U.S. patent application number 11/473280 was filed with the patent office on 2007-07-26 for biomarker for ovarian cancer.
This patent application is currently assigned to The Johns Hopkins University, a non-profit organization. Invention is credited to Daniel W. Chan, Eric T. Fung, Zheng Wang, Fujun Zhang, Zhen Zhang.
Application Number | 20070172902 11/473280 |
Document ID | / |
Family ID | 37595819 |
Filed Date | 2007-07-26 |
United States Patent
Application |
20070172902 |
Kind Code |
A1 |
Zhang; Zhen ; et
al. |
July 26, 2007 |
Biomarker for ovarian cancer
Abstract
The present invention provides a protein-based biomarker that is
useful in qualifying ovarian cancer status in a patient. In
particular, the biomarker of this invention is useful to classify a
subject sample as ovarian cancer or non-ovarian cancer. The
biomarker can be detected by SELDI mass spectrometry.
Inventors: |
Zhang; Zhen; (Dayton,
MD) ; Chan; Daniel W.; (Clarksville, MD) ;
Fung; Eric T.; (Los Altos, CA) ; Wang; Zheng;
(Fremont, CA) ; Zhang; Fujun; (Fremont,
CA) |
Correspondence
Address: |
EDWARDS ANGELL PALMER & DODGE LLP
P.O. BOX 55874
BOSTON
MA
02205
US
|
Assignee: |
The Johns Hopkins University, a
non-profit organization
Ciphergen Biosystems, Inc., a small entity
|
Family ID: |
37595819 |
Appl. No.: |
11/473280 |
Filed: |
June 21, 2006 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60693324 |
Jun 22, 2005 |
|
|
|
Current U.S.
Class: |
435/7.23 ;
702/19 |
Current CPC
Class: |
G01N 33/57449 20130101;
G01N 2333/52 20130101; A61P 35/00 20180101 |
Class at
Publication: |
435/007.23 ;
702/019 |
International
Class: |
G01N 33/574 20060101
G01N033/574; G06F 19/00 20060101 G06F019/00 |
Claims
1. A method for qualifying ovarian cancer status in a subject
comprising: (a) measuring at least one biomarker including a
CTAP3-related protein in a biological sample from the subject; and
(b) correlating the measurement with ovarian cancer status.
2. The method of claim 1, wherein the CTAP3-related protein is
CTAP3 (residues 44-128 of SEQ ID NO:1).
3. The method of claim 2 wherein CTAP3 is measured by capturing the
biomarker on an adsorbent surface of a SELDI probe and detecting
the captured biomarkers by laser desorption-ionization mass
spectrometry.
4. The method of claim 2 wherein CTAP3 is measured by
immunoassay.
5. The method of claim 2 wherein the sample is serum.
6. The method of claim 2 wherein the correlating is performed by a
software classification algorithm.
7. The method of claim 2 wherein ovarian cancer status is selected
from ovarian cancer and non-ovarian cancer.
8. The method of claim 2 wherein ovarian cancer status is selected
from early stage and late stage 2.
9. The method of claim 2, further comprising: (c) reporting the
status to the subject.
10. The method of claim 2, further comprising: (c) recording the
status on a tangible medium.
11. The method of claim 2, further comprising: (c) managing subject
treatment based on the status.
12. The method of claim 11, further comprising: (d) measuring CTAP3
after managing treatment and correlating the measurement with
disease progression.
13. The method of claim 3, wherein the adsorbent is an IMAC copper
adsorbent.
14. The method of claim 3, wherein the adsorbent is a biospecific
adsorbent.
15. The method of claim 2, further comprising measuring and
correlating at least one biomarker selected from the group
consisting of CA125, transferrin, haptoglobin, ApoA1,
transthyretin, ITIH4 internal fragment, beta 2-microglobulin,
hepcidin, prostatin, osteopontin, esoinophil-derived neurotoxin,
leptin, prolactin, IGF-II, hemoglobin and modified forms
thereof.
16. The method of claim 2, further comprising measuring and
correlating at least one biomarker selected from the group
consisting of CA125 II, CA15-3, CA19-9, CA72-4, CA 195, tumor
associated trypsin inhibitor (TATI), CEA, placental alkaline
phosphatase (PLAP), Sialyl TN, galactosyltransferase, macrophage
colony stimulating factor (M-CSF, CSF-1), lysophosphatidic acid
(LPA), 110 kD component of the extracellular domain of the
epidermal growth factor receptor (p110EGFR), tissue kallikreins,
e.g., kallikrein 6 and kallikrein 10 (NES-1), prostasin, HE4,
creatine kinase B (CKB), LASA, HER-2/neu, urinary gonadotropin
peptide, Dianon NB 70/K, Tissue peptide antigen (TPA), SMRP,
osteopontin, and haptoglobin, leptin, prolactin, insulin-like
growth factor I and insulin-like growth factor II.
17. A method for determining the course of ovarian cancer
comprising: (a) measuring, at a first time, at least one biomarker
including a CTAP3-related protein in a biological sample from the
subject; (b) measuring, at a second time, the CTAP3-related protein
in a biological sample from the subject; and (c) comparing the
first measurement and the second measurement; wherein the
comparative measurements determine the course of the ovarian
cancer.
18. The method of claim 17, wherein the CTAP3-related protein is
CTAP3 (amino acid residues 44-128 of SEQ ID NO:1).
19. A kit comprising: (a) a solid support comprising at least one
capture reagent attached thereto, wherein the capture reagent binds
to a CTAP3-related protein; and (b) instructions for using the
solid support to detect the CTAP3-related protein.
20. The kit of claim 19, wherein the CTAP3-related protein is CTAP3
(amino acid residues 44-128 of SEQ ID NO:1).
21. The kit of claim 19, wherein the solid support comprising a
capture reagent is a SELDI probe.
22. The kit of claim 19, wherein the capture reagent is an IMAC
copper adsorbent.
23. The kit of claim 19, additionally comprising: (c) a container
containing the CTAP3 biomarker.
24. The kit of claim 19, additionally comprising: (c) an
immunospecific chromatography sorbent.
25. A kit comprising: (a) a solid support comprising at least one
capture reagent attached thereto, wherein the capture reagents
binds a CTAP3-related protein; and (b) a container containing the
CTAP3-related protein.
26. The kit of claim 25, wherein the CTAP3-related protein is CTAP3
(amino acid residues 44-128 of SEQ ID NO:1).
27. The kit of claim 26, wherein the capture reagent is an IMAC
capture reagent or a biospecific capture reagent.
28. A software product comprising: (a) code that accesses data
attributed to a sample, the data comprising measurement of at least
one biomarker including a CTAP3-related protein in the sample; and
(b) code that executes a classification algorithm that classifies
the ovarian cancer status of the sample as a function of the
measurement.
29. A method comprising communicating to a subject a diagnosis
relating to ovarian cancer status determined from the correlation
of 3 a CTAP3-related protein in a sample from the subject.
30. The method of claim 29, wherein the diagnosis is communicated
to the subject via a computer-generated medium.
31. A method for identifying a compound that interacts with a
CTAP3-related protein, wherein said method comprises: a) contacting
the CTAP3-related protein with a test compound; and b) determining
whether the test compound interacts with the CTAP3-related
protein.
32. The method of claim 31, wherein the CTAP3-related protein is
CTAP3 (amino acid residues 44-128 of SEQ ID NO:1).
33. A method for modulating the concentration of a CTAP3-related
protein in a cell, wherein said method comprises: a) contacting
said cell with a small molecule which inhibits the expression of
the CTAP3-related protein.
34. The method of claim 33, wherein the CTAP3-related protein is
CTAP3 (amino acid residues 44-128 of SEQ ID NO:1).
35. A method of treating ovarian cancer in a subject, wherein said
method comprises: administering to a subject a therapeutically
effective amount of a small molecule, wherein said small molecule
inhibits expression of CTAP3.
Description
RELATED APPLICATIONS
[0001] The instant application claims the benefit of U.S.
Provisional Application No. 60/693,324, filed Jun. 22, 2005, the
entire contents of which provisional application are expressly
incorporated herein by reference.
FIELD OF THE INVENTION
[0002] The invention provides for a biomarker important in the
detection of ovarian cancer. The marker was identified by
distinguishing the serum protein profile in ovarian cancer patients
from healthy individuals using SELDI analysis. The present
invention relates the biomarker to a system and method in which the
biomarker is used for the qualification of ovarian cancer status.
The present invention also identifies a biomarker as a known
protein, CTAP3.
BACKGROUND OF THE INVENTION
[0003] Ovarian cancer is among the most lethal gynecologic
malignancies in developed countries. Annually in the United States
alone, approximately 23,000 women are diagnosed with the disease
and almost 14,000 women die from it. (Jamal, A., et al., CA Cancer
J. Clin, 2002; 52:23-47). Despite progress in cancer therapy,
ovarian cancer mortality has remained virtually unchanged over the
past two decades. (Id.) Given the steep survival gradient relative
to the stage at which the disease is diagnosed, early detection
remains the most important factor in improving long-term survival
of ovarian cancer patients.
[0004] The poor prognosis of ovarian cancer diagnosed at late
stages, the cost and risk associated with confirmatory diagnostic
procedures, and its relatively low prevalence in the general
population together pose extremely stringent requirements on the
sensitivity and specificity of a test for it to be used for
screening for ovarian cancer in the general population.
[0005] The identification of tumor markers suitable for the early
detection and diagnosis of cancer holds great promise to improve
the clinical outcome of patients. It is especially important for
patients presenting with vague or no symptoms or with tumors that
are relatively inaccessible to physical examination. Despite
considerable effort directed at early detection, no cost effective
screening tests have been developed (Paley P J., Curr Opin Oncol,
2001; 13(5):399-402) and women generally present with disseminated
disease at diagnosis. (Ozols R F, et al., Epithelial ovarian
cancer. In: Hoskins W J, Perez C A, Young R C, editors. Principles
and Practice of Gynecologic Oncology. 3rd ed. Philadelphia:
Lippincott, Williams and Wilkins; 2000. p. 981-1057).
[0006] The best-characterized tumor marker, CA125, is negative in
approximately 30-40% of stage I ovarian carcinomas and its levels
are elevated in a variety of benign diseases. (Meyer T, et al., Br
J Cancer, 2000; 82(9):1535-8; Buamah P., J Surg Oncol, 2000;
75(4):264-5; Tuxen M K, et al., Cancer Treat Rev, 1995;
21(3):215-45). Its use as a population-based screening tool for
early detection and diagnosis of ovarian cancer is hindered by its
low sensitivity and specificity. (MacDonald N D, et al., Eur J
Obstet Gynecol Reprod Biol, 1999; 82(2):155-7; Jacobs I, et al.,
Hum Reprod, 1989; 4(1):1-12; Shih I-M, et al., Tumor markers in
ovarian cancer. In: Diamandis E P, Fritsche, H., Lilja, H., Chan,
D. W., and Schwartz, M., editor. Tumor markers physiology,
pathobiology, technology and clinical applications. Philadelphia:
AACC Press; in press). Although pelvic and more recently vaginal
sonography has been used to screen high-risk patients, neither
technique has the sufficient sensitivity and specificity to be
applied to the general population. (MacDonald N D, et al., supra).
Recent efforts in using CA125 in combination with additional tumor
markers (Woolas R P X F, et al., J Natl Cancer Inst, 1993;
85(21):1748-51; Woolas R P, et al., Gynecol Oncol, 1995;
59(1):111-6; Zhang Z, et al., Gynecol Oncol, 1999; 73(1):56-61;
Zhang Z, et al., Use of Multiple Markers to Detect Stage I
Epithelial ovarian cancers: Neural Network Analysis Improves
Performance. American Society of Clinical Oncology 2001; Annual
Meeting, Abstract) in a longitudinal risk of cancer model (Skates S
J, et al., Cancer, 1995; 76(10 Suppl):2004-10), and in tandem with
ultrasound as a second line test (Jacobs I D A, et al., Br Med J,
1993; 306(6884):1030-34; Menon U T A, et al., British Journal of
Obstetrics and Gynecology, 2000; 107(2):165-69) have shown
promising results in improving overall test specificity, which is
critical for a disease such as ovarian cancer that has a relatively
low prevalence.
[0007] Due to the dismal prognosis of late stage ovarian cancer, it
is the general consensus that a physician will accept a test with a
minimal positive predictive value of 10%. (Bast, R. C., et al.,
Cancer Treatment and Research, 2002; 107:61-97). Extending this to
the general population, a general screening test would require a
sensitivity greater than 70% and a specificity of 99.6%. Currently,
none of the existing serologic markers, such as CA125, CA72-4, or
M-CSF, individually delivers such a performance. (Bast, R. C., et
al., Int J Biol Markers, 1998; 13:179-87).
[0008] Thus, there is a critical need for new serological markers
that individually or in combination with other markers or
diagnostic modalities deliver the required sensitivity and
specificity for early detection of ovarian cancer. (Bast R C, et
al., Early detection of ovarian cancer: promise and reality ovarian
cancer: ISIS Medical Media Ltd., Oxford, UK; 2001. in press).
Without an acceptable screening test, early detection remains the
most critical factor in improving long-term survival of patients
with ovarian cancer.
[0009] Thus, it is desirable to have a reliable and accurate method
of determining the ovarian cancer status in patients, the results
of which can then be used to manage subject treatment.
SUMMARY OF THE INVENTION
[0010] It has been found that CTAP3 is up-regulated in the serum of
patients with ovarian cancer. Accordingly, the level of CTAP3 is
useful as a biomarker in making a determination of ovarian cancer
in a subject. Likewise, CTAP3-related proteins are useful as
biomarkers for making a determination of ovarian cancer in a
subject
[0011] The present invention provides sensitive and quick methods
and kits that are useful for determining the ovarian cancer status
by measuring one or more specific biomarkers, e.g., CTAP3-related
molecules such as CTAP3. The measurement of this marker in patient
samples provides information that diagnosticians can correlate with
a probable diagnosis of human cancer or a negative diagnosis (e.g.,
normal or disease-free). The marker is characterized by molecular
weight and its known protein identity. The marker can be resolved
from other proteins in a sample by using a variety of fractionation
techniques, e.g., chromatographic separation coupled with mass
spectrometry, protein capture using immobilized antibodies or by
traditional immunoassays. In preferred embodiments, the method of
resolution involves Surface-Enhanced Laser Desorption/Ionization
("SELDI") mass spectrometry, in which the surface of the mass
spectrometry probe comprises adsorbents that bind the markers.
[0012] The present invention provides a method of qualifying
ovarian cancer status in a subject comprising measuring at least
one biomarker including a CTAP3-related peptide, e.g., CTAP3, in a
biological sample from the subject; and (b) correlating the
measurement with ovarian cancer status. In certain methods, the
measuring step comprises detecting the presence or absence of the
marker in the sample. In other methods, the measuring step
comprises quantifying the amount of marker in the sample. In other
methods, the measuring step comprises qualifying the type of
biomarker in the sample.
[0013] The invention also relates to methods wherein the measuring
step comprises: providing a subject sample of blood or a blood
derivative; fractionating proteins in the sample on an anion
exchange resin and collecting fractions that contain a
CTAP3-related peptide, e.g., CTAP3, from the fractions on a surface
of a substrate comprising capture reagents that bind this protein
biomarkers. The blood derivative is, e.g., serum or plasma. In
preferred embodiments, the substrate is a SELDI probe comprising an
IMAC copper surface and wherein the protein biomarkers are detected
by SELDI. In other embodiments, the substrate is a SELDI probe
comprising biospecific affinity reagents that bind, for example,
CTAP3 and wherein the protein biomarkers are detected by SELDI. In
other embodiments, the substrate is a microtiter plate comprising
biospecific affinity reagents that bind CTAP3 and the protein
biomarker is detected by immunoassay.
[0014] In certain embodiments, the methods further comprise
managing subject treatment based on the status determined by the
method. For example, if the result of the methods of the present
invention is inconclusive or there is reason that confirmation of
status is necessary, the physician may order more tests.
Alternatively, if the status indicates that surgery is appropriate,
the physician may schedule the patient for surgery. Furthermore, if
the results show that treatment has been successful, no further
management may be necessary.
[0015] The invention also provides for such methods where the
CTAP3-related biomarker is measured again after subject management,
i.e., treatment. In these instances, the step of managing subject
treatment is then repeated and/or altered depending on the result
obtained. In further embodiments, the measurement is correlated
with disease progression.
[0016] The term "ovarian cancer status" refers to the status of the
disease in the patient. Examples of types of ovarian cancer
statuses include, but are not limited to, the subject's risk of
cancer, the presence or absence of disease, the stage of disease in
a patient, and the effectiveness of treatment of disease. Other
statuses and degrees of each status are known in the art.
[0017] In certain preferred embodiments, the method for qualifying
ovarian cancer status further comprises measuring and correlating
at least one biomarker selected from the group consisting of CA125,
transferrin, haptoglobin, ApoA1, transthyretin, ITIH4 internal
fragment, beta 2-microglobulin, hepcidin, prostatin, osteopontin,
esoinophil-derived neurotoxin, leptin, prolactin, IGF-II,
hemoglobin and modified forms thereof with ovarian cancer
status.
[0018] In other preferred embodiments, the method for qualifying
ovarian cancer status further comprises measuring and correlating
at least one biomarker selected from the group consisting of CA125
II, CA15-3, CA19-9, CA72-4, CA 195, tumor associated trypsin
inhibitor (TATI), CEA, placental alkaline phosphatase (PLAP),
Sialyl TN, galactosyltransferase, macrophage colony stimulating
factor (M-CSF, CSF-1), lysophosphatidic acid (LPA), 110 kD
component of the extracellular domain of the epidermal growth
factor receptor (p110EGFR), tissue kallikreins, e.g., kallikrein 6
and kallikrein 10 (NES-1), prostasin, HE4, creatine kinase B (CKB),
LASA, HER-2/neu, urinary gonadotropin peptide, Dianon NB 70/K,
Tissue peptide antigen (TPA), SMRP, osteopontin, and haptoglobin,
leptin, prolactin, insulin-like growth factor I and insulin-like
growth factor II with ovarian cancer status.
[0019] In one embodiment, the invention provides methods that
comprise measuring the CTAP3 marker in conjunction with measuring
CA125 or CA125 II in a biological sample from a subject and
correlating the coordinated measurement with ovarian cancer status.
We have found that measurement of CTAP3 in conjunction or
combination with CA125 can provide enhanced results (e.g., enhanced
discrimination or detection) than measurement of either marker
alone.
[0020] In another embodiment, the invention provides methods that
comprise measuring the CTAP3 marker in a biological sample from a
subject and correlating the measurement with ovarian cancer status,
wherein the status is borderline ovarian cancer versus invasive
ovarian cancer. We have found that CTAP3 can discriminate between
borderline ovarian cancer versus invasive ovarian cancer
[0021] The CTAP3-related biomarkers of the invention can be
characterized in one or more of several respects. In particular, in
one aspect, this marker is characterized by molecular weight under
the conditions specified herein, particularly as determined by mass
spectral analysis. In another aspect, the marker can be
characterized by features of the marker's mass spectral signature
such as size (including area) and/or shape of the marker's spectral
peaks, features including proximity, size and shape of neighboring
peaks, etc. In yet another aspect, the marker can be characterized
by affinity binding characteristics, particularly ability to
binding to an IMAC copper adsorbent under specified conditions,
however, other metals, e.g., nickel, may also be used. In preferred
embodiments, marker of the invention may be characterized by each
of such aspects, i.e. molecular weight, mass spectral signature and
IMAC-Cu absorbent binding.
[0022] For the mass values of the marker disclosed herein, the mass
accuracy of the spectral instrument is considered to be about
within +/-0.15 percent of the disclosed molecular weight value.
Additionally, to such recognized accuracy variations of the
instrument, the spectral mass determination can vary within
resolution limits of from about 400 to 1000 m/dm, where m is mass
and dm is the mass spectral peak width at 0.5 peak height. Those
mass accuracy and resolution variances associated with the mass
spectral instrument and operation thereof are reflected in the use
of the term "about" in the disclosure of the mass of the marker I.
It is also intended that such mass accuracy and resolution
variances and thus meaning of the term "about" with respect to the
markers disclosed herein is inclusive of variants of the marker as
may exist due to sex, genotype and/or ethnicity of the subject and
the particular cancer or origin or stage thereof.
[0023] The present invention further provides a method of
qualifying ovarian cancer status in a subject comprising (a)
measuring the CTAP3 biomarker in a sample from the subject, and (b)
correlating the measurement with ovarian cancer status. In certain
methods, the measuring step comprises detecting the presence or
absence of the marker in the sample. In other methods, the
measuring step comprises quantifying the amount of marker in the
sample.
[0024] The accuracy of a diagnostic test is characterized by a
Receiver Operating Characteristic curve ("ROC curve"). An ROC is a
plot of the true positive rate against the false positive rate for
the different possible cutpoints of a diagnostic test. An ROC curve
shows the relationship between sensitivity and specificity. That
is, an increase in sensitivity will be accompanied by a decrease in
specificity. The closer the curve follows the left axis and then
the top edge of the ROC space, the more accurate the test.
Conversely, the closer the curve comes to the 45-degree diagonal of
the ROC graph, the less accurate the test. The area under the ROC
is a measure of test accuracy. The accuracy of the test depends on
how well the test separates the group being tested into those with
and without the disease in question. An area under the curve
(referred to as "AUC") of 1 represents a perfect test, while an
area of 0.5 represents a less useful test. Thus, biomarker and
diagnostic methods of the present invention have an AUC greater
than 0.50, more preferred tests have an AUC greater than 0.60, more
preferred tests have an AUC greater than 0.70.
[0025] Preferred methods of measuring biomarkers include use of a
biochip array. Biochip arrays useful in the invention include
protein and nucleic acid arrays. One or more markers are captured
on the biochip array and subjected to laser ionization to detect
the molecular weight of the marker. Analysis of the marker is, for
example, by molecular weight of the marker against a threshold
intensity that is normalized against total ion current. Preferably,
logarithmic transformation is used for reducing peak intensity
ranges to limit the number of markers detected.
[0026] In preferred methods of the present invention, the step of
correlating the measurement of the biomarkers with ovarian cancer
status is performed by a software classification algorithm.
Preferably, data is generated on immobilized subject samples on a
biochip array, by subjecting said biochip array to laser ionization
and detecting intensity of signal for mass/charge ratio; and,
transforming the data into computer readable form; and executing an
algorithm that classifies the data according to user input
parameters, for detecting signals that represent marker present in
ovarian cancer patients and are lacking in non-cancer subject
controls.
[0027] Preferably the biochip surfaces are, for example, ionic,
anionic, comprised of immobilized nickel ions, comprised of a
mixture of positive and negative ions, comprised of one or more
antibodies, single or double stranded nucleic acids, proteins,
peptides or fragments thereof, amino acid probes, or phage display
libraries.
[0028] In other preferred methods one or more of the marker is
measured using laser desorption/ionization mass spectrometry,
comprising providing a probe adapted for use with a mass
spectrometer comprising an adsorbent attached thereto, and
contacting the subject sample with the adsorbent, and; desorbing
and ionizing the marker from the probe and detecting the
deionized/ionized markers with the mass spectrometer.
[0029] Preferably, the laser desorption/ionization mass
spectrometry comprises: providing a substrate comprising an
adsorbent attached thereto; contacting the subject sample with the
adsorbent; placing the substrate on a probe adapted for use with a
mass spectrometer comprising an adsorbent attached thereto; and,
desorbing and ionizing the marker from the probe and detecting the
desorbed/ionized marker with the mass spectrometer. The adsorbent
can for example be hydrophobic, hydrophilic, ionic or metal chelate
adsorbent, such as, nickel or an antibody, single- or double
stranded oligonucleotide, amino acid, protein, peptide or fragments
thereof.
[0030] The methods of the present invention can be performed on any
type of patient sample that would be amenable to such methods,
e.g., blood, serum and plasma.
[0031] The present invention also provides kits comprising (a) a
capture reagent that binds the CTAP3-related biomarker; and (b) a
container comprising the biomarker. In preferred embodiments, the
capture reagent binds the biomarker. While the capture reagent can
be any type of reagent, preferably the reagent is a SELDI probe.
The capture reagent may also bind other known biomarkersas
disclosed herein. In certain preferred embodiments, the kit of
further comprises a second capture reagent that binds the biomarker
that the first capture reagent did not bind.
[0032] In certain kits of the present invention, the capture
reagent comprises an immobilized metal chelate ("IMAC").
[0033] Certain kits of the present invention further comprise a
wash solution that selectively allows retention of the bound
biomarker to the capture reagent as compared with other biomarkers
after washing.
[0034] The invention also provides kits comprising (a) a first
capture reagent that binds the CTAP3-related biomarker, and (b)
instructions for using the capture reagent to measure the
biomarker. In certain of these kits, the capture reagent comprises
an antibody. Furthermore, some kits further comprise an MS probe to
which the capture reagent is attached or is attachable. In some
kits, the capture reagent comprises an IMAC. The kits may also
contain a wash solution that selectively allows retention of the
bound biomarker to the capture reagent as compared with other
biomarkers after washing. Preferably, the kit comprises written
instructions for use of the kit for determining ovarian cancer
status and the instructions provide for contacting a test sample
with the capture reagent and measuring one or more biomarkers
retained by the capture reagent.
[0035] The kit also provides for a capture reagent, which is an
antibody, single or double stranded oligonucleotide, amino acid,
protein, peptide or fragments thereof. Measurement of the protein
biomarker using the kit, is by mass spectrometry or immunoassays
such as an ELISA.
[0036] Purified proteins for detection of ovarian cancer and/or
generation of antibodies for further diagnostic assays are also
provided for. Purified proteins include, for example, a purified
peptide of CTAP3. The invention also provides this purified peptide
further comprising a detectable label.
[0037] In another embodiment, non-invasive medical imaging
techniques such as transvaginal ultrasound, positron emisson
tomography (PET) or single photon emission computerized tomography
(SPECT) imaging are particularly useful for the detection of
cancer, coronary artery disease and brain disease. Ultrasound with
Doppler flow, PET, and SPECT imaging show the chemical functioning
of organs and tissues, while other imaging techniques--such as
X-ray, CT and MRI--primarily show structure. The use of ultrasound
with flow, PET and SPECT imaging has become increasingly useful for
qualifying and monitoring the development of diseases such as
ovarian cancer.
[0038] The CTAP3-related biomarkers disclosed herein, or fragments
thereof, can be used in the context of PET and SPECT imaging
applications. After modification with appropriate tracer residues
for PET or SPECT applications, the CTAP3-related biomarker that
interacts with tumor proteins can be used to image the deposition
of biomarkers in ovarian cancer patients.
[0039] Other aspects of the invention are described infra.
BRIEF DESCRIPTION OF THE DRAWINGS
[0040] FIG. 1 (includes F1A through 1E) shows mass spectra of the
CTAP3 marker. The mass spectral peak of the marker is designated
within the depicted spectra with a vertical line.
[0041] FIG. 2 represents the amino acid sequence of precursor
platelet basic protein (PPBP) (Swiss Prot Accession No.: P02775).
CTAP3 is an 85 amino acid protein comprising amino acid residues
44-128 of PPBP.
DETAILED DESCRIPTION OF THE INVENTION
1. Introduction
[0042] A biomarker is an organic biomolecule which is
differentially present in a sample taken from a subject of one
phenotypic status (e.g., having a disease) as compared with another
phenotypic status (e.g., not having the disease). A biomarker is
differentially present between different phenotypic statuses if the
mean or median expression level of the biomarker in the different
groups is calculated to be statistically significant. Common tests
for statistical significance include, among others, t-test, ANOVA,
Kruskal-Wallis, Wilcoxon, Mann-Whitney and odds ratio. Biomarkers,
alone or in combination, provide measures of relative risk that a
subject belongs to one phenotypic status or another. Therefore,
they are useful as markers for disease (diagnostics), therapeutic
effectiveness of a drug (theranostics) and drug toxicity.
2. Biomarkers for Ovarian Cancer
[0043] 2.1. Biomarkers
[0044] This invention provides polypeptide-based biomarkers,
CTAP3-related proteins (also called "CTAP-III related proteins"),
that are differentially present in subjects having ovarian cancer,
in particular, ovarian cancer versus normal (non-ovarian cancer).
The biomarker is characterized by mass-to-charge ratio as
determined by mass spectrometry, by the shape of their spectral
peak in time-of-flight mass spectrometry and by their binding
characteristics to adsorbent surfaces. These characteristics
provide one method to determine whether a particular detected
biomolecule is a biomarker of this invention. These characteristics
represent inherent characteristics of the biomolecules and not
process limitations in the manner in which the biomolecules are
discriminated. In one aspect, this invention provides this
biomarker in isolated form.
[0045] The biomarker was discovered using SELDI technology
employing ProteinChip arrays from Ciphergen Biosystems, Inc.
(Fremont, Calif.) ("Ciphergen"). Serum samples were collected from
subjects diagnosed with ovarian cancer and subjects diagnosed as
normal. The samples were fractionated by anion exchange
chromatography. Fractionated samples were applied to SELDI biochips
and spectra of polypeptides in the samples were generated by
time-of-flight mass spectrometry on a Ciphergen PCS 4000 mass
spectrometer. The spectra thus obtained were analyzed by Ciphergen
Express.TM. Data Manager Software with Biomarker Wizard and
Biomarker Pattern Software from Ciphergen Biosystems, Inc. The mass
spectra for each group were subjected to scatter plot analysis. A
Mann-Whitney test analysis was employed to compare ovarian cancer
and control groups for each protein cluster in the scatter plot,
and proteins were selected that differed significantly
(p<0.0001) between the two groups. This method is described in
more detail in the Example Section.
[0046] The invention provides biomarkers referred to herein as
CTAP3-related proteins or CTAP3-related biomarkers. The
CTAP3-related biomarkers include peptides and polypeptides that
comprise amino acid sequences derived from precursor platelet basic
protein (PPBP) (SEQ ID NO:1), including fragments thereof.
[0047] In specific embodiments, the invention provides the CTAP3
biomarker. CTAP3 is an 85 amino acid protein derived from PPBP
(SwissProt P02775). CTAP3 is recognized by antibodies available
from, e.g., Chemicon International (catalog 1484P)
(www.chemicon.com, Temecula, Calif.). CTAP3 is a fragment of
precursor platelet basic protein and consisting of amino acids
44-128 of SEQ ID NO:1.
[0048] The "ProteinChip assay" column refers to chromatographic
fraction in which the biomarker is found, the type of biochip to
which the biomarker binds and the wash conditions, as discussed
below. TABLE-US-00001 TABLE 1 Up or down regulated in ProteinChip
.RTM. Marker P-Value ovarian cancer assay CTAP3 <0.0001 Up
IMAC-Cu (M9313.9) 100 mM Na (predicted mass: phosphate, 9293) pH
7.0
[0049] The biomarker of this invention is characterized by their
mass-to-charge ratio as determined by mass spectrometry. The
mass-to-charge ratio of the biomarker is provided in Table 1 after
the "M." Thus, for example, CTAP has a measured mass-to-charge
ratio of 9313.9. The mass-to-charge ratios were determined from
mass spectra generated on a Ciphergen Biosystems, Inc. PBS II mass
spectrometer. This instrument has a mass accuracy of about +/-0.15
percent. Additionally, the instrument has a mass resolution of
about 400 to 1000 m/dm, where m is mass and dm is the mass spectral
peak width at 0.5 peak height. The mass-to-charge ratio of the
biomarkers was determined using Biomarker Wizard.TM. software
(Ciphergen Biosystems, Inc.). Biomarker Wizard assigns a
mass-to-charge ratio to a biomarker by clustering the
mass-to-charge ratios of the same peaks from all the spectra
analyzed, as determined by the PBSII, taking the maximum and
minimum mass-to-charge-ratio in the cluster, and dividing by two.
Accordingly, the masses provided reflect these specifications.
[0050] The biomarker of this invention can be further characterized
by the shape and/or location of its mass spectral speak(s). Mass
spectra showing peaks representing the biomarker are presented in
FIG. 1. In that FIG. 1, the spectra (FIGS. 1A through 1E) were
generated through SELDI analysis and show mass values differing
than the values set forth in Table 1. Those differing mass values
are attributed to differences in internal calibration of the
analysis tools.
[0051] The biomarker of this invention is further characterized by
its binding properties on chromatographic surfaces. CTAP3 binds to
IMAC-Cu after washing with sodium phosphate, pH 7.
[0052] The identity of a biomarker of this invention has been
determined and is indicated in Table 1. The method by which this
determination was made is described in the Example section
below.
[0053] The preferred biological source for detection of the
biomarkers is serum. However, in other embodiments, the biomarkers
can be detected in plasma or urine.
[0054] The biomarker of this invention is a polypeptide.
Accordingly, this invention provides this polypeptide in isolated
form. The biomarker can be isolated from biological fluids, such as
urine or serum. It can be isolated by any method known in the art,
based on both their mass and their binding characteristics. For
example, a sample comprising the polypeptide can be subject to
chromatographic fractionation, as described herein, and subject to
further separation by, e.g., acrylamide gel electrophoresis.
Knowledge of the identity of the biomarker also allows their
isolation by immunoaffinity chromatography.
[0055] A biomarker of this invention, CTAP3, has been demonstrated
to differentially present in several classes. It was found in a
comparison of samples from early stage ovarian cancer versus
healthy controls; early stage ovarian cancer versus post-operative
cancer free (serial samples from patients before and after
treatment), and early stage ovarian cancer versus benign disease,
either ovarian or non-ovarian disease.
3. Biomarkers and Different Forms of a Protein
[0056] Proteins frequently exist in a sample in a plurality of
different forms. These forms can result from either or both of pre-
and post-translational modification. Pre-translational modified
forms include allelic variants, splice variants and RNA editing
forms. Post-translationally modified forms include forms resulting
from proteolytic cleavage (e.g., cleavage of a signal sequence or
fragments of a parent protein), glycosylation, phosphorylation,
lipidation, oxidation, methylation, cysteinylation, sulphonation
and acetylation.
[0057] Accordingly, the invention provides biomarkers referred to
herein as "CTAP3-related proteins" or "CTAP3-related biomarkers".
The CTAP3-related biomarkers include peptides and proteins that
comprise amino acid sequences derived from precursor platelet basic
protein (PPBP), including fragments thereof. Exemplary
CTAP3-related biomarkers include polypeptides referred to in the
literature as B TG1, Beta TG, Beta thromboglobulin, Chemokine (C X
C motif) ligand 7, Connective tissue activating peptide III, CXC
chemokine ligand 7, CXCL7, LA PF4, LDGF, Low affinity platelet
factor IV, MDGF, NAP 2 L1, Neutrophil activating peptide 2, PBP,
Platelet basic protein precursor, Pro platelet basic protein,
SCYB7, Small inducible cytokine B7, Small inducible cytokine
subfamily B member 7, TC1, TC2, TGB, TGB1, THBGB, THBGB1,
Thrombocidin 1, Thrombocidin 2, and Thromboglobulin beta 1. Table 2
sets forth exemplary CTAP3-related molecules and the amino acid
residues of PPBP that each molecule contains. TABLE-US-00002 TABLE
2 Exemplary CTAP3-related molecules Amino Acid Residues of PBPP
Peptide Name (SEQ ID NO: 1) Macrophage-derived growth factor (MDGF)
1-128 Small inducible cytokine B7 (CXCL7) 1-128 Leukocyte-derived
growth factor (LDGF) 1-128 Platelet basic protein (Mature form)
35-128 Connective tissue-activating peptide III 44-128 (CTAP3)
Connective tissue-activating peptide 3a 44-124 (CTAP3a) TC-2 44-126
Beta-thromboglobulin 48-128 TC-1 59-126 Neutrophil-activating
peptide 2 55-128 Neutrophil-activating peptide 2 59-124
Neutrophil-activating peptide 2 59-121 Low-affinity platelet factor
IV (LA-PF4) 59-128 Chemokine_CXC 60-122
[0058] When detecting or measuring a protein in a sample, the
ability to differentiate between different forms of a protein
depends upon the nature of the difference and the method used to
detect or measure. For example, an immunoassay using a monoclonal
antibody will detect all forms of a protein containing the eptiope
and will not distinguish between them. However, a sandwich
immunoassay that uses two antibodies directed against different
epitopes on a protein will detect all forms of the protein that
contain both epitopes and will not detect those forms that contain
only one of the epitopes. In diagnostic assays, the inability to
distinguish different forms of a protein has little impact when the
forms detected by the particular method used are equally good
biomarkers as any particular form. However, when a particular form
(or a subset of particular forms) of a protein is a better
biomarker than the collection of different forms detected together
by a particular method, the power of the assay may suffer. In this
case, it is useful to employ an assay method that distinguishes
between forms of a protein and that specifically detects and
measures a desired form or forms of the protein. Distinguishing
different forms of an analyte or specifically detecting a
particular form of an analyte is referred to as "resolving" the
analyte.
[0059] Mass spectrometry is a particularly powerful methodology to
resolve different forms of a protein because the different forms
typically have different masses that can be resolved by mass
spectrometry. Accordingly, if one form of a protein is a superior
biomarker for a disease than another form of the biomarker, mass
spectrometry may be able to specifically detect and measure the
useful form where traditional immunoassay fails to distinguish the
forms and fails to specifically detect to useful biomarker.
[0060] One useful methodology combines mass spectrometry with
immunoassay. First, a biosepcific capture reagent (e.g., an
antibody, aptamer or Affibody that recognizes the biomarker and
other forms of it) is used to capture the biomarker of interest.
Preferably, the biospecific capture reagent is bound to a solid
phase, such as a bead, a plate, a membrane or an array. After
unbound materials are washed away, the captured analytes are
detected and/or measured by mass spectrometry. (This method also
will also result in the capture of protein interactors that are
bound to the proteins or that are otherwise recognized by
antibodies and that, themselves, can be biomarkers.) Various forms
of mass spectrometry are useful for dectecting the protein forms,
including laser desorption approaches, such as traditional MALDI or
SELDI, and electrospray ionization.
[0061] Thus, when reference is made herein to detecting a
particular protein or to measuring the amount of a particular
protein, it means detecting and measuring the protein with or
without resolving various forms of protein. For example, the step
of "measuring CTAP3" includes measuring CTAP3 by means that do not
differentiate between various forms of the protein (e.g., certain
immunoassays) as well as by means that differentiate some forms
from other forms or that measure a specific form of the protein. In
contrast, when it is desired to measure a particular form or forms
of a protein, e.g., a particular form of CTAP3, the particular form
(or forms) is specified. For example, "measuring M9313.9" means
measuring CTAP3 in a way that distinguishes M9313.9 from other
forms of CTAP3.
4. Detection of Biomarkers for Ovarian Cancer
[0062] The biomarkers of this invention can be detected by any
suitable method. Detection paradigms that can be employed to this
end include optical methods, electrochemical methods (voltametry
and amperometry techniques), atomic force microscopy, and radio
frequency methods, e.g., multipolar resonance spectroscopy.
Illustrative of optical methods, in addition to microscopy, both
confocal and non-confocal, are detection of fluorescence,
luminescence, chemiluminescence, absorbance, reflectance,
transmittance, and birefringence or refractive index (e.g., surface
plasmon resonance, ellipsometry, a resonant mirror method, a
grating coupler waveguide method or interferometry).
[0063] In one embodiment, a sample is analyzed by means of a
biochip. Biochips generally comprise solid substrates and have a
generally planar surface, to which a capture reagent (also called
an adsorbent or affinity reagent) is attached. Frequently, the
surface of a biochip comprises a plurality of addressable
locations, each of which has the capture reagent bound there.
[0064] Protein biochips are biochips adapted for the capture of
polypeptides. Many protein biochips are described in the art. These
include, for example, protein biochips produced by Ciphergen
Biosystems, Inc. (Fremont, Calif.), Zyomyx (Hayward, Calif.),
Invitrogen (Carlsbad, Calif.), Biacore (Uppsala, Sweden) and
Procognia (Berkshire, UK). Examples of such protein biochips are
described in the following patents or published patent
applications: U.S. Pat. No. 6,225,047 (Hutchens & Yip); U.S.
Pat. No. 6,537,749 (Kuimelis and Wagner); U.S. Pat. No. 6,329,209
(Wagner et al.); PCT International Publication No. WO 00/56934
(Englert et al.); PCT International Publication No. WO 03/048768
(Boutell et al.) and U.S. Pat. No. 5,242,828 (Bergstrom et
al.).
[0065] 4.1. Detection by Mass Spectrometry
[0066] In a preferred embodiment, the biomarkers of this invention
are detected by mass spectrometry, a method that employs a mass
spectrometer to detect gas phase ions. Examples of mass
spectrometers are time-of-flight, magnetic sector, quadrupole
filter, ion trap, ion cyclotron resonance, electrostatic sector
analyzer and hybrids of these.
[0067] In a further preferred method, the mass spectrometer is a
laser desorption/ionization mass spectrometer. In laser
desorption/ionization mass spectrometry, the analytes are placed on
the surface of a mass spectrometry probe, a device adapted to
engage a probe interface of the mass spectrometer and to present an
analyte to ionizing energy for ionization and introduction into a
mass spectrometer. A laser desorption mass spectrometer employs
laser energy, typically from an ultraviolet laser, but also from an
infrared laser, to desorb analytes from a surface, to volatilize
and ionize them and make them available to the ion optics of the
mass spectrometer. The analyis of proteins by LDI can take the form
of MALDI or of SELDI. The analyis of proteins by LDI can take the
form of MALDI or of SELDI.
[0068] Laser desorption/ionization in a single TOF instrument
typically is performed in linear extraction mode. Tandem mass
spectrometers can employ orthogonal extraction modes.
[0069] 4.1.1. SELDI
[0070] A preferred mass spectrometric technique for use in the
invention is "Surface Enhanced Laser Desorption and Ionization" or
"SELDI," as described, for example, in U.S. Pat. Nos. 5,719,060 and
6,225,047, both to Hutchens and Yip. This refers to a method of
desorption/ionization gas phase ion spectrometry (e.g., mass
spectrometry) in which an analyte (here, one or more of the
biomarkers) is captured on the surface of a SELDI mass spectrometry
probe.
[0071] SELDI also has been called is called "affinity capture mass
spectrometry" or "Surface-Enhanced Affinity Capture" ("SEAC"). This
version involves the use of probes that have a material on the
probe surface that captures analytes through a non-covalent
affinity interaction (adsorption) between the material and the
analyte. The material is variously called an "adsorbent," a
"capture reagent," an "affinity reagent" or a "binding moiety."
Such probes can be referred to as "affinity capture probes" and as
having an "adsorbent surface." The capture reagent can be any
material capable of binding an analyte. The capture reagent is
attached to the probe surface by physisorption or chemisorption. In
certain embodiments the probes have the capture reagent already
attached to the surface. In other embodiments, the probes are
pre-activated and include a reactive moiety that is capable of
binding the capture reagent, e.g., through a reaction forming a
covalent or coordinate covalent bond. Epoxide and acyl-imidizole
are useful reactive moieties to covalently bind polypeptide capture
reagents such as antibodies or cellular receptors. Nitrilotriacetic
acid and iminodiacetic acid are useful reactive moieties that
function as chelating agents to bind metal ions that interact
non-covalently with histidine containing peptides. Adsorbents are
generally classified as chromatographic adsorbents and biospecific
adsorbents.
[0072] "Chromatographic adsorbent" refers to an adsorbent material
typically used in chromatography. Chromatographic adsorbents
include, for example, ion exchange materials, metal chelators
(e.g., nitrilotriacetic acid or iminodiacetic acid), immobilized
metal chelates, hydrophobic interaction adsorbents, hydrophilic
interaction adsorbents, dyes, simple biomolecules (e.g.,
nucleotides, amino acids, simple sugars and fatty acids) and mixed
mode adsorbents (e.g., hydrophobic attraction/electrostatic
repulsion adsorbents).
[0073] "Biospecific adsorbent" refers to an adsorbent comprising a
biomolecule, e.g., a nucleic acid molecule (e.g., an aptamer), a
polypeptide, a polysaccharide, a lipid, a steroid or a conjugate of
these (e.g., a glycoprotein, a lipoprotein, a glycolipid, a nucleic
acid (e.g., DNA)-protein conjugate). In certain instances, the
biospecific adsorbent can be a macromolecular structure such as a
multiprotein complex, a biological membrane or a virus. Examples of
biospecific adsorbents are antibodies, receptor proteins and
nucleic acids. Biospecific adsorbents typically have higher
specificity for a target analyte than chromatographic adsorbents.
Further examples of adsorbents for use in SELDI can be found in
U.S. Pat. No. 6,225,047. A "bioselective adsorbent" refers to an
adsorbent that binds to an analyte with an affinity of at least
10.sup.-8 M.
[0074] Protein biochips produced by Ciphergen comprise surfaces
having chromatographic or biospecific adsorbents attached thereto
at addressable locations. Ciphergen's ProteinChip.RTM. arrays
include NP20 (hydrophilic); H4 and H50 (hydrophobic); SAX-2, Q-10
and (anion exchange); WCX-2 and CM-10 (cation exchange); IMAC-3,
IMAC-30 and IMAC-50 (metal chelate); and PS-10, PS-20 (reactive
surface with acyl-imidizole, epoxide) and PG-20 (protein G coupled
through acyl-imidizole). Hydrophobic ProteinChip arrays have
isopropyl or nonylphenoxy-poly(ethylene glycol)methacrylate
functionalities. Anion exchange ProteinChip arrays have quaternary
ammonium functionalities. Cation exchange ProteinChip arrays have
carboxylate functionalities. Immobilized metal chelate ProteinChip
arrays have nitrilotriacetic acid functionalities (IMAC 3 and IMAC
30) or O-methacryloyl-N,N-bis-carboxymethyl tyrosine funtionalities
(IMAC 50) that adsorb transition metal ions, such as copper,
nickel, zinc, and gallium, by chelation. Preactivated ProteinChip
arrays have acyl-imidizole or epoxide functional groups that can
react with groups on proteins for covalent binding.
[0075] Such biochips are further described in: U.S. Pat. No.
6,579,719 (Hutchens and Yip, "Retentate Chromatography," Jun. 17,
2003); U.S. Pat. No. 6,897,072 (Rich et al., "Probes for a Gas
Phase Ion Spectrometer," May 24, 2005); U.S. Pat. No. 6,555,813
(Beecher et al., "Sample Holder with Hydrophobic Coating for Gas
Phase Mass Spectrometer," Apr. 29, 2003); U.S. Patent Publication
No. U.S. 2003-0032043 A1 (Pohl and Papanu, "Latex Based Adsorbent
Chip," Jul. 16, 2002); and PCT International Publication No. WO
03/040700 (Um et al., "Hydrophobic Surface Chip," May 15, 2003);
U.S. Patent Publication No. US 2003-0218130 A1 (Boschetti et al.,
"Biochips With Surfaces Coated With Polysaccharide-Based
Hydrogels," Apr. 14, 2003) and U.S. Pat. No. 7,045,366 (Huang et
al., "Photocrosslinked Hydrogel Blend Surface Coatings," May 16,
2006).
[0076] In general, a probe with an adsorbent surface is contacted
with the sample for a period of time sufficient to allow the
biomarker or biomarkers that may be present in the sample to bind
to the adsorbent. After an incubation period, the substrate is
washed to remove unbound material. Any suitable washing solutions
can be used; preferably, aqueous solutions are employed. The extent
to which molecules remain bound can be manipulated by adjusting the
stringency of the wash. The elution characteristics of a wash
solution can depend, for example, on pH, ionic strength,
hydrophobicity, degree of chaotropism, detergent strength, and
temperature. Unless the probe has both SEAC and SEND properties (as
described herein), an energy absorbing molecule then is applied to
the substrate with the bound biomarkers.
[0077] In yet another method, one can capture the biomarkers with a
solid-phase bound immuno-adsorbent that has antibodies that bind
the biomarkers. After washing the adsorbent to remove unbound
material, the biomarkers are eluted from the solid phase and
detected by applying to a SELDI biochip that binds the biomarkers
and analyzing by SELDI.
[0078] The biomarkers bound to the substrates are detected in a gas
phase ion spectrometer such as a time-of-flight mass spectrometer.
The biomarkers are ionized by an ionization source such as a laser,
the generated ions are collected by an ion optic assembly, and then
a mass analyzer disperses and analyzes the passing ions. The
detector then translates information of the detected ions into
mass-to-charge ratios. Detection of a biomarker typically will
involve detection of signal intensity. Thus, both the quantity and
mass of the biomarker can be determined.
[0079] 4.1.2. SEND
[0080] Another method of laser desorption mass spectrometry is
called Surface-Enhanced Neat Desorption ("SEND"). SEND involves the
use of probes comprising energy absorbing molecules that are
chemically bound to the probe surface ("SEND probe"). The phrase
"energy absorbing molecules" (EAM) denotes molecules that are
capable of absorbing energy from a laser desorption/ionization
source and, thereafter, contribute to desorption and ionization of
analyte molecules in contact therewith. The EAM category includes
molecules used in MALDI, frequently referred to as "matrix," and is
exemplified by cinnamic acid derivatives, sinapinic acid (SPA),
cyano-hydroxy-cinnamic acid (CHCA) and dihydroxybenzoic acid,
ferulic acid, and hydroxyaceto-phenone derivatives. In certain
embodiments, the energy absorbing molecule is incorporated into a
linear or cross-linked polymer, e.g., a polymethacrylate. For
example, the composition can be a co-polymer of
.alpha.-cyano-4-methacryloyloxycinnamic acid and acrylate. In
another embodiment, the composition is a co-polymer of
.alpha.-cyano-4-methacryloyloxycinnamic acid, acrylate and
3-(tri-ethoxy)silyl propyl methacrylate. In another embodiment, the
composition is a co-polymer of
.alpha.-cyano-4-methacryloyloxycinnamic acid and
octadecylmethacrylate ("C18 SEND"). SEND is further described in
U.S. Pat. No. 6,124,137 and PCT International Publication No. WO
03/64594 (Kitagawa, "Monomers And Polymers Having Energy Absorbing
Moieties Of Use In Desorption/Ionization Of Analytes," Aug. 7,
2003).
[0081] SEAC/SEND is a version of laser desorption mass spectrometry
in which both a capture reagent and an energy absorbing molecule
are attached to the sample presenting surface. SEAC/SEND probes
therefore allow the capture of analytes through affinity capture
and ionization/desorption without the need to apply external
matrix. The C18 SEND biochip is a version of SEAC/SEND, comprising
a C18 moiety which functions as a capture reagent, and a CHCA
moiety which functions as an energy absorbing moiety.
[0082] 4.1.3. SEPAR
[0083] Another version of LDI is called Surface-Enhanced
Photolabile Attachment and Release ("SEPAR"). SEPAR involves the
use of probes having moieties attached to the surface that can
covalently bind an analyte, and then release the analyte through
breaking a photolabile bond in the moiety after exposure to light,
e.g., to laser light (see, U.S. Pat. No. 5,719,060). SEPAR and
other forms of SELDI are readily adapted to detecting a biomarker
or biomarker profile, pursuant to the present invention.
[0084] 4.1.4. MALDI
[0085] MALDI is a traditional method of laser desorption/ionization
used to analyte biomolecules such as proteins and nucleic acids. In
one MALDI method, the sample is mixed with matrix and deposited
directly on a MALDI array. However, the complexity of biological
samples such as serum and urine makes this method less than optimal
without prior fractionation of the sample. Accordingly, in certain
embodiments with biomarkers are preferably first captured with
biospecific (e.g., an antibody) or chromatographic materials
coupled to a solid support such as a resin (e.g., in a spin
column). Specific affinity materials that bind the biomarkers of
this invention are described above. After purification on the
affinity material, the biomarkers are eluted and then detected by
MALDI.
[0086] In another mass spectrometry method, the biomarkers can be
first captured on a chromatographic resin having chromatographic
properties that bind the biomarkers. In the present example, this
could include a variety of methods. For example, one could capture
the biomarkers on a cation exchange resin, such as CM Ceramic
HyperD F resin, wash the resin, elute the biomarkers and detect by
MALDI. Alternatively, this method could be preceded by
fractionating the sample on an anion exchange resin before
application to the cation exchange resin. In another alternative,
one could fractionate on an anion exchange resin and detect by
MALDI directly. In yet another method, one could capture the
biomarkers on an immuno-chromatographic resin that comprises
antibodies that bind the biomarkers, wash the resin to remove
unbound material, elute the biomarkers from the resin and detect
the eluted biomarkers by MALDI or by SELDI.
[0087] 4.1.5. Other Forms of Ionization in Mass Spectrometry
[0088] In another method, the biomarkers are detected by LC-MS or
LC-LC-MS. This involves resolving the proteins in a sample by one
or two passes through liquid chromatography, followed by mass
spectrometry analysis, typically electrospray ionization.
[0089] 4.1.6. Data Analysis
[0090] Analysis of analytes by time-of-flight mass spectrometry
generates a time-of-flight spectrum. The time-of-flight spectrum
ultimately analyzed typically does not represent the signal from a
single pulse of ionizing energy against a sample, but rather the
sum of signals from a number of pulses. This reduces noise and
increases dynamic range. This time-of-flight data is then subject
to data processing. In Ciphergen's ProteinChip.RTM. software, data
processing typically includes TOF-to-M/Z transformation to generate
a mass spectrum, baseline subtraction to eliminate instrument
offsets and high frequency noise filtering to reduce high frequency
noise.
[0091] Data generated by desorption and detection of biomarkers can
be analyzed with the use of a programmable digital computer. The
computer program analyzes the data to indicate the number of
biomarkers detected, and optionally the strength of the signal and
the determined molecular mass for each biomarker detected. Data
analysis can include steps of determining signal strength of a
biomarker and removing data deviating from a predetermined
statistical distribution. For example, the observed peaks can be
normalized, by calculating the height of each peak relative to some
reference.
[0092] The computer can transform the resulting data into various
formats for display. The standard spectrum can be displayed, but in
one useful format only the peak height and mass information are
retained from the spectrum view, yielding a cleaner image and
enabling biomarkers with nearly identical molecular weights to be
more easily seen. In another useful format, two or more spectra are
compared, conveniently highlighting unique biomarkers and
biomarkers that are up- or down-regulated between samples. Using
any of these formats, one can readily determine whether a
particular biomarker is present in a sample.
[0093] Analysis generally involves the identification of peaks in
the spectrum that represent signal from an analyte. Peak selection
can be done visually, but software is available, as part of
Ciphergen's ProteinChip.RTM. software package, that can automate
the detection of peaks. In general, this software functions by
identifying signals having a signal-to-noise ratio above a selected
threshold and labeling the mass of the peak at the centroid of the
peak signal. In one useful application, many spectra are compared
to identify identical peaks present in some selected percentage of
the mass spectra. One version of this software clusters all peaks
appearing in the various spectra within a defined mass range, and
assigns a mass (M/Z) to all the peaks that are near the mid-point
of the mass (M/Z) cluster.
[0094] Software used to analyze the data can include code that
applies an algorithm to the analysis of the signal to determine
whether the signal represents a peak in a signal that corresponds
to a biomarker according to the present invention. The software
also can subject the data regarding observed biomarker peaks to
classification tree or ANN analysis, to determine whether a
biomarker peak or combination of biomarker peaks is present that
indicates the status of the particular clinical parameter under
examination. Analysis of the data may be "keyed" to a variety of
parameters that are obtained, either directly or indirectly, from
the mass spectrometric analysis of the sample. These parameters
include, but are not limited to, the presence or absence of one or
more peaks, the shape of a peak or group of peaks, the height of
one or more peaks, the log of the height of one or more peaks, and
other arithmetic manipulations of peak height data.
[0095] 4.1.7. General Protocol for SELDI Detection of Biomarkers
for Ovarian Cancer
[0096] A preferred protocol for the detection of the biomarker of
this invention is as follows. The biological sample to be tested,
e.g., serum, preferably is subject to pre-fractionation before
SELDI analysis. This simplifies the sample and improves
sensitivity. A preferred method of pre-fractionation involves
contacting the sample with an anion exchange chromatographic
material, such as Q HyperD (BioSepra, SA). The bound materials are
then subject to stepwise pH elution using buffers at pH 9, pH 7, pH
5, pH 4, pH 3 and organic wash. Various fractions containing the
biomarker are collected. CTAP3 is found in organic wash fraction
and also can be found in the pH 4 fraction.
[0097] The sample to be tested (preferably pre-fractionated) is
then contacted with an affinity capture probe comprising an IMAC-Cu
biochip. The probe is washed with a buffer that will retain the
biomarker while washing away unbound molecules. A suitable wash for
each biomarker is the buffer identified in Table 1. The biomarkers
are detected by laser desorption/ionization mass spectrometry.
[0098] Alternatively, if antibodies that recognize CTAP3 the
biomarker are available, these can be attached to the surface of a
probe, such as a pre-activated PS10 or PS20 ProteinChip array
(Ciphergen Biosystems, Inc.). These antibodies can capture the
biomarkers from a sample onto the probe surface. Then the
biomarkers can be detected by, e.g., laser desorption/ionization
mass spectrometry.
[0099] 4.2. Detection by Immunoassay
[0100] In another embodiment of the invention, the biomarkers of
the invention are measured by a method other than mass spectrometry
or other than methods that rely on a measurement of the mass of the
biomarker. In one such embodiment that does not rely on mass, the
biomarkers of this invention are measured by immunoassay.
Immunoassay requires biospecific capture reagents, such as
antibodies, to capture the biomarkers. Antibodies can be produced
by methods well known in the art, e.g., by immunizing animals with
the biomarkers. Biomarkers can be isolated from samples based on
their binding characteristics. Alternatively, if the amino acid
sequence of a polypeptide biomarker is known, the polypeptide can
be synthesized and used to generate antibodies by methods well
known in the art.
[0101] This invention contemplates traditional immunoassays
including, for example, sandwich immunoassays including ELISA or
fluorescence-based immunoassays, as well as other enzyme
immunoassays. Nephelometry is an assay done in liquid phase, in
which antibodies are in solution. Binding of the antigen to the
antibody results in changes in absorbance, which is measured. In
the SELDI-based immunoassay, a biospecific capture reagent for the
biomarker is attached to the surface of an MS probe, such as a
pre-activated ProteinChip array. The biomarker is then specifically
captured on the biochip through this reagent, and the captured
biomarker is detected by mass spectrometry.
5. Determination of Subject Ovarian Cancer Status
[0102] The biomarkers of the invention can be used in diagnostic
tests to assess ovarian cancer status in a subject, e.g., to
diagnose ovarian cancer. The phrase "ovarian cancer status"
includes any distinguishable manifestation of the disease,
including non-disease. For example, ovarian cancer status includes,
without limitation, the presence or absence of disease (e.g.,
ovarian cancer v. non-ovarian cancer), the risk of developing
disease, the stage of the disease, the progression of disease
(e.g., progress of disease or remission of disease over time) and
the effectiveness or response to treatment of disease.
[0103] The correlation of test results with ovarian cancer status
involves applying a classification algorithm of some kind to the
results to generate the status. The classification algorithm may be
as simple as determining whether or not the amount of a
CTAP3-related protein, e.g., CTAP3, measured is above or below a
particular cut-off number. When multiple biomarkers are used, the
classification algorithm may be a linear regression formula.
Alternatively, the classification algorithm may be the product of
any of a number of learning algorithms described herein.
[0104] In the case of complex classification algorithms, it may be
necessary to perform the algorithm on the data, thereby determining
the classification, using a computer, e.g., a programmable digital
computer. In either case, one can then record the status on
tangible medium, for example, in computer-readable format such as a
memory drive or disk or simply printed on paper. The result also
could be reported on a computer screen.
[0105] 5.1. Single Markers
[0106] The biomarker of the invention can be used in diagnostic
tests to assess ovarian cancer status in a subject, e.g., to
diagnose ovarian cancer. The phrase "ovarian cancer status"
includes any distinguishable manifestation of the disease,
including non-disease. For example, disease status includes,
without limitation, the presence or absence of disease (e.g.,
ovarian cancer v. non-ovarian cancer), the risk of developing
disease, the stage of the disease, the progress of disease (e.g.,
progress of disease or remission of disease over time) and the
effectiveness or response to treatment of disease. Based on this
status, further procedures may be indicated, including additional
diagnostic tests or therapeutic procedures or regimens.
[0107] The power of a diagnostic test to correctly predict status
is commonly measured as the sensitivity of the assay, the
specificity of the assay or the area under a receiver operated
characteristic ("ROC") curve. Sensitivity is the percentage of true
positives that are predicted by a test to be positive, while
specificity is the percentage of true negatives that are predicted
by a test to be negative. An ROC curve provides the sensitivity of
a test as a function of 1-specificity. The greater the area under
the ROC curve, the more powerful the predictive value of the test.
Other useful measures of the utility of a test are positive
predictive value and negative predictive value. Positive predictive
value is the percentage of people who test positive that are
actually positive. Negative predictive value is the percentage of
people who test negative that are actually negative.
[0108] The biomarkers of this invention show a statistical
difference in different ovarian cancer statuses of at least
p.ltoreq.0.05, p.ltoreq.10.sup.-2, p.ltoreq.10.sup.-3,
p.ltoreq.10.sup.-4 or p.ltoreq.10.sup.-5. Diagnostic test that use
these biomarkers alone or in combination show a sensitivity and
specificity of at least 75%, at least 80%, at least 85%, at least
90%, at least 95%, at least 98% and about 100%.
[0109] CTAP3 is differentially present in ovarian cancer, and,
therefore, each is individually useful in aiding in the
determination of ovarian cancer status. The method involves, first,
measuring the selected biomarker in a subject sample using the
methods described herein, e.g:, capture on a SELDI biochip followed
by detection by mass spectrometry and, second, comparing the
measurement with a diagnostic amount or cut-off that distinguishes
a positive ovarian cancer status from a negative ovarian cancer
status. The diagnostic amount represents a measured amount of a
biomarker above which or below which a subject is classified as
having a particular ovarian cancer status. For example, if the
biomarker is up-regulated compared to normal during ovarian cancer,
then a measured amount above the diagnostic cutoff provides a
diagnosis of ovarian cancer. Alternatively, if the biomarker is
down-regulated during ovarian cancer, then a measured amount below
the diagnostic cutoff provides a diagnosis of ovarian cancer. As is
well understood in the art, by adjusting the particular diagnostic
cut-off used in an assay, one can increase sensitivity or
specificity of the diagnostic assay depending on the preference of
the diagnostician. The particular diagnostic cut-off can be
determined, for example, by measuring the amount of the biomarker
in a statistically significant number of samples from subjects with
the different ovarian cancer statuses, as was done here, and
drawing the cut-off to suit the diagnostician's desired levels of
specificity and sensitivity.
[0110] 5.2. Combinations of Markers
[0111] While individual biomarkers are useful diagnostic
biomarkers, it has been found that a combination of biomarkers can
provide greater predictive value of a particular status than
asingle biomarker alone. Specifically, the detection of a plurality
of biomarkers in a sample can increase the sensitivity and/or
specificity of the test. A combination of at least two biomarkers,
preferably at least three or more than three biomarkers, is
sometimes referred to as a "biomarker profile" or "biomarker
fingerprint." Accordingly, a CTAP3-related protein, e.g., CTAP3,
can be combined with other biomarkers for ovarian or endometrial
cancer to improve the sensitivity and/or specificity of the
diagnostic test.
[0112] In particular, a diagnostic test for ovarian cancer status
involving the measurement of CTAP3 and any of the following
biomarkers for ovarian cancer identified in Table 3 (including
their modified forms where appropriate) can have greater predictive
power than the measurement of a CTAP3-related protein, e.g., CTAP3,
alone: TABLE-US-00003 TABLE 3 Marker Comments (up- or
down-regulated in cancer) Transferrin Down-regulated; 79 kD,
detected on IMAC ProteinChip array charged with nickel WO 03/057014
Haptoglobin Up-regulated; 9.2 kD detected on IMAC precursor
ProteinChip array charged with nickel protein WO 03/057014 fragment
ApoA1 Down-regulated; predicted mass 28078.62D; detected on IMAC or
H50 ProteinChip array. WO 2004/013609 Transthyretin Down-regulated;
predicted mass 13761D and and 12887 D, respectively; detected on
Q10 transthyretin ProteinChip array. delta N 10 WO 2004/013609
ITIH4 Up-regulated; among other fragments: internal
MNFRPGVLSSRQLGLPGPPDVPDHAAYHPF fragments (SEQ ID NO: 1), a fragment
spanning amino acids 660-689 of human Inter-alpha trypsin
inhibitor, heavy chain H4, predicted mass: 3273.72 D; detected on
IMAC ProteinChip array WO 2004/013609 and WO 2005/098447 Beta 2-
Up-regulated; detected at 11.7 KD on IMAC-Cu microglobulin
ProteinChip array US Provisional Application 60/693,679, filed Jun.
24, 2005 Hepcidin Up-regulated; detected by SELDI - and modified
co-precipitate with ITIH4 fragment. forms Hepcidin-25 (SEQ ID NO:
2): DTHFPICIFCCGCCHRSKCGMCCKT Hepcidin-24 (SEQ ID NO: 3):
THFPICIFCCGCCHRSKCGMCCKT Hepcidin-22 (SEQ ID NO: 3):
FPICIFCCGCCHRSKCGMCCKT Hepcidin-20 (SEQ ID NO: 4): ICIFCCGCCHRSKCGM
CCKT Haptoglobin Up-regulated. Detected at 11,600 D-11,700 D on
alpha an IMAC ProteinChip array charged with copper; WO 02/100242
Prostatin Up-regulated USP 6,846,642 Osteopontin Up-regulated In
urine - Glycosylated -- US 2005-0009120 A1 In serum - US
2005-0214826 Eosinophil- Up regulated in urine. Glycosylated
Detected at derived 17.4 KDa on a WCX2 ProteinChip array.
neurotoxin US 2005-0009120 A1 leptin Down-regulated; US
2005-0214826 prolactin Up-regulated; US 2005-0214826 IGF-II
Down-regulated; US 2005-0214826 Hemoglobin Up-regulated; (alpha- WO
2006-019906 hemoglobin, beta- hemoglobin) CA 125 Up-regulated
[0113] In a study on samples of a Japanese cohort, the combination
of hepcidin, ApoA1, .beta.2 microglobulin and CTAP-3 was found to
be a particularly effective diagnostic combination.
[0114] The diagnosis of ovarian cancer typically involves the
measurement of CA125, as increased levels of this marker are
correlated with ovarian cancer. Therefore, levels of CA125 can be
correlated with any combination of the above markers in determining
ovarian cancer status. CA125 is especially useful in that women
undergoing tests for ovarian cancer typically have CA125 tested as
routine part of the work-up.
[0115] Other biomarkers with which CTAP3 can be combined include,
but are not limited to, CA125 II, CA15-3, CA19-9, CA72-4, CA 195,
tumor associated trypsin inhibitor (TATI), CEA, placental alkaline
phosphatase (PLAP), Sialyl TN, galactosyltransferase, macrophage
colony stimulating factor (M-CSF, CSF-1), lysophosphatidic acid
(LPA), 110 kD component of the extracellular domain of the
epidermal growth factor receptor (p110OEGFR), tissue kallikreins,
e.g., kallikrein 6 and kallikrein 10 (NES-1), prostasin, HE4,
creatine kinase B (CKB), LASA, HER-2/neu, urinary gonadotropin
peptide, Dianon NB 70/K, Tissue peptide antigen (TPA), SMRP,
osteopontin, and haptoglobin, leptin, prolactin, insulin like
growth factor I or II.
[0116] 5.3. Determining Risk of Developing Disease
[0117] In one embodiment, this invention provides methods for
determining the risk of developing disease in a subject. Biomarker
amounts or patterns are characteristic of various risk states,
e.g., high, medium or low. The risk of developing a disease is
determined by measuring the relevant biomarker or biomarkers and
then either submitting them to a classification algorithm or
comparing them with a reference amount and/or pattern of biomarkers
that is associated with the particular risk level.
[0118] 5.4. Determining Stage of Disease
[0119] In one embodiment, this invention provides methods for
determining the stage of disease in a subject. Each stage of the
disease has a characteristic amount of a biomarker or relative
amounts of a set of biomarkers (a pattern). The stage of a disease
is determined by measuring the relevant biomarker Qr biomarkers and
then either submitting them to a classification algorithm or
comparing them with a reference amount and/or pattern of biomarkers
that is associated with the particular stage.
[0120] 5.5. Determining Course (Progression/Remission) of
Disease
[0121] In one embodiment, this invention provides methods for
determining the course of disease in a subject. Disease course
refers to changes in disease status over time, including disease
progression (worsening) and disease regression (improvement). Over
time, the amounts or relative amounts (e.g., the pattern) of the
biomarkers changes. For example, CTAP3 is increased with disease.
Therefore, the trend of these markers, either increased or
decreased over time toward diseased or non-diseased indicates the
course of the disease. Accordingly, this method involves measuring
one or more biomarkers in a subject at at least two different time
points, e.g., a first time and a second time, and comparing the
change in amounts, if any. The course of disease is determined
based on these comparisons.
[0122] 5.6. Reporting the Status
[0123] Additional embodiments of the invention relate to the
communication of assay results or diagnoses or both to technicians,
physicians or patients, for example. In certain embodiments,
computers will be used to communicate assay results or diagnoses or
both to interested parties, e.g., physicians and their patients. In
some embodiments, the assays will be performed or the assay results
analyzed in a country or jurisdiction which differs from the
country or jurisdiction to which the results or diagnoses are
communicated.
[0124] In a preferred embodiment of the invention, a diagnosis
based on the differential presence in a test subject of a
CTAP3-related protein is communicated to the subject as soon as
possible after the diagnosis is obtained. The diagnosis may be
communicated to the subject by the subject's treating physician.
Alternatively, the diagnosis may be sent to a test subject by email
or communicated to the subject by phone. A computer may be used to
communicate the diagnosis by email or phone. In certain
embodiments, the message containing results of a diagnostic test
may be generated and delivered automatically to the subject using a
combination of computer hardware and software which will be
familiar to artisans skilled in telecommunications. One example of
a healthcare-oriented communications system is described in U.S.
Pat. No. 6,283,761; however, the present invention is not limited
to methods which utilize this particular communications system. In
certain embodiments of the methods of the invention, all or some of
the method steps, including the assaying of samples, diagnosing of
diseases, and communicating of assay results or diagnoses, may be
carried out in diverse (e.g., foreign) jurisdictions.
[0125] 5.7. Subject Management
[0126] In certain embodiments of the methods of qualifying ovarian
cancer status, the methods further comprise managing subject
treatment based on the status. Such management includes the actions
of the physician or clinician subsequent to determining ovarian
cancer status. For example, if a physician makes a diagnosis of
ovarian cancer, then a certain regime of treatment, such as
prescription or administration of therapeutic agent, might follow.
Alternatively, a diagnosis of non-ovarian cancer or non-ovarian
cancer might be followed with further testing to determine a
specific disease that might the patient might be suffering from.
Also, if the diagnostic test gives an inconclusive result on
ovarian cancer status, further tests may be called for.
[0127] Additional embodiments of the invention relate to the
communication of assay results or diagnoses or both to technicians,
physicians or patients, for example. In certain embodiments,
computers will be used to communicate assay results or diagnoses or
both to interested parties, e.g., physicians and their patients. In
some embodiments, the assays will be performed or the assay results
analyzed in a country or jurisdiction which differs from the
country or jurisdiction to which the results or diagnoses are
communicated.
[0128] In a preferred embodiment of the invention, a diagnosis
based on the presence or absence in a test subject of the biomarker
is communicated to the subject as soon as possible after the
diagnosis is obtained. The diagnosis may be communicated to the
subject by the subject's treating physician. Alternatively, the
diagnosis may be sent to a test subject by email or communicated to
the subject by phone. A computer may be used to communicate the
diagnosis by email or phone. In certain embodiments, the message
containing results of a diagnostic test may be generated and
delivered automatically to the subject using a combination of
computer hardware and software which will be familiar to artisans
skilled in telecommunications. One example of a healthcare-oriented
communications system is described in U.S. Pat. No. 6,283,761;
however, the present invention is not limited to methods which
utilize this particular communications system. In certain
embodiments of the methods of the invention, all or some of the
method steps, including the assaying of samples, diagnosing of
diseases, and communicating of assay results or diagnoses, may be
carried out in diverse (e.g., foreign) jurisdictions.
[0129] 5.8. Determining Therapeutic Efficacy of Pharmaceutical
Drug
[0130] In another embodiment, this invention provides methods for
determining the therapeutic efficacy of a pharmaceutical drug.
These methods are useful in performing clinical trials of the drug,
as well as monitoring the progress of a patient on the drug.
Therapy or clinical trials involve administering the drug in a
particular regimen. The regimen may involve a single dose of the
drug or multiple doses of the drug over time. The doctor or
clinical researcher monitors the effect of the drug on the patient
or subject over the course of administration. If the drug has a
pharmacological impact on the condition, the amounts or relative
amounts (e.g., the pattern or profile) of the biomarkers of this
invention changes toward a non-disease profile. Therefore, one can
follow the course of the amounts of these biomarkers in the subject
during the course of treatment. Accordingly, this method involves
measuring one or more biomarkers in a subject receiving drug
therapy, and correlating the amounts of the biomarkers with the
disease status of the subject. One embodiment of this method
involves determining the levels of the one or more biomarkers at
least two different time points during a course of drug therapy,
e.g., a first time and a second time, and comparing the change in
amounts of the biomarkers, if any. For example, the one or more
biomarkers can be measured before and after drug administration or
at two different time points during drug administration. The effect
of therapy is determined based on these comparisons. If a treatment
is effective, then the one or more biomarkers will trend toward
normal, while if treatment is ineffective, the biomarkers will
trend toward disease indications. If a treatment is effective, then
the one or more biomarkers will trend toward normal, while if
treatment is ineffective, the one or more biomarkers will trend
toward disease indications.
6. Generation of Classification Algorithms for Qualifying Ovarian
Cancer Status
[0131] In some embodiments, data derived from the spectra (e.g.,
mass spectra or time-of-flight spectra) that are generated using
samples such as "known samples" can then be used to "train" a
classification model. A "known sample" is a sample that has been
pre-classified. The data that are derived from the spectra and are
used to form the classification model can be referred to as a
"training data set." Once trained, the classification model can
recognize patterns in data derived from spectra generated using
unknown samples. The classification model can then be used to
classify the unknown samples into classes. This can be useful, for
example, in predicting whether or not a particular biological
sample is associated with a certain biological condition (e.g.,
diseased versus non-diseased).
[0132] The training data set that is used to form the
classification model may comprise raw data or pre-processed data.
In some embodiments, raw data can be obtained directly from
time-of-flight spectra or mass spectra, and then may be optionally
"pre-processed" as described above.
[0133] Classification models can be formed using any suitable
statistical classification (or "learning") method that attempts to
segregate bodies of data into classes based on objective parameters
present in the data. Classification methods may be either
supervised or unsupervised. Examples of supervised and unsupervised
classification processes are described in Jain, "Statistical
Pattern Recognition: A Review", IEEE Transactions on Pattern
Analysis and Machine Intelligence, Vol. 22, No. 1, January 2000,
the teachings of which are incorporated by reference.
[0134] In supervised classification, training data containing
examples of known categories are presented to a learning mechanism,
which learns one or more sets of relationships that define each of
the known classes. New data may then be applied to the learning
mechanism, which then classifies the new data using the learned
relationships. Examples of supervised classification processes
include linear regression processes (e.g., multiple linear
regression (MLR), partial least squares (PLS) regression and
principal components regression (PCR)), binary decision trees
(e.g., recursive partitioning processes such as
CART--classification and regression trees), artificial neural
networks such as back propagation networks, discriminate analyses
(e.g., Bayesian classifier or Fischer analysis), logistic
classifiers, and support vector classifiers (support vector
machines).
[0135] A preferred supervised classification method is a recursive
partitioning process. Recursive partitioning processes use
recursive partitioning trees to classify spectra derived from
unknown samples. Further details about recursive partitioning
processes are provided in U.S. Patent Application No. 2002 0138208
A1 to Paulse et al., "Method for analyzing mass spectra."
[0136] In other embodiments, the classification models that are
created can be formed using unsupervised learning methods.
Unsupervised classification attempts to learn classifications based
on similarities in the training data set, without pre-classifying
the spectra from which the training data set was derived.
Unsupervised learning methods include cluster analyses. A cluster
analysis attempts to divide the data into "clusters" or groups that
ideally should have members that are very similar to each other,
and very dissimilar to members of other clusters. Similarity is
then measured using some distance metric, which measures the
distance between data items, and clusters together data items that
are closer to each other. Clustering techniques include the
MacQueen's K-means algorithm and the Kohonen's Self-Organizing Map
algorithm.
[0137] Learning algorithms asserted for use in classifying
biological information are described, for example, in PCT
International Publication No. WO 01/31580 (Barnhill et al.,
"Methods and devices for identifying patterns in biological systems
and methods of use thereof"), U.S. Patent Application No. 2002
0193950 A1 (Gavin et al., "Method or analyzing mass spectra"), U.S.
Patent Application No. 2003 0004402 A1 (Hitt et al., "Process for
discriminating between biological states based on hidden patterns
from biological data"), and U.S. Patent Application No. 2003
0055615 A1 (Zhang and Zhang, "Systems and methods for processing
biological expression data").
[0138] The classification models can be formed on and used on any
suitable digital computer. Suitable digital computers include
micro, mini, or large computers using any standard or specialized
operating system, such as a Unix, Windows.TM. or Linux.TM. based
operating system. The digital computer that is used may be
physically separate from the mass spectrometer that is used to
create the spectra of interest, or it may be coupled to the mass
spectrometer.
[0139] The training data set and the classification models
according to embodiments of the invention can be embodied by
computer code that is executed or used by a digital computer. The
computer code can be stored on any suitable computer readable media
including optical or magnetic disks, sticks, tapes, etc., and can
be written in any suitable computer programming language including
C, C++, visual basic, etc.
[0140] The learning algorithms described above are useful both for
developing classification algorithms for the biomarkers already
discovered, or for finding new biomarkers for ovarian cancer. The
classification algorithms, in turn, form the base for diagnostic
tests by providing diagnostic values (e.g., cut-off points) for
biomarkers used singly or in combination.
7. Compositions of Matter
[0141] In another aspect, this invention provides compositions of
matter based on the biomarker of this invention.
[0142] In one embodiment, this invention provides a biomarker of
this invention in purified form. Purified biomarkers have utility
as antigens to raise antibodies. Purified biomarkers also have
utility as standards in assay procedures. As used herein, a
"purified biomarker" is a biomarker that has been isolated from
other proteins and peptides, and/or other material from the
biological sample in which the biomarker is found. Biomarkers may
be purified using any method known in the art, including, but not
limited to, mechanical separation (e.g., centrifugation), ammonium
sulphate precipitation, dialysis (including size-exclusion
dialysis), size-exclusion chromatography, affinity chromatography,
anion-exchange chromatography, cation-exchange chromatography, and
methal-chelate chrmatography. Such methods may be performed at any
appropriate scale, for example, in a chromatography column, or on a
biochip.
[0143] In another embodiment, this invention provides a biospecific
capture reagent, optionally in purified form, that specifically
binds a biomarker of this invention. In one embodiment, the
biospecific capture reagent is an antibody. Such compositions are
useful for detecting the biomarker in a detection assay, e.g., for
diagnostics.
[0144] In another embodiment, this invention provides an article
comprising a biospecific capture reagent that binds a biomarker of
this invention, wherein the reagent is bound to a solid phase. For
example, this invention contemplates a device comprising bead,
chip, membrane, monolith or microtiter plate derivatized with the
biospecific capture reagent. Such articles are useful in biomarker
detection assays.
[0145] In another aspect this invention provides a composition
comprising a biospecific capture reagent, such as an antibody,
bound to a biomarker of this invention, the composition optionally
being in purified form. Such compositions are useful for purifying
the biomarker or in assays for detecting the biomarker.
[0146] In another embodiment, this invention provides an article
comprising a solid substrate to which is attached an adsorbent,
e.g., a chromatographic adsorbent or a biospecific capture reagent,
to which is further bound a biomarker of this invention. In one
embodiment, the article is a biochip or a probe for mass
spectrometry, e.g., a SELDI probe. Such articles are useful for
purifying the biomarker or detecting the biomarker.
8. Kits for Detection of Biomarkers for Ovarian Cancer
[0147] In another aspect, the present invention provides kits for
qualifying ovarian cancer status, which kits are used to detect the
biomarker according to the invention. In one embodiment, the kit
comprises a solid support, such as a chip, a microtiter plate or a
bead or resin having a capture reagent attached thereon, wherein
the capture reagent binds a biomarker of the invention. Thus, for
example, the kits of the present invention can comprise mass
spectrometry probes for SELDI, such as ProteinChip.RTM. arrays. In
the case of biospecfic capture reagents, the kit can comprise a
solid support with a reactive surface, and a container comprising
the biospecific capture reagent.
[0148] The kit can also comprise a washing solution or instructions
for making a washing solution, in which the combination of the
capture reagent and the washing solution allows capture of the
biomarker or biomarkers on the solid support for subsequent
detection by, e.g., mass spectrometry. The kit may include more
than type of adsorbent, each present on a different solid
support.
[0149] In a further embodiment, such a kit can comprise
instructions for suitable operational parameters in the form of a
label or separate insert. For example, the instructions may inform
a consumer about how to collect the sample, how to wash the probe
or the particular biomarkers to be detected.
[0150] In yet another embodiment, the kit can comprise one or more
containers with biomarker samples, to be used as standard(s) for
calibration.
9. Determining Therapeutic Efficacy of Pharmaceutical Drug
[0151] In another embodiment, this invention provides methods for
determining the therapeutic efficacy of a pharmaceutical drug.
These methods are useful in performing clinical trials of the drug,
as well as monitoring the progress of a patient on the drug.
Therapy or clinical trials involve administering the drug in a
particular regimen. The regimen may involve a single dose of the
drug or multiple doses of the drug over time. The doctor or
clinical researcher monitors the effect of the drug on the patient
or subject over the course of administration. If the drug has a
pharmacological impact on the condition, the amounts or relative
amounts (e.g., the pattern or profile) of the biomarkers of this
invention changes toward a non-disease profile. For example,
hepcidin is increased with disease, while transthyretin is
decreased in disease. Therefore, one can follow the course of the
amounts of these biomarkers in the subject during the course of
treatment. Accordingly, this method involves measuring one or more
biomarkers in a subject receiving drug therapy, and correlating the
amounts of the biomarkers with the disease status of the subject.
One embodiment of this method involves determining the levels of
the biomarkers for at least two different time points during a
course of drug therapy, e.g., a first time and a second time, and
comparing the change in amounts of the biomarkers, if any. For
example, the biomarkers can be measured before and after drug
administration or at two different time points during drug
administration. The effect of therapy is determined based on these
comparisons. If a treatment is effective, then the biomarkers will
trend toward normal, while if treatment is ineffective, the
biomarkers will trend toward disease indications. If a treatment is
effective, then the biomarkers will trend toward normal, while if
treatment is ineffective, the biomarkers will trend toward disease
indications.
10. Use of Biomarkers for Ovarian Cancer in Screening Assays and
Methods of Treating Ovarian Cancer
[0152] The methods of the present invention have other applications
as well. For example, the biomarker can be used to screen for
compounds that modulate the expression of the biomarker in vitro or
in vivo, which compounds in turn may be useful in treating or
preventing ovarian cancer in patients. In another example, the
biomarkers can be used to monitor the response to treatments for
ovarian cancer. In yet another example, the biomarkers can be used
in heredity studies to determine if the subject is at risk for
developing ovarian cancer.
[0153] Thus, for example, the kits of this invention could include
a solid substrate having a hydrophobic function, such as a protein
biochip (e.g., a Ciphergen H50 ProteinChip array, e.g., ProteinChip
array) and a sodium acetate buffer for washing the substrate, as
well as instructions providing a protocol to measure the biomarkers
of this invention on the chip and to use these measurements to
diagnose ovarian cancer.
[0154] Compounds suitable for therapeutic testing may be screened
initially by identifying compounds which interact with one or more
CTAP3-related proteins. By way of example, screening might include
recombinantly expressing a CTAP3-related protein, e.g., CTAP3,
purifying the biomarker, and affixing the biomarker to a substrate.
Test compounds would then be contacted with the substrate,
typically in aqueous conditions, and interactions between the test
compound and the biomarker are measured, for example, by measuring
elution rates as a function of salt concentration. Certain proteins
may recognize and cleave CTAP3-related proteins, in which case the
proteins may be detected by monitoring the digestion of one or more
biomarkers in a standard assay, e.g., by gel electrophoresis of the
proteins.
[0155] In a related embodiment, the ability of a test compound to
inhibit the activity of one or more of the CTAP3-related proteins
may be measured. One of skill in the art will recognize that the
techniques used to measure the activity of a particular biomarker
will vary depending on the function and properties of the
biomarker. For example, an enzymatic activity of a biomarker may be
assayed provided that an appropriate substrate is available and
provided that the concentration of the substrate or the appearance
of the reaction product is readily measurable. The ability of
potentially therapeutic test compounds to inhibit or enhance the
activity of a given biomarker may be determined by measuring the
rates of catalysis in the presence or absence of the test
compounds. The ability of a test compound to interfere with a
non-enzymatic (e.g., structural) function or activity of a
CTAP3-related proteins may also be measured. For example, the
self-assembly of a multi-protein complex which includes one of the
CTAP3-related proteins may be monitored by spectroscopy in the
presence or absence of a test compound. Alternatively, if the
biomarker is a non-enzymatic enhancer of transcription, test
compounds which interfere with the ability of the biomarker to
enhance transcription may be identified by measuring the levels of
biomarker-dependent transcription in vivo or in vitro in the
presence and absence of the test compound.
[0156] Test compounds capable of modulating the activity of a
CTAP3-related protein may be administered to patients who are
suffering from or are at risk of developing ovarian cancer or other
cancer. For example, the administration of a test compound which
increases the activity of a particular biomarker may decrease the
risk of ovarian cancer in a patient if the activity of the
particular biomarker in vivo prevents the accumulation of proteins
for ovarian cancer. Conversely, the administration of a test
compound which decreases the activity of a particular biomarker may
decrease the risk of ovarian cancer in a patient if the increased
activity of the biomarker is responsible, at least in part, for the
onset of ovarian cancer.
[0157] In an additional aspect, the invention provides a method for
identifying compounds useful for the treatment of disorders such as
ovarian cancer which are associated with increased levels of
modified forms of CTAP3. For example, in one embodiment, cell
extracts or expression libraries may be screened for compounds
which catalyze the cleavage of full-length CTAP3 to form truncated
forms of CTAP3. In one embodiment of such a screening assay,
cleavage of CTAP3 may be detected by attaching a fluorophore to
CTAP3 which remains quenched when CTAP3 is uncleaved but which
fluoresces when the protein is cleaved. Alternatively, a version of
full-length CTAP3 modified so as to render the amide bond between
amino acids x and y uncleavable may be used to selectively bind or
"trap" the cellular protesase which cleaves full-length CTAP3 at
that site in vivo. Methods for screening and identifying proteases
and their targets are well-documented in the scientific literature,
e.g., in Lopez-Ottin et al. (Nature Reviews, 3:509-519 (2002)).
[0158] In yet another embodiment, the invention provides a method
for treating or reducing the progression or likelihood of a
disease, e.g., ovarian cancer, which is associated with the
increased levels of truncated CTAP3. For example, after one or more
proteins have been identified which cleave full-length CTAP3,
combinatorial libraries may be screened for compounds which inhibit
the cleavage activity of the identified proteins. Methods of
screening chemical libraries for such compounds are well-known in
art. See, e.g., Lopez-Otin et al. (2002). Alternatively, inhibitory
compounds may be intelligently designed based on the structure of
CTAP3.
[0159] N-terminal truncations of CTAP3 are thought to diminish
CTAP3's protease inhibitory activity. See, e.g., Abrahamson et al.
(Biochem. J. 273:621-626 (1991)). Compounds which impart truncated
CTAP3 with the functionality of full-length CTAP3 are likely
therefore to be useful in treating conditions, such as ovarian
cancer, which are associated with the truncated form of CTAP3.
Therefore, in a further embodiment, the invention provides methods
for identifying compounds which increase the affinity of truncated
CTAP3 for its target proteases. For example, compounds may be
screened for their ability to impart truncated CTAP3 with the
protease inhibitory activity of full-length CTAP3. Test compounds
capable of modulating the inhibitory activity of CTAP3 or the
activity of molecules which interact with CTAP3 may then be tested
in vivo for their ability to slow or stop the progression of
ovarian cancer in a subject. One of ordinary skill in the art would
understand that the methods exemplified above using CTAP3 are
applicable to any of the CTAP3-related proteins described
herein.
[0160] At the clinical level, screening a test compound includes
obtaining samples from test subjects before and after the subjects
have been exposed to a test compound. The levels in the samples of
one or more of the CTAP3-related proteins, e.g., CTAP3, 2 may be
measured and analyzed to determine whether the levels of the
biomarkers change after exposure to a test compound. The samples
may be analyzed by mass spectrometry, as described herein, or the
samples may be analyzed by any appropriate means known to one of
skill in the art. For example, the levels of a CTAP3-related
proteins may be measured directly by Western blot using radio- or
fluorescently-labeled antibodies which specifically bind to the
biomarkers. Alternatively, changes in the levels of mRNA encoding
the one or more biomarkers may be measured and correlated with the
administration of a given test compound to a subject. In a further
embodiment, the changes in the level of expression of one or more
of the biomarkers may be measured using in vitro methods and
materials. For example, human tissue cultured cells which express,
or are capable of expressing, one or more of the CTAP3-related
proteins may be contacted with test compounds. Subjects who have
been treated with test compounds will be routinely examined for any
physiological effects which may result from the treatment. In
particular, the test compounds will be evaluated for their ability
to decrease disease likelihood in a subject. Alternatively, if the
test compounds are administered to subjects who have previously
been diagnosed with ovarian cancer, test compounds will be screened
for their ability to slow or stop the progression of the
disease.
11. EXAMPLES
11.1. Example 1
Discovery of a Biomarker for Ovarian Cancer
Example 1
Profiling Methods:
[0161] Serum fractionation: 25 ul of supernatant were mixed with
37.5 ul of a denaturing buffer (U9: 9 M urea, 2% CHAPS, 50 mM Tris
pH 9.0) and vortexed for 30 minutes at 4 degrees. Add 37.5 ul of 50
mM Tris (pH9) to give a total volume of 100 ul. For each sample,
100 ul of Q Ceramic HyperD 20 anion exchange resin as 50%
suspension was equilibrated in 100 ul of 50 mM Tris (pH9) first,
then U1 buffer (U9 that was diluted 1:9 in 50 mM Tris pH 9.0) three
times. 100 ul of the denatured serum was applied to the resin and
allowed to bind for thirty minutes at 4 degrees. The unbound
material was collected and then 75 ul of wash buffer 1 (50 mM
Tris-HCl+0.1% OGP+50 mM Sodium Chloride, pH 9) was added to the
resin. The resin was agitated for 10 minutes on a Micromix. This
wash was collected and combined with the unbound material (flow
through; fraction 1). Fractions were then collected in a stepwise
pH gradient using two times 75 ul each aliquots of wash buffers at
pH 7, 5, 4, 3, and organic solvent. Each time the resin was
agitated for 10 minutes on a Micromix. This led to the collection
of a total of six fractions. The buffers are as follows: Wash
Buffer 2: 50 mM HEPES+0.1% OGP 50 mM Sodium Chloride (pH 7); Wash
Buffer 3: 100 mM Sodium Acetate+0.1% OGP+50 mM Sodium Chloride (pH
5); Wash Buffer 4: 100 mM Sodium Acetate+0.1% OGP+50 mM Sodium
Chloride (pH 4); Wash Buffer 5: 50 mM Sodium Citrate+0.1% OGP+50 mM
Sodium Chloride (pH 3); Wash Buffer 6: 33.3% 2-propanol/16.7%
acetonitrile/0.1% trifluoroacetic acid. Fractionation was performed
on a Tecan Aquurius 96 (Tecan) and a Micromix shaker (DPC). A
sample of control pooled human serum (Intergen) was processed
identically in one well of each column of samples.
Chip Binding:
[0162] 20 ul of each fraction was first pH adjusted with 20 ul of
different buffers and then bound to IMAC and CM10 ProteinChip
arrays. For IMAC arrays, the spots were charged with copper and
rinsed and equilibrated. Fractions 1 and 2 were mixed with 20 ul of
IMAC binding buffer (100 mM sodium phosphate pH 7.0 containing 500
mM NaCl); fractions 3-6 were mixed with 100 mM Tris HCl pH 10. For
CM10, fractions 1, 2 and 3 were mixed with 20 ul of 100 mM acetic
acid; fractions 4, 5 and 6 were mixed with 20 ul of CM10 binding
buffer (100 mM Na Acetate, pH 4.0). Binding was allowed to occur
for 120 minutes at room temperature. Chips were then washed two
times with 150 ul binding buffer and then twice with 200 ul water.
The matrix used was SPA (add 400 ul of 50% acetonitrile and 0.5%
TFA to one tube, mix 5 minutes). Each spot was deposited with 1 ul
of matrix twice. Chip binding was performed on a Tecan Aquurius 96
(Tecan) and a Micromix shaker (DPC).
Data Acquisition and Analysis:
[0163] ProteinChip arrays were read on PCS4000 instruments using
CiphergenExpress software version 3.0. Instruments were monitored
weekly for performance using insulin and immunoglobulin standards.
Each chip was read at two laser energies, low and high. Spectra
were organized and baseline subtracted. Spectra were externally
calibrated using a set of calibrants. Spectra were then normalized
to total ion current according to the following parameters: for
chips containing SPA, the low energy starting mass was 2000 M/Z;
the high energy starting mass was 10000 M/Z. For peak clustering,
the signal to noise ratio was set at 3.
Example 2
Chromatographic Assay of Ovarian Cancer Markers
[0164] Add 50 ul 50% suspension of IDA-Ni (II) beads (Biosepra IMAC
Hypercel, charged with 0.1M NiSO4) to 96-well filter plate.
Transfer IDA-Ni slurry to a plastic beaker and keep slurry well
mixed by hand or on magnetic stir plate at low speed during
dispensing. Use pipet tips with large orifice to dispense
beads.
[0165] Wash beads three times, each with 200 ul 0.02% (w/v) Triton
X100 PBS (2.times.). 5 ul serum+7.5 ul 9M urea 2% (w/v) CHAPS 50 mM
Tris HCl pH9 in v-bottom 96-well plate, RT vortex 2 min.
[0166] Dilute with 150 ul 0.02% (w/v) Triton X100 PBS (2.times.)
with protease inhibitor cocktail (Roche, no EDTA, 1 tablet per 50
ml).
[0167] Add to IDA-Ni plate, RT shake for 30 min on Micromix shaker
(settings: 15, 6, 30).
[0168] Vacuum filter on vacuum manifold.
[0169] Wash plate with 200 ul of 0.02% TX100 PBS (2.times.) eight
times, no mixing.
[0170] Gently blot dry bottom of filter plate on Kimwipe (remember
to dry edges of filter membrane).
[0171] Elute with 75 ul 10 mM imidazole 1M urea 0.1% (w/v) CHAPS
0.3M KCl 100 mM TrisHCl pH7.5 four times, mix 10 min each time (15,
6, 10). Vacuum filter to collect in 0.45 ml v-bottom 96-well
plate.
IMAC30 Chip Binding in Bioprocessor:
[0172] Add 50 ul 50 mM CuSO4 to each well. Shake for 10 min on
Micromix (15, 5, 10).
[0173] Empty wells. Add 150 ul water and empty wells.
[0174] Add 50 ul 50 mM NaOAc pH4 to each well. Shake for 5 min.
[0175] Empty wells. Add 150 ul water and empty wells.
[0176] Equilibrate 2 times 200 ul 1M urea 0.1% CHAPS 0.3M KCl 100
mM Tris HCl pH7.5.
[0177] Shake for 5 min each (15, 5, 5) on Micromix.
[0178] Mix eluate plate at low speed for 1 minute on Micromix.
[0179] Add 40 ul of eluate to 150 ul (final imidazole 2.1 mM) of 1M
urea 0.1% CHAPS 0.3M KCl 100 mM Tris HCl pH7.5 on IMAC30-Cu (II)
chip. Seal with tape and vortex 60 min 15, 6, 60) at RT.
[0180] Wash 1 time with 200 ul 1M urea 0.1% (w/v) CHAPS 0.3M KCl 50
mM TrisHCl pH7.5, mix 5 min.
[0181] Wash 2 times with 200 ul water, mix 1 min each.
[0182] Add 2 times 1 ul sinapinic acid. 1 tube SPA+200 ul ACN+200
ul 1% TFA.
[0183] Read chips using low laser intensity. Focus at 14 KDa.
Q10 Chip Binding in Bioprocessor:
[0184] Equilibrate 2 times 200 ul 0.1M sodium phosphate buffer
pH7.5. Shake for 5 min each (15, 5, 5).
[0185] Mix eluate plate at low speed for 1 minute on Micromix.
[0186] Add 40 ul of eluate to 150 ul (final imidazole 2.1 mM) of
0.1M sodium phosphate buffer pH7.5 on Q10 chip. Seal with tape and
vortex 60 min (15, 5, 60) at RT.
[0187] Wash 2 times with 200 ul 0.1M sodium phosphate pH7.5, mix 5
min each.
[0188] Wash 1 time with 200 ul water, mix 1 min.
[0189] Add 2 times 1 ul sinapinic acid. 1 tube SPA+200 ul ACN+200
ul 1% TFA.
[0190] Read chips using low and high laser intensity. Focus at 14
KDa.
Preparation of IDA-Ni Beads:
[0191] Measure 100 ml IMAC Hypercel (Biosepra) packed gel in a
graduated cylinder.
[0192] Transfer to a filter unit (0.45 .mu.m cellulose acetate).
Wash beads with 500 ml water.
[0193] Transfer packed beads to a 500 ml round bottle.
[0194] Add 100 ml 0.1M NiSO.sub.4 to beads and mix on rotator for 2
hour at RT.
[0195] Wash beads with 1000 ml water in filter unit.
[0196] Wash with 500 ml 2.times.PBS pH7.2.
[0197] Store IDA-Ni beads in 2.times.PBS pH7.2 as 50% slurry at
4.degree. C.
Materials and Reagents:
[0198] IMAC Hypercel (Biosepra)
[0199] Pipet tips with large orifice 1-200 ul (VWR 53503-612)
[0200] NiSO4.7H2O [0201] PBS pH7.2, 10.times. (GIBCO, dilute to
2.times.) [0202] Urea [0203] CHAPS (prepare 10% (w/v) CHAPS stock
solution in water) [0204] Tris base (to prepare U9CHAPS TrisHCl
pH9) [0205] HCl for adjusting pH of Tris base [0206] Triton X100
(prepare 1% (w/v) TX100 stock solution in water, dilute in
2.times.PBS to make 0.02%)
[0207] Complete protease inhibitor cocktail tablets, EDTA-free
(Roche, 1 873 580)
[0208] Silent Screen filter plate 96 well, Loprodyne membrane 1.2
.mu.m pore (Nalge Nunc, 256065)
[0209] Vacuum Manifold for 96-Well Plates [0210] Immidazole [0211]
KCl [0212] 1M Tris HCl pH7.5 (Invitrogen) [0213]
CuSO.sub.4.5H.sub.2O [0214] Sodium acetate and acetic acid (to make
50 mM sodium acetate buffer pH4) [0215] Sodium phosphate monobasic
and dibasic (to make 0.1M sodium phosphate buffer pH7.5) [0216]
Sinapinic acid (Ciphergen Biosystems) [0217] Acetonitrile [0218]
TFA [0219] IMAC30 chips [0220] Q10 chips
[0221] It is understood that the examples and embodiments described
herein are for illustrative purposes only and that various
modifications or changes in light thereof will be suggested to
persons skilled in the art and are to be included within the spirit
and purview of this application and scope of the appended claims.
All publications, patents, and patent applications cited herein are
hereby incorporated by reference in their entirety for all
purposes.
Sequence CWU 1
1
6 1 128 PRT Homo sapiens 1 Met Ser Leu Arg Leu Asp Thr Thr Pro Ser
Cys Asn Ser Ala Arg Pro 1 5 10 15 Leu His Ala Leu Gln Val Leu Leu
Leu Leu Ser Leu Leu Leu Thr Ala 20 25 30 Leu Ala Ser Ser Thr Lys
Gly Gln Thr Lys Arg Asn Leu Ala Lys Gly 35 40 45 Lys Glu Glu Ser
Leu Asp Ser Asp Leu Tyr Ala Glu Leu Arg Cys Met 50 55 60 Cys Ile
Lys Thr Thr Ser Gly Ile His Pro Lys Asn Ile Gln Ser Leu 65 70 75 80
Glu Val Ile Gly Lys Gly Thr His Cys Asn Gln Val Glu Val Ile Ala 85
90 95 Thr Leu Lys Asp Gly Arg Lys Ile Cys Leu Asp Pro Asp Ala Pro
Arg 100 105 110 Ile Lys Lys Ile Val Gln Lys Lys Leu Ala Gly Asp Glu
Ser Ala Asp 115 120 125 2 25 PRT Homo sapiens 2 Asp Thr His Phe Pro
Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg 1 5 10 15 Ser Lys Cys
Gly Met Cys Cys Lys Thr 20 25 3 24 PRT Homo sapiens 3 Thr His Phe
Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser 1 5 10 15 Lys
Cys Gly Met Cys Cys Lys Thr 20 4 20 PRT Homo sapiens 4 Ile Cys Ile
Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys Gly Met 1 5 10 15 Cys
Cys Lys Thr 20 5 30 PRT Homo sapiens 5 Met Asn Phe Arg Pro Gly Val
Leu Ser Ser Arg Gln Leu Gly Leu Pro 1 5 10 15 Gly Pro Pro Asp Val
Pro Asp His Ala Ala Tyr His Pro Phe 20 25 30 6 22 PRT Homo sapiens
6 Phe Pro Ile Cys Ile Phe Cys Cys Gly Cys Cys His Arg Ser Lys Cys 1
5 10 15 Gly Met Cys Cys Lys Thr 20
* * * * *