and are associated with metastasis (Liotta, L. A., et al. (1991)
Cell 64:327-336). Examples of MMPs are the type IV collagenases,
e.g., MMP-2 (gelatinase A. EC 3.4.24.24) and MMP-9 (gelatinase B,
3.4.24.35), and stromelysins (EC 3.4.24.17 and 3.4.24.22). Some
MMPs are specifically inhibited by molecules called tissue
inhibitors of metalloproteinases (TIMPs, Woessner, J. F., Jr.
(1995)Ann. New York Acad. Sci., 732:11-21), which also may be
overproduced by tumor cells, however under certain conditions
enzyme activity is in molar excess over the TIMPs (Freeman, M. R.
et al. (1993) J. Urol. 149:659; Lu, X. et al. (1991) Cancer Res.
51:6231-6235; Kossakowska, A. E. et al. (1991) Blood 77:2475-2481).
Accordingly, in one embodiment of this invention, the enzyme
complexes of the present methods comprise an inhibitor of the
enzyme (TIMPS, e.g., TIMP-1 or TIMP-2). The detection of an
inhibitor can be accomplished using art-recognized techniques. Many
of MMPs are translated as pro-enzymes, and may be found in a
variety of structures, with ranges of molecular weights including
smaller forms (45 kDa, 55 kDa, 62 kDa), and larger forms (72 kDa,
82 kDa, 92 kDa, and higher polymers such as 150 kDa and
greater).
[0026] In another embodiment of the invention, the high molecular
weight enzyme complex comprises a protein binding molecule, e.g., a
lipocalin. Lipocalins are small secreted proteins that bind small,
hydrophobic molecules to form molecular complexes. Lipocalins are
implicated in a variety of functions including, among others,
regulation of the immune response, e.g., lipocalins can exert
certain immunomodulatory effects in vitro. It has been shown that
neutrophil lipocalin covalently attaches to human neutrophil
gelatinase (type IV collagenase) thus forming Neutrophil
Gelatinase-Associated Lipocalin (NGAL) (Treibel et al. (1992) and
Kjelsen et al. (1993)) although most of the protein is secreted in
uncomplexed form. These authors prose a regulatory role for NGAL on
the action of the gelatinase.
[0027] In another embodiment, the present invention includes
methods of detecting a lipocalin, e.g., NGAL, as an indicator of a
TRAC. Such lipocalins can be detected in a biological sample as an
isolated lipocalin or as multimers of lipocalins, e.g., dimers and
trimers.
[0028] The tissue remodelling conditions that can be monitored by
the methods of this invention include a variety of types of cancer;
moreover, the enzymes are suitable for diagnosis of other tissue
remodelling conditions, such as arthritis, degenerative conditions,
and obstructive conditions. The invention provides non-invasive
methods for diagnosing these conditions by assay for enzyme
complexes, e.g., hMW enzyme complexes, in biological fluids.
[0029] The methods of this invention embody detection of enzymes in
urine, for diagnosis and prognosis of cancer. The invention also
relates to diagnosis and prognosis of metastatic prostate cancer.
The varieties of cancer suitable for diagnosis by the methods of
this invention include, among others, cancers of epithelial origin,
for example, cancers of the nervous system, breast, retina, lung,
skin, kidney, liver, pancreas, genito-urinary tract, ovarian,
uterine and vaginal cancers, and gastrointestinal tract cancers,
which form in cells of epithelial origin. Using the methods
described here, cancers of mesodermal and endodermal origin, for
example, cancers arising in bone or in hematopoietic cells, are
also diagnosed.
[0030] The term "subject," as used herein, includes a living animal
or human in need of diagnosis or prognosis for, or susceptible to,
a condition, in particular an "tissue remodelling-associated
condition" as defined below. The subject is an organism capable of
responding to tissue remodelling signals such as growth factors,
under some circumstances, the subject is susceptible to cancer and
to arthritis. In one embodiment, the subject is a mammal, including
humans and non-human mammals such as dogs, cats, pigs, cows, sheep,
goats, horses, rats, and mice. In one embodiment, the subject is a
human. The term "subject" does not preclude individuals that are
entirely normal with respect to tissue remodelling-associated
conditions or normal in all respects. The subject may formerly have
been treated surgically or by chemotherapy, and may be under
treatment by hormone therapy or have been treated by hormone
therapy in the past.
[0031] The term "patient," as used herein, includes a human subject
who has presented at a clinical setting with a particular symptom
or symptoms suggesting one or more diagnoses. A patient may be in
need of further categorization by clinical procedures well-known to
medical practitioners of the art (or may have no further disease
indications and appear to be in any or all respects normal). A
patient's diagnosis may alter during the course of disease
progression, such as development of further disease symptoms, or
remission of the disease, either spontaneously or during the course
of a therapeutic regimen or treatment. Thus, the term "diagnosis"
does not preclude different earlier or later diagnoses for any
particular patient or subject. The term "prognosis" includes an
assessment for a subject or patient of a probability of developing
a condition associated with or otherwise indicated by presence of
one or more enzymes in a biological sample, e.g., in urine.
[0032] The term "biological sample" includes biological samples
obtained from a subject. Examples of such samples include urine,
blood taken from a prick of the finger or other source such as
intravenous, blood fractions such as serum and plasma, feces and
fecal material and extracts, saliva, cerebrospinal fluid, amniotic
fluid, mucus, and cell and tissue material such as cheek smear, Pap
smear, fine needle aspiration, sternum puncture, and any other
biopsied material taken during standard medical and open surgical
procedures.
[0033] The term "invasiveness" as used here with respect to
metastatic cancer (Darnell, J. (1990) Molecular Cell Biology, Third
Ed., W. H. Freeman, NY) is distinct from the use of the term
"invasive" to describe a medical procedure, and the distinction is
made in context. "Invasive" for a medical procedure pertains to the
extent to which a particular procedure interrupts the integrity of
the body. "Invasiveness" ranges from fully non-invasive, such as
collection of urine or saliva; to mildly invasive, for example a
Pap smear, a cheek scrape or blood test, which requires trained
personnel in a clinical setting; to more invasive, such as a
sternum marrow collection or spinal tap; to extensively invasive,
such as open surgery to detect the size and nature of tumors by
biopsy of material, taken for example during brain surgery, lung
surgery, or transurethral resection in the case of prostate
cancer.
[0034] Cancer or neoplasia is characterized by deregulated cell
growth and division. A tumor arising in a tissue originating from
endoderm or exoderm is called a carcinoma, and one arising in
tissue originating from mesoderm is known as a sarcoma (Darnell, J.
(1990) Molecular Cell Biology, Third Ed., W. H. Freeman, NY). A
current model of the mechanism for the origin of a tumor is by
mutation in a gene known as an oncogene, or by inactivation of a
second tumor-suppressing genes (Weinberg, R. A. (September 1988)
Scientific Amer. 44-51). The oncogenes identified thus far have
arisen only in somatic cells, and thus have been incapable of
transmitting their effects to the germ line of the host animal. In
contrast, mutations in tumor-suppressing genes can be identified in
germ line cells, and are thus transmissible to an animal's progeny.
Examples of cancers include cancers of the nervous system, breast,
retina, lung, skin, kidney, liver, pancreas, genito-urinary tract,
gastrointestinal tract, cancers of bone, and cancers of
hematopoietic origin such as leukemias and lymphomas. In one
embodiment of the present invention, the cancer is not a cancer of
the bladder.
[0035] An arthritic condition such as rheumatoid arthritis is an
example of a TRAC since the disease when chronic is characterized
by disruption of collagenous structures (J. Orten et al. (1982)
Human Biochemistry, Tenth Ed., C. V. Mosby, St. Louis, Mo.). Excess
collagenase is produced by cells of the proliferating synovium.
Other TRAC conditions such as ulcerative, obstructive and
degenerative diseases are similarly characterized by alterations in
the enzymes of metabolism of structural proteins.
[0036] The term "electrophoresis" is used to indicate any
separation system of molecules in an electric field, generally
using an inert support system such as paper, starch gel, or
polyacrylamide. The electrophoresis methods with polyacrylamide
gels and the sodium dodecyl sulfate denaturing detergent are
described in the Examples below. The protocols are not intended to
exclude equivalent procedures known to the skilled artisan. Other
SDS polyacrylamide procedures, known to the skilled artisan, may be
used, e.g., a single polyacrylamide concentration such as 10%, may
be substituted for the gradient in the separation gel. The physical
support for the electrophoretic matrix may be capillary tubes
rather than glass plates. Details of several SDS-polyacrylamide gel
electrophoresis systems are described in many review articles and
biotechnology manuals (e.g., Maniatis, T., Molecular Cloning: A
Laboratory Manual, 2nd Edition, Cold Spring Harbor Press, Cold
Spring Harbor, N.Y.). The method is not limited to use of SDS and
other detergents. Further, electrophoresis in the absence of
detergents may be employed. Proteins may be separated under
non-denaturing conditions, for example in the presence of urea on a
polyacrylamide matrix (Maniatis, supra), or by charge, for example
by the procedure of iso-electric focusing.
[0037] In using an electrophoretic technique for separation of
enzymes, the electrophoretogram may be developed as a zymogram. The
term "zymography" is meant here to include any separations system
utilizing a chemically inert separating or support matrix, that
allows detection of an enzyme following electrophoresis, by
exposing the matrix of the separations system to conditions that
allow enzyme activity and subsequent detection. More narrowly, the
term zymography designates incorporation of an appropriate
substrate for the enzyme of interest into the inert matrix, such
that exposing the matrix to the conditions of activity after the
electrophoresis stop yields a system to visualize the precise
location, and hence the mobility, of the active enzyme. By
techniques well-known to the skilled artisan, the molecular weights
of proteins are calculated based on mobilities derived from
positions on a zymogram. Such techniques include comparison with
molecular weight standards, the mobilities of which are determined
from general protein stains or from pre-stains specific to those
standards, and comparison with positive controls of purified
isolated enzymes of interest, which are visualized by the technique
of the zymogram, i.e., enzyme activity.
[0038] In particular, substrates for detection of proteases by
zymography are included in the electrophoresis matrix. For type IV
collagenases, the natural substrate is a type IV collagen and
gelatin, a type I collagen derivative, is used for the zymography
substrate in the Examples presented herein. However other proteins
that are suitable for detection of further proteases of interest in
TRAC diagnosis, for example, include fibronectin; vitronectin;
collagens of types I through III and V through XII; procollagens;
elastin; laminin; plasmin; plasminogen; entactin; nidogen;
syndecan; tenascin; and sulfated proteoglycans substituted with
such saccharides as hyaluronic acid, chondroitin-6-sulfate,
condroitin-4-sulfate, heparan sulfate, keratan sulfate, and
dermatan sulfate and heparin. Further, convenient inexpensive
substrate proteins such as casein, which may not be the natural
target of a protease of interest, but are technically appropriate,
are included as suitable substrate components of the zymography
techniques of the present invention. Chemically synthesized
mimetics of naturally occurring protein substrates are also
potential zymography substrates, and may even be designed to have
favorable properties, such chromogenic or fluorogenic ability to
produce a color or fluorescent change upon enzymatic cleavage.
[0039] Zymography may be adapted to detection of a protease
inhibitor in the biological sample. Since a variety of natural MMP
inhibitors are elaborated, such as TIMP-1 and TIMP-2, and are found
to be deregulated during TRAC situations, the present invention
includes detection of enzyme complexes which comprise enzyme
inhibitors, e.g., TIMPs. Thus for example, a "reporter enzyme" for
which an enzyme inhibitory activity is being measured, may be
incubated with each biological sample obtained by subjects and
patients, in one or more quantities corresponding to one or more
aliquots of sample, prior to electrophoresis. This enzyme is
omitted from one aliquot of the biological sample. The inhibitory
presence in the sample is detected as disappearance or decrease of
the reporter enzyme band from the developed zymogram.
Alternatively, functional enzyme activity assays which include in
the reaction mix a known level of active enzyme, to which is added
aliquots of experimental samples with putative inhibitory activity,
can detect the presence of inhibitors.
[0040] Further, the enzymes of tissue remodelling extend to enzyme
activities beyond those of proteolytic activity. For example,
enzymes that are substituted with residues such as glycosyl,
phosphate, sulfate, lipids and nucleotide residues (e.g. adenyl)
are well-known to those skilled in the art. These residues are in
turn added or removed by other enzymes, e.g., glycosidases,
kinases, phosphatases, adenyl transferases, etc. Convenient
detection methods for the presence of such activities for TRAC
diagnosis and prognosis are readily developed by those with skill
in the art, and are intended to comprise part of the invention
here.
[0041] The zymogram as described in the Examples herein is
developed by use of a general stain for protein, in this case,
Coomassie Blue dye. The development is possible with general
protein stains, e.g., Amido Black dye, and SYPRO Orange stain
(Biorad Laboratories, Hercules, Calif. 94537). Further, enzyme
activity may be detected by additional techniques beyond that of a
clear zone of digestion in a stained matrix, for example, by
absence of areas of radioactivity with a radio-labelled substrate,
by change in mobility of a radio-labelled substrate, or by absence
of or change in mobility of bands of fluorescence or color
development with use of fluorogenic or chromogenic substrates,
respectfully.
[0042] Quantitative densitometry can be performed with zymograms by
placing the gel directly on an activated plate of a Molecular
Dynamics phosphorimager (Molecular Dynamics, 928 East Argues Ave.,
Sunnyvale, Calif. 94086), or with a Datacopy G8 plate scanner
attached to a MacIntosh computer equipped with an 8-bit videocard
and McImage (Xerox Imaging Systems). Background measurements, areas
of the gel separate from sample lanes, can similarly be scanned,
and values subtracted from the readings for enzyme activities.
[0043] Another electrophoretically-based technique for analysis of
a biological sample for presence of specific proteins is an
affinity-based-mobility alteration system (Lander, A. (1991) Proc.
Natl. Acad. Sci. U.S., 88(7):2768-2772). An MMP or other type of
enzyme of interest might be detected, for example, by inclusion of
a substrate analog that binds essentially irreversibly to the
enzyme, hence decreasing the mobility. The affinity material is
present during electrophoresis, and is incorporated into the
matrix, so that detection of the enzyme of interest occurs as a
result of alteration of mobility in contrast to mobility in the
absence of the material. Yet another technique of electrophoretic
protein separation is based on the innate charge of a protein as a
function of the pH of the buffer, so that for any protein species,
there exists a pH at which that protein will not migrate in an
electric field, or the isoelectric point, designated pI. Proteins
of a biological sample, such as a urine sample, may be separated by
isoelectric focusing, then developed by assaying for enzymatic
activity for example by transfer to material with substrate, i.e.,
zymography. Electrophoresis is often used as the basis of
immunological detections, in which the separation step is followed
by physical or electrophoretic transfer of proteins to an inert
support such as paper or nylon (known as a "blot"), and the blotted
pattern of proteins may be detected by use of a specific primary
binding (Western blot) by an antibody followed by development of
bound antibodies by secondary antibodies bound to a detecting
enzyme such as horse radish peroxidase. Additional immunological
detection systems for TRAC enzyme complexes are now described in
detail below.
[0044] The term "antibody" as used herein is intended to include
fragments thereof which are also specifically reactive with one of
the components in the methods and kits of the invention. Antibodies
can be fragmented using conventional techniques and the fragments
screened for utility in the same manner as described above for
whole antibodies. For example, F(ab).sub.2 fragments can be
generated by treating an antibody with pepsin. The resulting
F(ab).sub.2 fragment can be treated to reduce disulfide bridges to
produce Fab fragments. The term "antibody" is further intended to
include single chain, bispecific and chimeric molecules. The term
"antibody" includes possible use both of monoclonal and polyclonal
antibodies (Ab) directed against a target, according to the
requirements of the application.
[0045] Polyclonal antibodies can be obtained by immunizing animals,
for example rabbits or goats, with a purified form of the antigen
of interest, or a fragment of the antigen containing at least one
antigenic site. Conditions for obtaining optimal immunization of
the animal, such as use of a particular immunization schedule, and
using adjuvants e.g. Freund's adjuvant, or immunogenic substituents
covalently attached to the antigen, e.g. keyhole limpet hemocyanin,
to enhance the yield of antibody titers in serum, are well-known to
those in the art. Monoclonal antibodies are prepared by procedures
well-known to the skilled artisan, involving obtaining clones of
antibody-producing lymphocyte, i.e. cell lines derived from single
cell line isolates, from an animal, e.g. a mouse, immunized with an
antigen or antigen fragment containing a minimal number of
antigenic determinants, and fusing said clone with a myeloma cell
line to produce an immortalized high-yielding cell line. Many
monoclonal and polyclonal antibody preparations are commercially
available, and commercial service companies that offer expertise in
purifying antigens, immunizing animals, maintaining and bleeding
the animals, purifying sera and IgG fractions, or for selecting and
fusing monoclonal antibody producing cell lines, are available.
[0046] Specific high affinity binding proteins, that can be used in
place of antibodies, can be made according to methods known to
those in the art. For example, proteins that bind specific DNA
sequences may be engineered (Ladner, R. C., et. al., U.S. Pat. No.
5,096,815), and proteins that bind a variety of other targets,
especially protein targets (Ladner, R. C., et. al., U.S. Pat. No.
5,233,409; Ladner, R. C., et al., U.S. Pat. No. 5,403,484) may be
engineered and used in the present invention for covalent linkage
to a chelator molecule, so that a complex with a radionuclide may
be formed under mild conditions. Antibodies and binding proteins
can be incorporated into large scale diagnostic or assay protocols
that require immobilizing the compositions of the present invention
onto surfaces, for example in multi-well plate assays, or on beads
for column purifications.
[0047] General techniques to be used in performing various
immunoassays are known to those of ordinary skill in the art.
Moreover, a general description of these procedures is provided in
U.S. Pat. No. 5,051,361 which is incorporated herein by reference,
and by procedures known to the skilled artisan, and described in
manuals of the art (Ishikawa, E., et. al. (1988) Enzyme Immunoassay
Igaku-shoin, Tokyo, NY; Hallow, E. and D. Lane, Antibodies: A
Laboratory Manual, CSH Press, NY). Examples if several immunoassays
are given discussed here.
[0048] Radioimmunoassays (RIA) utilizing radioactively labeled
ligands, for example, antigen directly labeled with .sup.3H, or
.sup.14C, or .sup.125I, measure presence of MMP's as antigenic
material. A fixed quantity of labeled MMP antigen competes with
unlabeled antigen from the sample for a limited number of antibody
binding sites. After the bound complex of labeled antigen-antibody
is separated from the unbound (free) antigen, the radioactivity in
the bound fraction, or free fraction, or both, is determined in an
appropriate radiation counter. The concentration of bound labeled
antigen is inversely proportional to the concentration of unlabeled
antigen present in the sample. The antibody to MMP can be in
solution, and separation of free and bound antigen MMP can be
accomplished using agents such as charcoal, or a second antibody
specific for the animal species whose immunoglobulin contains the
antibody to MMP. Alternatively, antibody to MMP can be attached to
the surface of an insoluble material, which in this case,
separation of bound and free MMP is performed by appropriate
washing.
[0049] Immunoradiometric assays (IRMA) are immunoassays in which
the antibody reagent is radioactively labeled. An IRMA requires the
production of a multivalent MMP conjugate, by techniques such as
conjugation to a protein e.g., rabbit serum albumin (RSA). The
multivalent MMP conjugate must have at least 2 MMP residues per
molecule and the MMP residues must be of sufficient distance apart
to allow binding by at least two antibodies to the MMP. For
example, in an IRMA the multivalent MMP conjugate can be attached
to a solid surface such as a plastic sphere. Unlabeled "sample" MMP
and antibody to MMP which is radioactively labeled are added to a
test tube containing the multivalent MMP conjugate coated sphere.
The MMP in the sample competes with the multivalent MMP conjugate
for MMP antibody binding sites. After an appropriate incubation
period, the unbound reactants are removed by washing and the amount
of radioactivity on the solid phase is determined. The amount of
bound radioactive antibody is inversely proportional to the
concentration of MMP in the sample.
[0050] Other immunoassay techniques use enzyme labels such as
horseradish peroxidase, alkaline phosphatase, luciferase, urease,
and .beta.-galactosidase. For example, MMP's conjugated to
horseradish peroxidase compete with free sample MMP's for a limited
number of antibody combining sites present on antibodies to MMP
attached to a solid surface such as a microtiter plate. The MMP
antibodies may be attached to the microtiter plate directly, or
indirectly, by first coating the microtiter plate with multivalent
MMP conjugates (coating antigens) prepared for example by
conjugating MMP with serum proteins such as rabbit serum albumin
(RSA). After separation of the bound labeled MMP from the unbound
labeled MMP, the enzyme activity in the bound fraction is
determined colorimetrically, for example by a multi-well microtiter
plate reader, at a fixed period of time after the addition of
horseradish peroxidase chromogenic substrate.
[0051] Alternatively, the antibody, attached to a surface such as a
microtiter plate or polystyrene bead, is incubated with an aliquot
of the biological sample. MMP present in the fluid will be bound by
the antibody in a manner dependent upon the concentration of MMP
and the association constant between the two. After washing, the
antibody/MMP complex is incubated with a second antibody specific
for a different epitope on MMP distal enough from the MMP-specific
antibody binding site such that stearic hindrance in binding of two
antibodies simultaneously to MMP may be accomplished. For example,
the second antibody may be specific for a portion of the proenzyme
sequence. The second antibody can be labeled in a manner suitable
for detection, such as by radioisotope, a fluorescent compound or a
covalently linked enzyme. The amount of labeled secondary antibody
bound after washing away unbound secondary antibody is proportional
to the amount of MMP present in the biological sample.
[0052] The above examples of immunoassays describe the use of
radioactively and enzymatically labeled tracers. Assays also may
include use of fluorescent materials such as fluorescein and
analogs thereof, 5-dimethylaminonaphthalene-1-sulfonyl derivatives,
rhodamine and analogs thereof, coumarin analogs, and
phycobiliproteins such as allophycocyanin and R-phycoerythrin;
phosphorescent materials such as erythrosin and europium;
luminescent materials such as luminol and luciferin; and sols such
as gold and organic dyes. In one embodiment of the present
invention, the biological sample is treated to remove low molecular
weight contaminants.
[0053] In one embodiment of the present invention, the biological
sample is treated to remove low molecular weight contaminants, for
example, by dialysis. By the term "dialysis" this invention
includes any technique of separating the enzymes in the sample from
low molecular weight contaminants. The Examples use Spectra/Por
membrane dialysis tubing with a molecular weight cut-off (MWCO) of
3,500, however other products with different MWCO levels are
functionally equivalent. Other products include hollow fiber
concentration systems consisting of regenerated cellulose fibers
(with MWCO of 6,000 or 9,000) for larger volumes; a multiple
dialyzer apparatus with a sample size for one to 5 ml; and multiple
microdialyzer apparatus, convenient for samples in plates with 96
wells and MWCOs at 5,000, 8,000 and 10,000, for example. These
apparatuses are available from PGC Scientific, Gaithersburg, Md.,
20898. Those with skill in the art will appreciate the utility of
multiple dialysis units, and especially suitable for kits for
reference lab and clinic usage. Other equivalent techniques include
passage through a column holding a resin or mixture of resins
suitable to removal of low molecular weight materials. Resins such
as BioGel (BioRad, Hercules, Calif.) and Sepharose (Pharmacia,
Piscataway, N.J.) and others are well-known to the skilled artisan.
The technique of dialysis, or equivalent techniques with the same
function, are intended to remove low molecular weight contaminants
from the biological fluids. While not an essential component of the
present invention, the step of removal of such contaminants
facilitates detection of the disorder-associated enzymes in the
biological samples.
[0054] The invention is further illustrated by the following
examples, which should not be construed as further limiting. The
contents of all references, pending patent applications and
published patents, cited throughout this application are hereby
expressly incorporated by reference.
EXAMPLES
[0055] The following materials and methods were used throughout
these Examples, set forth below.
Materials and Methods
[0056] Urine Sample Collection and Preparation--Urine sample
collection was performed as described in Moses, M. A., et al.
(1998) Cancer Res. 58:1395-9, the contents of which are herein
incorporated by reference in their entirety. Samples were
immediately frozen after collection and stored frozen at
-20.degree. C. until assay. Prior to analysis, specimens containing
blood or leukocytes were excluded by testing for the presence of
blood and leukocytes using Multistix 9 Urinalysis Strips (Bayer,
Elkhart, Ind.). The creatine concentrations of urine samples were
determined using a commercial kit (Sigma Chemical Co., St. Louis,
Mo.) according to manufacturer's instructions.
[0057] Substrate Gel Electrophoresis--Substrate gel electrophoresis
was performed based on a previously described in U.S. Ser. No.
09/469,637 with modifications. Original urine samples (50 .mu.l)
were mixed with non-reducing sample buffer [4% sodium dodecyl
sulfate (SDS), 0.15 M Tris pH 6.8, 20% v/v glycerol, and 0.5% v/v
bromphenol blue] and were separated on a 10% polyacrylamide gel
containing 0.1% gelatin (Bio-Rad, Hercules, Calif.). After
electrophoresis, gels were washed twice with 2.5% Triton X-100 (15
minutes/each wash). Substrate digestion was carried out by
incubating the gel in 50 mM Tris-HCl (pH7.6) containing 5 mM
CaCl.sub.2, 1 .mu.M ZnCl.sub.2, 1% Triton X-100, and 0.02%
NaN.sub.3 at 37.degree. C. for 24 hours. The gel was stained with
0.1% Coomassie Brilliant Blue R250 (BioRad, Hercules, Calif.), and
the location of gelatinolytic activities were detected as clear
bands on the background of a uniform blue staining.
[0058] Protein Electrophoresis and Western Blot Analysis--Urine
samples were concentrated using an UltraFree-4 centrifugal filter
device with molecular weight cut off (MWCO) of 50 kDa (Millipore,
Bedford, Mass.). Protein concentrations of the concentrated urine
samples were determined using the MicroBCA method (Pierce,
Rockford, Ill. 61105). Equal amount of proteins (20 .mu.g) was
loaded onto 4-15% gradient gels and separated by SDS-PAGE under
non-reducing conditions. Resolved proteins were electrophoretically
transferred to nitrocellulose membranes (TransBlot, Bio-Rad,
Hercules, Calif.). The membranes were blocked with 5% low fat dry
milk in TBS-T (10 mM Tris, pH 7.2, 50 mM NaCl, 0.5% Tween 20) for 1
hour at room temperature, followed by incubating with primary
antibody at 4.degree. C. for 18 hours. Blots were washed 8 times
with TBS-T (5 minutes/wash) and incubated with 1:5000 dilution of
horseradish peroxidase (HRP) conjugated secondary antibody (Vector
Laboratories, Burlingame, Calif.) diluted in TBS-T containing 3%
BSA for 1 hour at room temperature. Labeled proteins were
visualized with enhanced chemiluminescence (Amersham, Arlington
Heights, Ill.). Purified polyclonal antibodies against human NGAL
were used at 1:100 dilution (Kjeldsen, L., et al. (1993)). Purified
human neutrophil MMP-9/NGAL complex was used as positive control
(CalBiochem, La Jolla, Calif.).
[0059] Immunoprecipitation--Original urine samples containing the
125 kDa MMP activity were mixed with equal volumes of RIPA buffer
(150 mM NaCl, 1.0% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, 50 mM
Tris pH 8.0, 0.02% sodium azide). 50 .mu.l diluted urine samples
were mixed with increasing amount of the rabbit anti-human NGAL
antibody or a control antibody. After incubating on ice for thirty
minutes, samples were mixed with 5 .mu.l RIPA-buffered Zysorbin
(ZyoMed Laboratories, South San Francisco, Calif.). Followed an
additional incubation on ice for thirty minutes, the
antibody-antigen complexes were removed with a centrifugation at
10,000 g for 5 minutes. The supernatants were subjected to
substrate gel electrophoresis to detect the remaining MMP
activities.
[0060] In vitro reconstitution of MMP-NGAL complexes--Recombinant
human proMMP-9 (Oncogene, Cambridge, Mass.) was diluted with
gelatinase buffer [50 mM Sodium Acetate (pH=5.5) or 50 mM Tris-HCl
(pH=7.0, 7.6, or 8.0) containing 5 mM CaCl.sub.2, 1 .mu.M
ZnCl.sub.2], to a final concentration of 10 .mu.M. Recombinant
human NGAL was purified as previously described and was diluted to
70 .mu.M in the gelatinase buffer. ProMMP-9 was mixed with NGAL in
a molar ratio of 1:20 and was incubated at 37.degree. C. for one
hour. The formation of MMP-9/NGAL complex was analyzed using
substrate gel electrophoresis. ProMMP-9 and NGAL were also
individually diluted in normal control urine with no MMP activity.
The possibility of MMP-9/NGAL complex formation in urine was
investigated by mixing proMMP-9 and NGAL in moral ratios of 2:1,
1:5, 1:10 and 1:20. MMP-9/NGAL complex was detected using substrate
gel electrophoresis.
Example 1
Substrate Gel Electrophoresis of MMP Activities in Urine
Samples
[0061] MMP activities contained in urine samples were assayed using
substrate gel electrophoresis. 50 .mu.l of freshly thawed urine
sample was used for analysis. At least four major MMP activities
were readily detected in these urine samples, with apparent
molecular mass of 200,000, 125,000, 92,000, and 72,000 (FIG. 1A).
The 92 kDa and the 72 kDa MMP activities have previously been
determined to be MMP-9 and MMP-2 respectively. The 200 kDa MMP
activities is in correspondence with the predicted molecular size
of MMP-9 dimer. The identity of the 125 kDa MMP is unclear. When
analyzed together with purified human MMP-9/NGAL complex from
neutrophil, the 125 kDa urinary MMP activity migrated in the same
position as that of human neutrophil MMP-9/NGAL (FIG. 2A). This 125
kDa urinary MMP is an active complex of MMP-9 and NGAL. The
identity of these gelatinolytic activities of being MMPs was
confirmed by inhibition studies using 1,10-phenanthroline at a
final concentration of 10 mM (data not shown).
Example 2
[0062] Western Blot Analysis of Urine Samples with Anti-NGAL
Antibody
[0063] To further demonstrate the identity of the 125 kDa urinary
MMP as a complex of MMP-9 and -NGAL, concentrated urine samples
were subjected to Western blot analysis using a purified antibody
against human NGAL (Kjeldsen, L. (1993)). Under non-reducing
conditions, a protein band of 125 kDa was detected in urine samples
containing the 125 kDa MMP activity (FIG. 1B). Screening of urine
samples from cancer patients established a correlationship between
the detection of MMP-9/NGAL protein complex and the presence of the
125 kDa MMP activity (FIG. 1B). Using the purified anti-NGAL
antibody, a 125 kDa protein band was consistently detected in urine
samples containing the 125 kDa MMP activity. The antibody also
detected the presence of NGAL monomer (25 kDa), dimer (50 kDa), and
trimer forms (75 kDa) in all of the urine samples analyzed. The
specificity of the NGAL antibody was confirmed using purified human
neutrophil MMP-9/NGAL complex. Under non-reducing conditions, the
antibody recognized the 125 kDa MMP-9/NGAL complex in the
concentrated urine sample, as well as the MMP-9/NGAL complex
purified from neutrophil (FIG. 2B). In addition to the MMP-9/NGAL
complex and the NGAL monomer, dimer and trimer complexes, several
minor protein bands with approximate molecular sizes of 150 kDa
were also detected in the concentrated urine sample. Although their
identities are currently unclear, they are most likely to be
proteins that non-specifically cross-reacted with anti-NGAL
antibody.
Example 3
Immunoprecipitation-Zymography
[0064] To further verify the identity of the 125 kDa MMP activity
in urine, anti-NGAL antibody was used to immunoprecipitate any MMP
activities that exist in the complex form with NGAL in urine. As
shown in FIG. 3, anti-NGAL antibody specifically immunoprecipitated
the 125 kDa urinary MMP activity, in a concentration-dependent
manner. Increasing amounts of the 125 kDa urinary MMP activity was
removed by the treatment with increasing amounts of anti-NGAL
antibody. When treated with 1.0 .mu.l of anti-NGAL antibody, the
125 kDa MMP activity was completely removed. The anti-NGAL antibody
had no effect on any other MMP activities, e.g., the 200 kDa MMP-9
dimer, the 92 kDa MMP-9, or the 72 kDa MMP-2. The specificity of
immunoprecipitation was also confirmed using a control antibody
which did not immunoprecipitate any of the MMP activities, even at
the highest concentration. The increase in MMP-2 activity in the
sample treated with 1.0 .mu.l of control antibody resulted from
endogenous MMP-2 activity contained in the serum. Taken together
these data support our finding that the 125 kDa MMP activity in
urine samples of cancer patients is a complex of MMP-9 and
NGAL.
Example 4
Re-Constitution of MMP-9/NGAL Complex In Vitro
[0065] The formation of MMP-9/NGAL complex was first investigated
using gelatinase buffer that contains cationic ions. Recombinant
human proMMP-9 and human NGAL were first diluted in gelatinase
buffers with different pH values (5.5, 7.0, 7.6 and 8.0). Diluted
proMMP-9 and NGAL were subsequently mixed in a molar ratio of 1:10,
to final concentrations of 2.6 .mu.M and 26 .mu.M respectively.
After one hour incubation at 37.degree. C., the formation of
MMP-9/NGAL complexes was monitored using substrate gel
electrophoresis. Mixing proMMP-9 and NGAL generated a predominant
MMP activity with a molecular size of approximately 115 kDa (FIG.
4A). Formation of the 115 kDa MMP-9/NGAL complex occurred in
buffers with pH values ranging from 5.5 to 8.0, the pH range of
normal urine. However, the size of this predominant MMP activity is
not the same as that of purified human neutrophil MMP-9/NGAL. There
is a minor MMP activity of 125 kDa, observed in pH 7.0, 7.6 and 8.0
buffers. The possibility of MMP-9/NGAL complex formation in urine
was directly studied by diluting proMMP-9 and NGAL in normal
control urine. Diluted proMMP-9 and NGAL were mixed in different
molar ratios (proMMP-9/NGLA=2:1, 1:5, 1:10 and 1:20) and incubated
at 37.degree. C. for one hour. The formation of a 115 kDa
MMP-9/NGAL complex was readily detected in all mixing ratios (FIG.
4B). No MMP activity was detected in the control urine used as a
diluent.
Example 5
Modulation of MMP-9 Degradation by NGAL In Vitro
[0066] The effect of NGAL on MMP-9 degradation in vitro was studied
by mixing MMP-9 (0.1.mu.) and NGAL (1/0.mu.) prior to incubation.
MMP-9 degradation was inhibited in the presence of NGAL resulting
in a decrease in the enzymatic degradation rate as evidenced by an
increase in the remaining amounts of enzyme at each time point
compared with MMP-9 incubated by itself. Immunodepleted NGAL had no
apparent protection of MMP-9. In the presence of increasing amount
of NGAL, degradation of MMP-9 decreased and resulted in an increase
in the remaining MMP-9 activity. NGAL appears to be capable of
protecting MMP-9 from degradation in a dose-dependent manner,
resulting in the preservation of MMP-9 activity. These data suggest
a potential regulatory role for NGAL in modulating MMP-9 activity,
for example, NGAL may be involved in tumor progression via its
interaction with MMP-9.
Example 6
Modulation of MMP-9 Degradation by NGAL in Cell Culture
[0067] The protective effect of NGAL on MMP-9 degradation was
studied in cell culture using MDA-MB-231 human breast carcinoma
cells. MMP-9 activity was detected in cells overexpressing NGAL
(N-2 and N-5). Thus, it appeared that elevated NGAL expression
resulted in an increase in MMP-9 activity. Steady state MMP-9 mRNA
levels were determined using RT-PCT analysis and no apparent
differences were detected. Expression levels of endogenous MMP-9
inhibitor, TIMP-1, and a house-keeping gene, GAPGH, were determined
and overexpression of NGAL had no apparent influence on mRNA levels
of TIMP-1 or GAPDH. Overexpression of NGAL in human breast
carcinoma cells resulted in an increase in MMP-9 activity
independent of changes in MMP-9 gene transcription.
Discussion
[0068] Identification of hMW enzyme complexes in the urine of
cancer patients, e.g., enzyme complexes comprising MMP-9 and NGAL,
is predictive of TRAC and is supported by the following findings:
(a) the 125 kDa MMP activity in urine migrates at the same position
as human neutrophil MMP-9/NGAL does; (b) anti-NGAL antibody
successfully detected a 125 kDa protein band in most of the
concentrated urine samples that contain the 125 kDa MMP activity;
(c) the same antibody was able to specifically immunoprecipitate
the 125 kDa MMP activity in urine in a concentration-dependent
manner, without affecting any other MMP activities. Such evidence
agrees with the findings described in U.S. Ser. No. 09/469,637,
which is incorporated herein by reference in its entirety, that the
detection of hMW MMPs, as well as MMP-9 and MMP-2, serves as
independent predictors of metastatic or organ-confined cancers,
respectively.
[0069] NGAL was first identified as a 25 kDa protein that was
co-purified with human neutrophil gelatinase (Kjeldsen, L., et al:
(1993) J Biol Chem. 268: 10425-32). Binding of NGAL and MMP-9
results in a gelatinase activity of 135 kDa detected in specific
granules of human neutrophil stimulated with phorbol myristate
acetate (PMA) (Kjeldsen, L. et al. (1993)). NGAL and MMP-9 are
stored in specific granules, while MMP-9 is also present
independently in gelatinase granules (Morel, F., et al., (1994)
Biochim Biophys Acta. 1201: 373-80; Kjeldsen, L., et al. (1994)
Blood. 83: 799-807; and Borregaard, N. and Cowland, J. B. (1997)
Blood. 89: 3503-21). However, the MMP-9/NGLA complex detected in
urine of cancer patients are not derived from leukocytes since we
have specifically excluded the urine samples that contain
leukocytes.
[0070] Interestingly, human NGAL contains sequence similarities to
mouse 24p3 and rat neu/HER2/c-erbB-2 related lipocalin (NRL), both
overexpressed in oncogene mediated cell transformation (Cowland, J.
B. and Borregaard, N. (1997) Genomics. 45: 17-23; Hraba-Renevey,
S., et al. (1989) Oncogene. 4: 601-8; Stoesz, S. P. and Gould, M.
N. (1995) Oncogene. 11: 2233-41). Under normal conditions,
expression of human NGAL is restricted to breast, lung, trachea,
and bone marrow (Cowland, J. B. and Borregaard, N. (1997) Genomics.
45: 17-23; Stoesz, S. P., et al. (1998) Int. J Cancer. 79: 565-72).
However, elevated levels of NGAL expression has been observed in
human breast tumors as well as in adenocarcinomas of lung, colon
and pancreas (Stoesz (1998); Friedl, A., et al. (1999) Histochem J.
31: 433-41). An increased production of NGAL can be closely
associated with cancer disease status, which subsequently
contribute to the elevated levels of MMP-9/NGAL complex in urine.
This complex can be detected with substrate gel electrophoresis as
well as antibody-based assays. As described in U.S. Ser. No.
09/469,637, the presence of the 125 kDa MMP activity in urine can
serve as an independent multivariate predictor of cancer
metastasis, the identification of this activity as MMP-9/NGAL
complex will facilitate the development of a non-invasive prognosis
tool to assess disease status of various cancers.
[0071] The origin of the 125 kDa MMP-9/NGAL activity in urine of
cancer patients remains unclear. Given that the glomerular
filtration limit is only 45 kDa, it is unlikely that this large
protein complex is directly filtered from serum into urine. The
possibility that MMP-9/NGAL complex forms after each component was
separately filtrated into urine was investigated using in vitro
reconstitution assay. The results demonstrate the feasibility of
MMP-9/NGAL complex formation in gelatinase buffers with different
pH values, as well as, in normal urine. Therefore, it is likely
that MMP-9 and NGAL are separately executed into urine where they
form the 125 kDa MMP-9/NGAL complex.
[0072] The existence of MMP-9 and NGAL complex in urine was
supported by a recent independent study (Monier, F., Clin Chim
Acta. 299: 11-23, 2000). Under reducing conditions, MMP-9 and NGAL
were separately detected in a continuous-elution electrophoresis
fraction that contains a 115 kDa gelatinase activity. The detection
of MMP-9 and NGAL in the same fraction shows the observed 115 kDa
gelatinase activity as a complex of MMP-9 and NGAL.
[0073] Recent studies have also confirmed that NGAL appears to
exert a protective effect on MMP-9 and prevents MMP-9 from
degradation both in vitro and in cells. Examples 5 and 6 suggest
that the MMP-9-NGAL complex likely plays an active role in tumor
progression.
Equivalents
[0074] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims.
* * * * *